Self-assembly and switching of the bacterial flagellum

Keiichi Namba

Graduate School of Frontier Biosciences, Osaka University and Dynamic NanoMachine Project, ICORP, JST

3-4 Hikaridai, Seika, Kyoto 619-0237 Japan


The bacterial flagellum is made of a rotary motor and a long, thin helical propeller by means of which bacteria swim. The helical propeller is call the flagellar filament, which is a tubular structure with a diameter of about 20 nm but grows up to about 15 m long by self-assembly of about 30,000 copies of a single protein flagellin, and yet forms left- and right-handed helical forms, switching between these two in response to the reversal of the motor rotation, allowing bacteria to tumble and change their swimming direction for taxis. The motor is about 30 nm in diameter, spans the cell membrane, peptidoglycan layer, and outer membrane, and is made of a rotor, approximately eight stator units that surround it, a rotation switching regulator, a drive shaft, and a bushing. The hook, which connects the filament to the motor, is a short, highly flexible segment that functions as a universal joint. The axial structures of the flagellum are constructed by proteins translocated from the cytoplasm to the distal end, where different cap complexes help efficient self-assembly of these proteins. We have been trying to visualize the structure of the flagellum in atomic detail to understand how it self-assembles and works. X-ray crystal structures of core fragments of axial component proteins are now available. X-ray fiber diffraction gave high-resolution structural information. Electron cryomicroscopy also visualized the structures of the filament, the rotor and cap-filament complex, and recently enabled us to build an atomic model of the filament based on a 4 Å resolution map. All these structures have provided interesting implications for and insights into the mechanisms of each part of the flagellum, demonstrating the importance of dual nature of protein molecules, namely, flexibility and precision.




Non-Specific Nucleases Involved in Cell Defense

Hanna S. Yuan1*, Chia-Lung Li1,2, Kuo-Chiang Hsia1, Kin-Fu Chak3 and Lien-I Hor4

1Institute of Molecular Biology, Academia Sinica, Nan-Kang, Taipei, Taiwan.

2Institute of Biochemistry and Molecular Biology, National Taiwan University, Taipei, Taiwan.

3Institute of Biochemistry, National Yang-Ming University, Taipei, Taiwan.

4Department of Microbiology and Immunology, National Cheng-Kung University, Tainan, Taiwan


In bacteria, there are many non-specific nucleases participated in cell defense, such as the Escherichia coli toxin ColE7.  This toxin is expressed and released to kill other closely related bacterial cells by degrading their chromosomal DNA when the host bacteria encounter environmental stresses.  ColE7 bears an HNH motif that has been identified in more than two hundreds of prokaryotic and eukaryotic endonucleases, involved in DNA homing, restriction, repair or chromosome degradation.  The crystal structures of the endonuclease domain of ColE7 and its complex with DNA demonstrate for the first time how the HNH motif mediates its functions in DNA binding and hydrolysis.  The Vvn from Vibrio vulnificus is another example of a non-specific endonuclease involved in cell protection.  Vvn is capable of digesting both DNA and RNA.  It functions in the periplasm of host cells, preventing the uptake of foreign DNA during transformation.  The crystal structures of Vvn and Vvn in complex with a duplex DNA suggest the structural basis for its sequence-independent recognition of DNA and RNA.  Our data provide a solid foundation for a better understanding of the molecular mechanisms of non-specific endonucleases involved in the protection of bacterial cells and the recognition of DNA by sequence-independent DNA-binding proteins.




The crystal structure of the B. subtilis CodW, a new N-terminal Ser nucleophile peptidase, and its complex structure with E. coli HslV

Seong-Hwan Rho*, Hyun Ho Park*, Young Jun Lim*, Min Suk Kang#, Byung Kook Lim#, Ihn Sik Seong#,

Chin Ha Chung#, Jimin Wang, and Soo Hyun Eom*

*Department of Life Science, Kwangju Institute of Science and Technology, Gwangju 500-712, Korea

#School of Biological Sciences, Seoul National University, Seoul 151-742, Korea

Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue,

New Haven, CT06520-8114


Auto activation of the proteasome/HslV-like peptidases is self-cleaving to generate the N-terminal Thr nucleophile upon assembly. However, the homologous Bacillus subtilis CodW does not self-cleave at the highly conserved site; instead, it is processed with 5 extra residues at the N-terminus and is a new type of peptidases with the N-terminal Ser nucleophile. We have determined the 2.5Å-resolution crystal structure of the Bacillus subtilis CodW. After several cycles of refinement using NCS and TLS restraints, the structure reveals that the extra 5 residues occupy the putative substrate-binding site of the proteasome/HslV-like peptidases. The N-terminal Ser is stabilized by a hydrogen-bonding network of Thr116, Glu118 and a water molecule. A possible catalytic mechanism was anticipated. The structure also explains its inability of auto-cleaving at the usual site, providing insight into auto-processing of the proteasome/HslV-like peptidases. The hybrid complex structure of CodW-HslV (E. coli) was determined by molecular replacement at the 4.1Å-resolution. The typical double-donut configuration (U6W6W6U6) of the structure supports the previous observation that CodW may have a similar activation mechanism with that of HslV, despite its unique catalytic mechanism.




Study signaling mechanisms of programmed cell death in a single living cell

Donald C. Chang

Department of Biology, Hong Kong University of Science and Technology

Clear Water Bay, Hong Kong, China


Apoptosis (programmed cell death) is a very important cellular process that is essential for maintaining our normal physiological function. Due to its newly discovered roles in a variety of pathological disorders, including cancer, Alzheimer’s disease and auto-immune diseases, its study has attracted tremendous scientific interests in the past five years. Results from recent investigations have revealed that apoptosis is controlled by a complex network of signal transduction pathways. So far, most of the information obtained was based on in vitro studies using conventional biochemical methods. Due to the lack of synchrony in the apoptotic events within a cell population, sometimes it was difficult to determine the temporal relationship between two signaling steps based on the study of cell extracts. A new challenge now is to study the mechanisms of signal transduction using a biophysical approach, in which cellular events within an intact living cell can be directly monitored. Such an in vivo study is very difficult to do; it became feasible only recently due to the development of several new technologies, including the availability of color mutants of fluorescence proteins and advancement of digital imaging techniques. In this talk, I will summarize some of our research in this area. For example, we have used the GFP gene-fusion technique and living-cell imaging methods to examine the dynamic process of release of mitochondrial proteins during programmed cell death. This work has provided important insight on the regulation mechanisms of apoptosis.  Furthermore, we have developed novel molecular bio-sensors based on the fluorescence resonance energy transfer (FRET) method for detecting caspase activation within a single living cell. These sensors not only enable us to study the regulating mechanisms of enzyme activation in an in vivo condition, they also provide a powerful tool for discovering new drugs that are targeted to controlling the apoptotic process. Finally, using the FRET technique, we can now investigate protein-protein interaction within a living cell during various steps of programmed cell death. (Supported by RGC grants HKUST6109/01M and 6104/02M and UGC grant AoE/B-15/01).




Diffusion barrier in the initial segment membrane of neurons as revealed

by single molecule nanotechnologies

Chieko Nakada1, Kenneth Ritchie1, Yuichi Oba2, Mitsuhiro Nakamura2,3, Yoko Hotta1,2, Ryota Iino1,4,

Rinshi S. Kasai1, Kazuhiko Yamaguchi5, Takahiro Fujiwara1, & Akihiro Kusumi1

1Kusumi Membrane Organizer Project, ERATO/SORST , JST. Department of Biological Science and Institute for Advanced Research, Nagoya University. 2Graduate School of Bioagricultural Sciences, Nagoya University. 3Laboratory for Memory and Learning, Brain Science Institute, RIKEN. Present addresses: 4Department of Applied Physics and Chemistry, The University of Electro-Communications. 5Yoshida ATP System Project, ERATO, JST.

Address: Chikusa-ku, Furo-cho, Nagoya 464-8602, Japan


Formation and maintenance of polarized distributions of membrane proteins in the cell membrane are the absolute necessity for the function of polarized cells. In polarized neurons, various membrane proteins are localized either in the somatodendritic domain or the axon. However, since the plasma membrane is one continuous membrane, while neurons undergo polarized delivery of membrane proteins to each domain, they must somehow block diffusional mixing of delivered proteins between these domains. However, the presence of a diffusion barrier in the cell membrane of the axonal initial segment (IS), which separates these two domains, has been controversial: it is difficult to conceive a barrier mechanism by which diffusion in a fluid bilayer could be blocked. By studying the dynamics of a phospholipid, a major component of the cell membrane, at a single-molecule level in the plasma membrane of cultured neurons, we addressed the following questions: 1) does a diffusion barrier exist in the IS membrane; 2) if it exists, when is it formed during neuronal development; and 3) what is the mechanism by which it restricts diffusion. We have established that a diffusion barrier does exist in the IS membrane, and that the diffusion barrier is formed in neurons during 7-10 days after birth by the accumulation of various transmembrane proteins that are anchored to the dense actin-based membrane skeleton meshes being formed under the IS membrane.




Purification and Ion Channel Formation of a Pseudomonas

Peptide Toxin, Tolaasin


Young-Kee Kim*

Department of Agricultural Chemistry, Chungbuk National University,

Gaeshin-dong, Heungduk-gu, Cheongju, Chungbuk 361-763, Korea


Tolaasin is an antimicrobial peptide toxin produced by Pseudomonas tolaasii and consists of 18 amino acids with a molecular mass of 1,985 Da. It is a pore-forming toxin, and pore formation on the plasma membrane results in the disruption of various cell membranes. P. tolaasii causes a disease, known as brown blotch, on the cultivated mushrooms. P. tolaasii 6264 was isolated as a pathogenic bacterium from the dark brown and sunken caps of cultivated mushrooms with disease symptom. Bacteria were cultivated and tolaasin molecules were purified from the culture supernatant by several steps of chromatographies. Two isomers of tolaasin, Tol I and Tol II, were isolated from the purified tolaasin preparation as major and minor components, respectively. When the purified tolaasin was incorporated into lipid bilayer, two types of ion channels were identified based on the gating behaviors and conductances. The slope conductance of type 1 tolaasin channel was 150 pS with linear current vs. voltage relationship. The type 2 tolaasin channel had two subconductance states of 300 and 500 pS. Ion channel formation of tolaasin was concentration-dependent, and single channel current was successfully obtained at 0.6 unit tolaasin. At higher concentration than 0.8 units, membrane became unstable and highly fluctuating currents were observed. However, spike-shaped currents by incomplete and brief openings were obtained at the concentrations below 0.6 units. Tolaasin-induced microsomal 45Ca2+ leakages were measured from 45Ca2+-stored microsomes. Microsomal 45Ca2+ release was reached maximal level at 0.8 units. Interestingly, a small amount of release was also observed at low concentrations of tolaasin without forming a complete channel. These results show that tolaasin forms two types of ion channel and the formation of membrane channel is dose-dependent.




Structure and interactions of a malarial vaccine candidate, AMA1, from the parasite Plasmodium falciparum


Raymond S. Norton

The Walter and Eliza Hall Institute of Medical Research, Parkville, 3050, AUSTRALIA


Apical membrane antigen 1 (AMA1), a merozoite surface protein found in all species of Plasmodium, is a strong candidate for inclusion in a malarial vaccine. Recombinant AMA1 protected against P. fragile in monkeys and P. chabaudi adami in mice. P. falciparum AMA1 is a target of antibodies that inhibit merozoite invasion in vitro.

The 62-kDa ectodomain of AMA1 consists of three disulfide-stabilised domains. We have determined the solution structure of domain III of this ectodomain (14 kDa) using 15N- and 13C/15N-labelled samples. It has a well-defined disulfide-stabilised core interrupted by a disordered loop, and both the N- and C-terminal regions of the molecule are unstructured. The structured region includes all three disulfide bonds. Naturally-occurring mutations across 121 different P. falciparum strains in domain III that are located far apart in the primary sequence tend to cluster in the region of the disulfide core in the 3D structure. Nearly all the polymorphic sites have high solvent accessibility, consistent with their location in epitopes recognised by protective antibodies. The disulfide-bond stabilised conformation of the ectodomain was essential for protection, as the antigen was not an effective vaccine after reduction and alkylation.

Possible interactions of domain III with other parts of the ectodomain will be discussed, along with strategies for determining the structure of the full ectodomain.

The structures of a series of peptides identified by phage display as binding to AMA1 and blocking merozoite invasion into red blood cells will also be described. Secondary structure elements observed for these peptides in solution correlate remarkably well with their potency in binding to AMA1 and inhibiting merozoite invasion. As the key residues of these peptides are located in beta-turns they represent promising candidates for mimetic design.




Open-closed conformational change of H+-ATPase b subunit revealed by down-sizing NMR


Hideo Akutsu*, Hiromasa Yagi, Toshio Yamazaki1 and Masasuke Yoshida2

Institute for Protein Research, Osaka University, 1RIKEN, GSC and 2Tokyo Institute of Technology 3-2 Yamadaoka,

Suita 565-0871, Japan


F1-ATPase is a sophisticated molecular motor. It is suggested that a driving force of the F1 rotation is the conformational change of the b subunit, which carries the catalytic site, from open to closed forms on ligand binding. A new insight into the mechanism of the F1 rotation has emerged through an application of a novel NMR methodology to this large protein based on segmental isotope labeling and TROSY. The segmental isotope labeling was carried out using intein. 92% of the backbone signals were assigned. We made some mutants, which are likely to be related to the structural change, and analyzed those NMR spectra and ATPase activities. The movements of the side-chains of Lys-164 and Thr-165 have turned to be essential to complete closed form. The ligand binding gets the adenine pocket compact, which induces switching of the hydrogen bond of D252 with K164 to T165, resulting in the closed form. The flexibility in the nucleotide-binding domain may also play an important role. The two step conformational change would elucidate the molecular mechanism of the piston movement of the b subunit in the 90º-30º rotation of F1 motor.




Clean SEA-HSQC: a method to map solvent exposed amides in large non-deuterated proteins with gradient-enhanced HSQC

Donghai LIN, Kong Hung SZE, and Guang ZHU*
Dept. of Biochemistry, The Hong Kong University of Science and Technology,

Clear Water Bay, Kowloon, Hong Kong


The SEA-HSQC method selectively observes solvent exposed amide protons with a SEA element. This experiment can effectively eliminate both NOE contributions from aliphatic protons and exchange-relayed NOE contributions from fast exchanging hydroxyl or amine protons, and suppress artifacts due to longitudinal relaxation contributions during the mixing period. The SEA-HSQC experiment can be used to map the solvent exposed residues in proteins to resolve the problem of resonance overlap, facilitate ligand binding studies, and measure exchange rates, kex. We have successfully applied this method to map a binding site on a protein-ligand complex and study the protein folding processes.




Proteome Imaging of Model Macromolecular Systems


Li Xing, Leif Bergman, Josefina Nilsson, Lars Haag, Sunny Wu, Nicklas Lof, Joseph Wang, Anders Wallande,

Bomu Wu, Jan Sedzik, Lena Hammar, Kjell Hultenby, Henrik Garoff, and R Holland Cheng*

Karolinska Institute Structural Virology, Department of Biosciences Novum, 14157 Stockholm, Sweden.

*holland.cheng@biosci.ki.se; http://www.biosci.ki.se/kisv


Dynamic biological processes are thought to be a consequence of synergistic cooperativity and metastability. Principles describing these events have been demonstrated by many of these large molecular machines. By means of structural analyses, critical submolecular components of these complex machines often include local environments of non-equivalence where dynamic pathways of action can only be characterized by including less ordered transition states. With rapid-freezing procedure, it is now feasible to quantify chemical descriptors of many biological processes at various degrees of detail.

Proteome imaging provided snapshots of large-scale molecular assemblies as they are in the dynamic events of biological process. Image data are collected at liquid Nitrogen or Helium temperature in vitreous ice to localize the function-specific domains. Robust algorithms have also been implemented based on various grid transformations to allow effective averaging of massive image data set.

To follow the conformation of subunit proteins in virus life cycles, the essences of lateral interaction between viral proteins will be demonstrated by a lipid-enveloped virus, where glycoprotein interactions above the lipid membrane was found to have a direct role in guiding the maturation process of the virus. Structures of another non-enveloped virus will be further demonstrated in how they utilize receptor to mediate capsid disassembly and the subsequent genome release to re-entering the host cell.




Free Fitness as a Measure of Biological Information, the Determinant of Molecular Evolution


Yuzuru Husimi1,2* and Takuyo Aita2,3

1Dept. of Functional Materials Science, Saitama University, Saitama 338-8570 Japan, 2REDS group/JST,

Saitama Small Enterprise Promotion Corporation, SKIP-city, Kawaguchi 333-0844 Japan and 3Computational Biology Research Center, National Institute of Advanced Industrial Science and Technology, Tokyo 135-0064 Japan.


If the mutation rate is not zero (as in a realistic situation), the survivor in molecular evolution is not necessarily the fittest. We were able to present a quasi-species model in which the fittest vanished. The model suggested the effectiveness of the concept of free fitness that is an analogous variable to free energy in thermodynamics as the determinant of a spontaneous process in the presence of mutational or thermal fluctuation. The small population size is another cause of fluctuation. We formulated the dynamical behavior of an adaptive walk on various fitness landscapes, which is a theoretical model for an experiment of directed evolution (or evolutionary molecular engineering). We discovered a variable, DG= DW+TDS, as a Lyapunov function of the process in a simple case, where DW, T and DS are the fitness change, a temperature-like variable representing fluctuation force (dependent on the mutation rate and the screened population size), and Shannon’s entropy change, respectively. We called W free fitness. The adaptive walker tends to the maximum free fitness point rather than to the maximum fitness point. We also found that Einstein relation-like formula holds near the maximum free fitness point for this stochastic molecular evolution and DG/T is a Lyapunov function in a more complex case. Manfred Eigen has commented two aspects of information concepts, that is, extent and content. Our finding suggested that the Shannon’s information gain -DS corresponds to the extent, and DW/T, which can be called the fitness information gain, corresponds to the content. Thus DG/T can be called the biological information gain, which increases during molecular evolution process.




Optical visualization and measurement of biomolecule monolayer and biomolecule interaction as well as Proteinchip applications


Gang Jin

Institute of Mechanics, Chinese Academy of Sciences,

15, Bei-si-huan west Rd., Beijing 100080, China


With the development of nanoscience and nanotechnology, the visualization and measurement in nanoscale is of increasing importance. Mostly, the scanning electron microscope (SEM), the scanning tunnel microscope (STM), the atomic force microscope (AFM) and the scanning near-field optical microscope (SNOM), etc. with the resolution in the order of nanometer even better would be used. The conventional physical optical methods would hardly be considered since the restriction of optical diffraction to the order of wavelength, but it should not be an obstacle for measurements in nanoscale, maybe we are pleasantly surprised by its behavior.

The recently developed imaging ellipsometry based on the principle of polarized light which were recognized about 100 years ago, and its applications in visualization of biomolecule monolayers and biomolecule interaction process are presented in this report. When light illuminates on an object surface, the interaction between the light and the object will produce the reflection, the transmission and the absorption. In other words, the reflection wave and the transmission wave carry the physical optical information of the object. The classic theory of optics predicts that the analysis of polarized state of the reflection and transmission would characterize the properties of the object. Ellipsometry can detect the very small variation of amplitude and phase of light wave introduced by micro-amount of material on a surface. It’s a powerful technique for thin film analysis with the thickness resolution in the order of angstrom. Imaging ellipsometry is combined with conventional ellipsometry, CCD imaging technique and image processing of computer. It is able to analyze an entire surface simultaneously with the high resolution (vertical in angstrom and lateral in micron). It has become a powerful tool for the visualization and analysis of biomolecule monolayers and biomolecule interaction.

Biomolecule monolayers such as protein layers, physically being the phase object, which does not introduce any variation of the amplitude of the probe light, but a slight phase shift.  Further theoretical analysis shows that the thickness (or surface concentration) of biomolecule layers on the substrates with high refractive index is proportional to the square root of the detected light intensity. Therefore, the spatial distribution of biomolecule monolayers could be shown in 3-dimension image. A lot of experiments proved that the visualization of biomolecule monolayers was performed with the resolution of angstrom within a dynamic thickness range of several tens nanometer. Not only the macromolecule monolayers like albumin, but also small biomolecule monolayer like biotin could be detected. Therefore, it could be foreseen a prospect of wide applications in visualization in nanscale and bio-medicine. Some results such as protein competitive adsorption, real-time detection with label-free for bio-molecule interaction, biosensor and proteinchip, etc. are presented here.




Prolyl Cis-Trans Isomerization of Proline, Pseudoproline, and Oligoprolines

Young Kee Kang

Department of Chemistry, Chungbuk National University

Cheongju, Chungbuk 361-763, Korea


The proline (Pro) residue is unique in that its side chain is covalently bonded to the nitrogen atom of the peptide backbone.  This leads the backbone not to form a hydrogen bond and the N-Ca rotation to be rigid.  The pyrrolidine of Pro residue is a five-membered ring, which may adopt two distinct down- and up-puckered conformations that are almost equally favorable.  Pro residue has a relatively high intrinsic probability (~5-9%) of having the cis peptide bond preceding proline as compared with other amino acids (~0.05%) from the analysis of X-ray protein structures.

It has been suggested that the cis-trans isomerization of the X-Pro peptide bond is often involved in the rate-determining steps for folding and refolding of various proteins.  Recently, the peptidyl prolyl isomerases (PPIases) have been known to be involved in the cell replication and implicated in severe diseases such as cancer, AIDS, and Alzheimer’s disease.  From chemical and enzymatic catalysis of peptide bond isomerization, the attacks of nucleophile to the carbonyl carbon and of elctrophile to the prolyl imide nitrogen have been suggested to lower the rotational barrier by forming a tetrahedral skeleton at the imide nitrogen.  The correlations of the cis-trans isomerization with the prolyl puckering were reported by analyzing X-ray structures of proteins and peptides.

We report here the results on proline (Ac-Pro-NHMe), pseudoproline (Ac-Oxa-NHMe), and oligoprolines (Ac-(Pro)n-Nme2, n = 1, 2) calculated using the ab initio method with the CPCM reaction field theory at the HF/6-31+G(d) level to investigate conformational preferences and thermodynamic properties of the proline residue along the cis-trans isomerization in the gas phase and in solutions.




Jumping-Among-Minima Model: Unified View for Energy

Landscape of Native Proteins

Akio Kitao

Institute of Molecular and Cellular Biosciences, The University of Tokyo

Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan


A native protein has a large number of substates that are energetically comparable with one another. These substates are distinguishable by their conformations and are termed conformational substates. A protein fluctuates among these substates in the native state. For the understanding of the protein dynamics, it is essential to study the energy landscape. In this work, the protein energy landscape is investigated based on the concept of the Jumping-Among-Minima (JAM) model. In this concept, protein motion is divided into intra-substate and inter-substate motions. The following results have been shown already from the JAM model; Intra-substate motions are relatively fast motions. Local energy surfaces of individual conformational substates are nearly harmonic and mutually similar. Inter-substate motions, jumps from a certain energy minimum to others, take place as stochastic processes. The collective modes involved with the inter-substate motions dominantly determine the magnitude of the atomic fluctuations, although they occupy only few percents in the number of the degrees of freedom.

The concept of the JAM model is shown to be very useful to understand protein dynamics in the native state. We will demonstrate that the JAM model can explain various results obtained not by only molecular simulations but also by experiments, e.g., NMR, X-ray crystallography, and inelastic neutron scattering




Multiscale molecular simulations of protein kinase C

membrane targeting mechanism

Jung-Hsin Lin

School of Pharmacy, National Taiwan University, Taipei 100, Taiwan

No.1 Jen-Ai Road Section 1, Taipei, 100, Taiwan


Protein kinase C (PKC) family is an essential component of many signaling pathways that control growth, differentiation and malignant transformation. The C2 domain of PKCb has been identified to be responsible for the membrane targeting process. Recent kinetics experiments have shown that the membrane association of the PKCb C2 domain is a diffusion-controlled process, and therefore it will be suitable to conduct Brownian dynamics simulations for association rate constant evaluation, under the framework of Northup–Allison-McCammon scheme. However, due to the uncertainty about the calcium binding and about the reaction criteria of this bimolecular encounter, it is also desirable to conduct molecular dynamics to clarify the roles of individual calcium binding sites on the PKCb C2 domain.

In order to have a more realistic picture of the PKCb C2 membrane association, an atomic model of a small unilamellar vesicle (SUV) has been constructed for the Brownian dynamics simulation. This SUV was first built using a coarse-grained lipid model to determine the average location of each lipid in the inner and outer monolayer of the vesicle. Subsequently, atomic models of POPS and POPC lipids were then placed in these previously determined locations with ratios fulfilling experimental conditions, and Monte Carlo simulations with atomic force fields were then performed to reach an equilibrated structure of the whole vesicle. The solution of Poisson-Boltzmann equation was used to evaluate the PKCb C2/vesicle binding free energy in the Monte Carlo docking simulations and the systemic force in the Brownian dynamics simulations.

It is found that the optimal orientation and location of the PKCb C2 domain with respect to the vesicle are very similar to the binding mode of the homologous PKCα C2 domain, which is recently determined by X-ray crystallography. The simulated association kinetics is also in good agreement with experiments.




Non-Canonical Watson-Crick Base Pairs

Shan-Ho Chou

Institute of Biochemistry, National Chung-Hsing University, Taichung, 40227, Taiwan

Institute of Life Science, National Central University, Jung-Li, Taiwan


Canonical Watson-Crick G·C and A·T base pairs have been the milestone in modern Biochemistry. However, such canonical base pairs may not be the only stable forms in life, since in human, more than 97% of the genome is consisted of so-called “junk DNA” or tandem repeats, which may separate in vivo and form stable structures other than the familiar duplex structure. In the past few years, we have found three such stable non-canonical Watson-Crick base pairs. Stable existences of such non-canonical structures raise the possibility that they exhibit unusual biological functions. 

            Sheared Ganti·Csyn Base Pair: Stable single-residue DNA d(GXC) loop structures closed by a G·C base pair have been determined. The closing G·C base pair in these loops is not of the canonical Watson-Crick type, but adopts instead a unique sheared mode. The cytidine residue in the closing base pair is transformed into the rare syn conformation. The facile formation of compact d(GXC) loops closed by a unique sheared Ganti·Csyn base pair demonstrates the great potential of the single-stranded d(GXC) triplet repeats to fold into stable hairpins.

            Zipper-like Watson-Crick Base Pairs: In the 5’-d(GXA)/(AYG)-5’ motifs, the potential canonical X·Y H-bonding is not present. Instead, the central X/Y pairs are transformed into inter-strand stacks that are bracketed by sheared G·A base pairs. Such motifs are analogous to the previously studied (GGA)2 motif presumably present in the human centromeric (TGGAA)n tandem repeat sequence.

            Looped-Out and Perpendicular: In the novel Actinomycin D / 5’-(GXC/CYG)2-5’ complex structure, the central Watson-Crick X·Y base pair is completely disrupted. The looped-out bases are not disordered but interact perpendicularly with the stacked bases/chromophore and form specific hydrogen bonds with DNA. Such a dramatic manipulation of Watson-Crick base pair conformation by a potent anticancer drug provides interesting potential for discovering new type of DNA/protein interaction.


Shan-Ho Chou*, Ko-Hsin Chin & Andrew H.-J. Wang, (2003) Unusual DNA Hairpin and Duplex Motifs (review), Nucleic Acids Research, 31, 2461-2474.




Advanced Electron Paramagnetic Resonance in Metalloenzyme Research

Hong-In Lee1*, Jin-won Lee2, Tran-Chen Yang3, Robert Igarashi4, Mikhail Laryukhin3, Peter E. Doan3, Joshua Telser4,

Lance C. Seefeldt5, Dennis R. Dean6, Sa-Ouk Kang2, and Brian M. Hoffman3

1Department of Chemistry Education, Kyungpook National University, Daegu, 702-701, Korea, 2Department of Microbiology, College of Natural Science and Research Center for Microbiology, Seoul National University, Seoul, 151-742, Korea, 3Department of Chemistry, Northwestern University, Evanston, Illnois 60208, USA, 4Department of Chemistry, Roosevelt University, Chicago, Illnois 60605, USA, 5Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, USA, and 6Department of Biochemistry, Virginia Tech, Blacksburg, Virginia 24061, USA


Transition metals found in metalloenzymes often form the active sites to perform the key functions of the proteins. The information to be gathered in studying a metalloenzyme includes the composition, structure, bonding, and substrate/inhibitor/product interaction at the active site throughout the enzymatic cycle. Such information can be obtained by using electron paramagnetic resonance (EPR), which can detect the magnetic couplings between metals, ligands, and bound molecules. However, the couplings are in general not resolved in most metalloenzymes because of the ensemble signals of randomly distributed samples, and the chemical information is lost. Electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) recover this information by selectively detecting the couplings from the 'crystal-like' subsets of the samples. In this talk, antioxidant Ni-containing Superoxide Dismutase (Ni-SOD) and nitrogen-fixing Nirogenase are exampled to demonstrate how the structural and mechanistic information of the enzymes can be extracted by ENDOR and ESEEM.




NMR snap shots of a fluctuating protein structure.

Ubiquitin at 3 kbar

Ryo Kitahara, Shigeyuki Yokoyama‡†, and Kazuyuki Akasaka#*

Cellular Signaling Laboratory, RIKEN Harima Institute at Spring-8, 1-1-1 Kouto, Mikazuki-cho,

Sayo, Hyogo 679-5148, Japan

Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo,

Bunkyo-ku, Tokyo 113-0033, Japan

#Department of Biotechnological Science, Faculty of Biology-Oriented Science and Technology, Kinki University,

 930 Nishimitani, Uchita-cho, Wakayama 649-6493, Japan


Conformational fluctuation is a key to understanding protein function, but we know little about the actual structural or shape changes associated with the fluctuation. By using variable-pressure NMR, a general method is planned for elucidating the shape change of a protein molecule in solution associated with a conformational fluctuation in atomic coordinates (1). Here we utilize multi-dimensional NMR spectroscopy and determine average coordinates at different pressures, which are to give “NMR snap shots” of a fluctuating protein structure because of the intimate relationship between conformation and volume of a protein. The first target chosen is ubiquitin (pH 4.6 at 20 °C), whose average coordinates were determined at 30 bar and at 3 kbar using NOE distance and torsion angle constraints. The structure at 3 kbar revealed that the helix swings by ~3 Å outwardly with a simultaneous reorientation of the C-terminal segment carrying the reactive-site residue 76, forming an “open” platform suitable for enzyme recognition. Spin relaxation analysis at 3 kbar indicates that the conformational fluctuation takes place in the 10 microsecond time range.


(1) K. Akasaka, Highly fluctuating protein structures revealed by variable-pressure Nuclear Magnetic Resonance, Biochemistry (Current Topics) 42, 10875-10885 (2003).




Single Molecular and Cellular Fluorescence-Absorption Spectroscopy of Photosynthetic Systems at 4-300K


Shigeru Itoh1, Kana Sugiura1, Yuzuru Shibata1, Hiroyuki Mino1, Hitoshi Tamiaki2 and Yoshitaka Saga2

 1Division of Material Science, Graduate School of Science, Nagoya University, Chikusa, Nagoya 464-8602 Japan, 2Department of Bioscience and Biotechnology, Ritsumeikan University, Kusatsu, Shiga 525-8577 Japan


Fluorescence spectra of photosynthetic pigments in a single antenna complex and in a living cell are studied by a micro-spectrophotometry system.  The system is composed of an Ar laser, a 56cm monochrometer, a galvano (XY)-piezo (Z) scan system, a microscope, a liq-He cryostat and cooled CCD/photomultiplier with 0.2 micron spacial resolution and 0.1nm wavelength resolution.  Two approaches as below will be presented.

(1) Detection of molecular fluctuation at 10K of the single chlorosome complex isolated from a filamentous green bacterium Chloroflexus aurantiacus. The chlorosome contains rod-shaped aggregates of bacteriochlorophyll c that accumulate photon energy into bacteriochlorophyll a in base plate proteins in its inside.  Single chlorosomes showed different fluorescence peaks in correlation with the energy transfer ability showing the strucutral fluctuation.

(2) Evaluation of fluctuation of photosystem I and II pigment systems among different spots in a single cell, different cells in a single filament of cells and among whole number of cells in a living culture of a cyanobacterium Nostoc sp. The whole single cell analysis of chlorophyll a fluorescence revealed wide variability of living cells even with a same genetic background.




Structure-based Focal Adhesion Signaling Mediated by Syndecan Proteoglycans

Weontae Lee*, Jun Shin, Bon-Kyoung Koo, Hye-Seo Park

Department of Biochemistry and Protein Network Research Center, College of Science, Yonsei University, Korea

134 Shinchon-dong, Seodaemoon-Gu, Seoul 120-740, Korea


The syndecan, transmembrane proteoglycans are involved in the organization of cytoskeleton and/or actin microfilaments and they have important roles as cell surface receptors during cell-matrix interactions. Syndecans contain highly conserved transmembrane and cytoplasmic domain, however, each syndecan takes a part in their unique function(s) on cytoskeleton organization.  Syndecan-1 is involved in cell spreading at early stage, but not in focal adhesions and stress fiber formation. Syndecan-4 is mainly participated in cytoskeletal and membrane reorganization to form stress fibers and focal adhesions at the later stage of primary fibroblast spreading.  In contrast, syndecan-2 plays a role selectively in the induction of stress fiber formation and focal adhesion formation in Lewis lung carcinoma-derived P29 cells, but not in primary fibroblasts. Therefore, it is likely that the cytoplasmic domain of each syndecan has a unique regulatory mechanism for cytoskeleton organization. Especially, syndecan-4 interacts with P+DINS (4,5)P2. We have demonstrated that both PKCa and the cytoplasmic domain of syndecan-4 are essential components involved in the signaling process of focal adhesion formation. In addition, we have observed a dramatic conformational change in the C2 region via phosphorylation at Ser183, resulting regulation of its binding ability to PKC by preventing P+DINS (4,5)P2-dependent oligomerization. We have also performed NMR structural studies on PKCa and syndecan cytoplasmic domains interacts with P+DINS (4,5)P2. In this presentation, structure-functions of all four syndecans based on their solution structures will be discussed in detail.




Functional regulation of the main light-harvesting chlorophyll a/b-protein complex through conformational rearrangement

 Jing Leng, Hui Chen, Liangbi Li *

Photosynthesis Research Center, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, China


The light-harvesting chlorophyll a/b-protein complex of photosystem II(LHC II) is the most abundant pigment protein complex in green plants. The complex mainly serves as antenna to capture and transfer light energy to photosynthetic reaction center, and participates in the regulation of excitation energy distribution between two photosystems, the formation of stacking grana in thylakoid membrane, light protection and adaptation to environment. Therefore it is very important to study the relationship between the structure and function of LHCII. It has been shown that LHC II mainly exists in trimeric form in the thylakoid membrane of higher plants. The organization of LHC II trimer needs membrane lipids, while there is less knowledge regarding contribution of the membrane lipids in structure and function to the formation of LHC II trimer at present.

In this study, LHC II was isolated from spinach chloroplasts by high-speed centrifugation after a non-ionic detergent treatment. Native and SDS denaturing polyacrylamide gel electrophoresis analysis indicated that the isolated complex was a heterotrimer and consisted of three polypeptides with molecular weights of 29 KDa, 28KDa and 26KDa, respectively. The determination of membrane lipid and fatty acid compositions in the isolated LHCII showed that the LHCII contained the same four membrane lipids (MGDG, DGDG, PG and SQDG) as photosystem II. But the content of PG(phosphatidylglycerol) with unusual 16:1-trans-hexadecenoic acid as the predominant fatty acid chains was two times of that in photosystem II. The treatment of the isolated LHCII with phospholipase A2(PLA2) induced the dissociation of the complex to monomer. Re-formation of trimer from the monomer happened after adding PG into the dissociated LHCII. This phenomenon showed that PG was directly involved in the trimer organization and stabilazation. In addition, the absorption band at 475nm, 655nm and the fluorescence excitation peak at 480nm decreased obviously in the monomer, indicating that the energy transfer from Chl b to Chl a in the LHCII was effected  after deletion of PG. From above results, it was suggested that PG not only played an important role in LHCII trimer formation and stabilization but also effected the pigments binding and the energy transfer within the LHCII.

* Author for correspondence,E-mail:lbli@ns.ibcas.ac.cn




Torque-speed relationship of the Na+-driven flagellar motor

Akihiko Ishijima1* and  Yoshiyuki Sowa2

1Department of Applied Physics, School of Engineering, Nagoya University, 

2Department of Biophysical Engineering, Osaka University,

Furocho, Chikusa-Ku, Nagoya, Aichi 464-8603, Japan


The bacterial flagellar motor is a rotary molecular machine driven by the ion flux across the cytoplasmic membrane.  Each motor rotates a long (~10 mm) helical filament as propel that extends from a cell body, and the rotating filament supplies propulsion of locomotion for a cell.  The flagellar motors can be classified by coupling ion: H+-driven and Na+-driven. 

In this study, the torque-speed relationship of the Na+-driven flagellar motor of Vibrio alginolyticus was investigated.  To study mechanism of flagellar motor, using V. algyonolyticus is favorable in cotrolling rotational speed by changing external NaCl concentration or by adding of specific inhibitor for the Na+-channel, and in utilizing various mutants and gene manipulation.  The rotation rate of the motor was measured by following the position of a spherical bead, attached to a flagellar filament, using optical nanometry.  To estimate the generated torque in wide speed range, we changed the load exerting the flagellar motor from 0.5 to 20 pN nm s by using various-sized beads (0.46 – 1.65 mm in diameter).  In the presence of 50 mM NaCl, the torque is approximately constant (~4000 pN nm) at lower speeds (up to speeds of 300 Hz) and then decreases steeply, similar to the H+-driven flagellar motor of E. coli.  When external Na+ concentration is changed, the effect of NaCl concentration on the generated torque of motor in the high-speed region seems to be larger than that in low-speed.  This tendency could be reproduced by a simple kinetic model, which takes the association and dissociation of Na+ ion to the motor into consideration. 




Near-field and far-field single molecule fluorescence Spectroscopy

Wunshain Fann

Institute of Atomic and molecular Sciences, Academia Sinica and Dept. of Physics,

National Taiwan University


Single molecule detection is an emerging field not only important from a purely scientific point of view but also in light of its significant technological impact. The unique advantage of single molecule experiments is the ability to observe phenomena otherwise obscured in ensemble measurements, such as the distribution of spectral positions and shapes, and discrete fluctuations in intensity. It is possible to check the statistical assumptions used in the ensemble of molecules. In addition, single molecule spectroscopy naturally implies ultra-sensitive measurements, which should play important role in future proteomics and genomic research. In this talk, we will use single fluorescence spectroscopy to illustrate the power of this technique.




Single Molecule Imaging and Quantitative Analysis of Molecular Interactions Inside Cells

Makio TOKUNAGA1,2,3*

1National Institute of Genetics, Japan, 2The Graduate Universtiy for Advanced Studies, Japan,

3Research Center for Allergy and Immunology, RIKEN, Japan,

Mishima, Shizuoka 411-8540, Japan


Objective-type total internal reflection fluorescence microscopy enabled us to visualize single molecules on or just below the cell surfaces.  We have further developed a new single-molecule method to visualize individual molecules inside cells, thin-layer illumination microscopy.  Illumination with a thin-layered laser beam minimized background, and allowed us to visualize single fluorescent molecules up to the depth of about 10 µm from the glass-medium interface. 

Single-molecule images of GFP-importin beta on nuclear pore complexes have been obtained almost as clearly as those on cover glass surfaces.  Both visualization of single pores and single molecules enabled us to analyze images quantitatively at the molecular level and to obtain kinetic parameters in cells, i.e., retention time at the pore, number of molecules bound to pores, and binding constant. 

It was found that there were two kinds of binding site of importin beta on NPC in the absence of cargo and Ran-GTP, weak binding sites and strong ones.  The former gathers many proteins on NPC.  The latter vanished in the presence of cargo and in the absence of Ran-GTP, suggesting that it is related with the active site of transport. 

Thus, single molecule imaging has opened a new way to obtain quantitative information on kinetics of molecular interactions and elucidate molecular mechanisms in cells. 




Rapid Action and Local Synthesis of Neurosteroids in the Brain Hippocampus:

Their Role in Synaptic Transmission and Learning and Memory


Suguru Kawato

Dep. of Biophysics and Life Sciences, Graduate School of Arts and Sciences, Univ. of Tokyo at Komaba,

Meguro, Tokyo 153, Japan


Neurosteroids in the hippocampus are promising intracrine neuromodulators which influence learning and memory. Neurosteroids(includinf sex steroids) are synthesized de novo in the brain, independent of steroids from blood circulation. We demonstrated the Ca-driven synthesis of neurosteroids in rat hippocampal neurons. In pathway of neurosteroidogenesis, cholesterol is converted to pregnenolone, dehydroepiandrosterone, androstenedione, testosterone, estrone, and finally to estradiol. Upon stimulation of NMDA, NMDA receptor-mediated Ca2+ influx occurred, then significant production of pregnenolone, pregnenolone sulfate and estradiol was observed in the hippocampus. Coa-localization of steroidogenic enzymes (such as cytochromes P450scc, P450c17, P450arom) was observed in pyramidal neurons in the CA1-CA3 and granule neurons in DG using immunohistochemical staining of hippocampal slices. Immunoelectron microscopic analysis demonstrated that P45017a and P450arom were localized at synapses of principal neurons in the hippocampus. Synaptic estrogen receptor was also localized in principal neurons in the hippocampus. Electrophysiological investigations indicated that both pregnenolone sulfate and estradiol at nM concentrations acutely (<30min) enhanced the induction of long-term potentiation in hippocampal slices. Ca2+ imaging analysis of hippocampal neurons demonstrated that the application of pregnenolone sulfate enhanced the NMDA-induced Ca2+ influx, and that the application of estradiol induced a single Ca2+ spike which was mediated by estrogen receptor a-like protein.  Taken together, neurosteroid in the brain can be 4th generation neuromessenger. Note that 1st generation neuromessenger. is neurotransmitter (glutamate, etc.), 2nd generation is catecholamine (dopamine, etc) and 3rd generation is neuropeptide.


(1) S. Kawato, et al., in Methods in Enzymol. (2002) Vol. 357, 241-249 "Histological and Metabolism Analysis of P450 in Brain"

(2) K. Shibuya, et al., Biochim. Biophys. Acta (2003) 1619, 301-316 Hippocampal cytochrome P450s synthesize brain neurosteroids which are paracrine neuromodulators of synaptic signal transduction

(3) Y. Hojo, Y., et al., Proc. Natl. Acad. Sci. U.S.A., in the press (2003) “Adult male rat hippocampus synthesizes estradiol from pregnenolone by cytochromes P45017 and P450 aromatase localized in neurons.”




Roles of Rho-associated kinase and myosin light chain kinase in morphological and migratory defects of focal adhesion kinase-null Cells

Hong-Chen Chen

Graduate Institute of Biomedical Sciences, National Chung Hsing University, Taiwan

No.250 Huo-Kuang road, Taichung 40227, Taiwan


Fibroblasts derived from focal adhesion kinase (FAK)-null mouse embryos have a reduced migration rate and an increase in the number and size of peripherally localized adhesions (Ilic et al., (1995), Nature 377, 539-544). In this study, we have found that Y27632, a specific inhibitor for Rho-associated kinase (Rho-kinase), dramatically reversed the round cell morphology of FAK-/- cells to a spread fibroblast-like shape in 30 min and significantly enhanced their motility. The effects of Y27632 on the FAK-/- cell morphology and motility were concomitant with reorganization of the actin cytoskeleton and redistribution of focal adhesions. Conversely, the expression of the constitutively active Rho-kinase in FAK+/+ cells led to round cell shape and inhibition of cell motility. Furthermore, coincident with the formation of cortical actin filaments, myosin light chain (MLC), Ser-19 phosphorylated MLC, and MLC kinase mainly accumulated at the FAK-/- cell periphery. We found that disruption of actin filaments by cytochalasin D prevented the peripheral accumulation of MLC kinase and that inhibition of myosin-mediated contractility by 2, 3-butanedione monoxime induced FAK-/- cells to spread. Taken together, our results suggest that Rho-kinase may mediate the formation of cortical actomyosin filaments at the FAK-/- cell periphery, which further recruits MLCK to the cell periphery and generates a non-polar contractile force surrounding the cell, leading to cell rounding and decreased motility.




Cross-talk between nicotinic and muscarinic ACh-receptors in rat superior cervical ganglion neurons

Lin-Ling He and Zhuan Zhou*

Institute of Neuroscience, Shanghai Institutes of Biological Sciences,

Chinese Academy of Sciences, Shanghai 200031, China.


There are two classes of classic acetylcholine receptors: nicotinic receptors (nAChRs) and muscarinic receptors (mAChRs).  Little is known about the interaction between the two AChR-classes.  Here we report that methacholine (MCh), a selective agonist of mAChRs, inhibited up to 80% of nicotine (Nic) induced nAChRs-currents in SCG neurons and chromaffin cells.  In on-cell patches, bath application of MCh reduced mean open-time of single nAChRs channels by 71 ± 7 %. Furthermore, the 'M-inhibition' substantially reduced ACh-induced Ca2+ influx through nAChRs and quantal neurotransmitter release. The 'M-inhibition' is time and temperature dependent.  The slow recovery of nAChR current after washout of MCh, as well as the high value of Q10 (3.2) suggest, instead of a physical process of open channel block, an intracellular chemical process is involved in the ‘M-inhibition’. The effects of GTP-γ-S, GDP-β-S and pertussis toxin suggest that ‘M-inhibition’ is mediated by G-protein signaling.  Taken together, these data suggested that mAChRs negatively modulate nAChRs via a G-protein mediated second messenger pathway.  The time dependence suggests that this cross talk may provide a novel mechanism for post-synaptic adaptation in all cells expressing both types of AChRs.


Supported by grants from China NSF and “973” program




Application of thin film phase plates in biological electron microscopy

Radostin Danev1*, Kuniaki Nagayama and Nobuteru Usuda2

1Center for Integrative Bioscience, Okazaki National Research Institutes, Okazaki , 444-8585, JAPAN;

2Department of Anatomy, Fujita Health University School Medicine, Toyoake 470-1192, JAPAN


We discuss two types of thin film phase plates and their corresponding contrast transfer properties in the Transmission Electron Microscope. The Zernike phase plate and the half-plane phase plate.

The Zernike phase plate [1,2] consists of a thin film with a small hole in the center. It is positioned at the back-focal plane of the objective lens. This phase plate introduces half pi phase shift to the scattered electrons leaving the central beam of unscattered electrons intact. We call this new imaging technique - Phase Transmission Electron Microscope (PTEM).

The half-plane phase plate [3] consists of a thin film covering one half of the diffraction plane. Only electrons scattered in this half will be phase-shifted. We call the resulting imager Difference-contrast Transmission Electron Microscope (DTEM). It produces images with topographic appearance very similar to that produced by Nomarski DIC contrast in light microscopy.

Both PTEM and DTEM provide considerable contrast improvement for biological specimens as demonstrated by the presented experimental images. Theoretical and practical aspects in the application of the phase plates will be discussed.

1. K. Nagayama, J. Phys. Soc. Jpn., 68 (1999) 811.

2. R. Danev, K. Nagayama, Ultramicroscopy 88 (2001) 243.

3. R. Danev, H. Okawara, N. Usuda, K. Kametani, K. Nagayama, J. Biol. Phys. 28 (2002) 627.




Optical heterodyne Surface Plasmon Resonance (SPR) biosensor

Chien Chou

Institute of Radiological Sciences National Yang-Ming University, Pei Tou, Taipei 112, Taiwan

155 sec.2 Li-Long st. Pei-Tou Taipei, 112, Taiwan


There are a number of different methods available for measuring equilibrium binding constants between two interactants, the most commonly used are ELISA, RIA, and affinity chromatography and fluorescence methods. However, many of these conventional methods are somewhat limited in determining the rate constants of the interactions. Surface plasmon resonance (SPR) biosensor technology has advanced to the point where it is possible to measure directly small molecules interacting with immobilized macromolecular targets providing detailed kinetics for optimization in drug discovery and many biomedical applications. Here, a novel optical heterodyne surface plasmon resonance (SPR) biosensor using Zeeman laser is demonstrated. There are two surface plasma waves (SPWs) being excited by two correlated P polarized waves in an SPR device of Kretschmann configuration.  The detection sensitivity and the dynamical range based on this amplitude sensitive method are enhanced. Base on the optical heterodyne SPR biosensor techniques, the aggregation of b-amyloid protein (bAP) is studied. The bAP plays a causative role in the etiology of Alzheimer’s disease (AD) which is one of the leading causes of death in the elderly of the developed country. SPR technology is capable of high sensitivity; therefore, it can be conducted to collect kinetic data for rapid binding events of small molecular weight peptide on the seconds time scale. Eventually, it can provide one of opportunities to understand the mechanisms associated with complex formation as well as new information to lead the optimization of drug candidates.




An eight-dimensional microscope and simultaneous structure & function measurements on living cells

Yao-Xiong Huang

Institute of Biomedical Engineering, Ji Nan University, Guang Zhou, China


A novel 8-dimensional microscope is developed to perform simultaneous measurement on the structure and function of single intact cell. The system is based on an inverted microscope, and capable of performing non-invasive, in situ, real time, and high sensitivity measurement on the parameters of single intact cells include: 1) the concentration, the chemical structures, and the hydrodynamic radius of the intracellular molecules, the morphology of the cell; 2) the functional change of the intracellular protein, and the flexibility and rigidity of the cell membrane; 3) the differentiation, proliferation and the activity, as well as the DNA content and the DNA damage of the cell. 4) the bright field true color optical sectioning of living cells and their 3-dimensional image reconstruction, 3-dimensional arbitrary image segmentation and analysis. A specially designed spatial filter setting in the optical path also allows the microscope obtain 3-dimensional relief images and observation of the minutest details of an unstained specimen.

The whole system consists of a microscope laser light scattering subsystem, a multi-channel fast micro-spectrophotometer, a bright field optical sectioning subsystem with function of micro-image processing and analysis, and a specially designed sample chamber. The sample chamber is under temperature control and with the function of air circulation to maintain the CO2 concentration constant inside and keep the biological cells alive for days, so that one can monitor the structure and function change of the cells in response to the variation of their physiological and biochemical conditions.

All the sub-systems are co-operated with each other for the structure and function measurements. In the meanwhile, each system can work independently from the whole system to perform specific functions.

Four model biological systems were used to illustrate the functions of our system. The first one was human red blood cell, for investigating its spontaneous structural and functional changes in responding to the variation of external conditions. The second one was to evaluate the in vitro cell activity and behavior of Swann cell cultured on scaffold for the purpose of tissue engineering. The third one was pollen spores, for bright field optical sectioning and 3-D image reconstruction, arbitrary segmentation. Living fibroblasts were used as the model system to check if the microscope can obtain 3-dimensional relief image on unstained cell. A similar effect as what obtained by Normarski DIC shows that the microscope is really an 8-dimensional one: 3 dimensions in space, one in timeone in wave lengthand the last 3 ones are the light styles: transmitted light, scattered light, and fluorescence.




Structural understanding of the allosteric conformational change of cyclic AMP receptor protein by cAMP binding

Hyung-Sik Won1, T. Yamazaki2, and Bong-Jin Lee1*

1College of Pharmacy, Seoul National University, Seoul 151-742, Korea and 2Institute for Protein Research,

Osaka University, Japan

1San 56-1, Shillim-Dong, Kwanak-Gu, Seoul, 151-742, Korea


Cyclic AMP Receptor Protein (CRP) is a homodimeric protein, which functions as a transcriptional regulator of more than 150 genes in prokaryotes. CRP is allosterically activated by cAMP binding, and functions by binding to specific DNA sites, as well as by interacting with RNA polymerase. A structural understanding of this allosteric conformational change, which is essential for its function, is still lacking because the structure of apo-CRP has not been solved. Therefore, we performed various NMR experiments to obtain apo-CRP structural data. Despite the high molecular mass of the apo-CRP dimer (47 kDa), which has been considered rather large to be assigned by NMR, we obtained a nearly complete set of backbone NMR assignments of the protein, by a series of multidimensional NMR spectra on the triply [13C, 15N, and 2H]-labeled apo-CRP. The secondary structure of apo-CRP was determined by analyses of the CSI and TALOS predictions, the NOE connectivities, the amide proton exchange rates, and the 1H-15N steady-state NOE values. This secondary structure of apo-CRP was compared with the known structure of cAMP-bound CRP. The results suggest that the cAMP-induced allosteric conformational change of CRP involves subunit realignment and domain rearrangement, resulting in the exposure of helix F onto the surface of the protein. In addition, the analyses of a series of [1H-15N]HSQC spectra of the protein in the absence and in the presence of cAMP or cGMP not only established for the first time that CRP possesses two cAMP-binding sites in each monomer, even in a solution without DNA, but also suggest that the syn-cAMP binding sites of the CRP dimer can be formed by an allosteric conformational change of the protein upon the binding of two anti-cAMPs at the N-terminal domain.


H.-S. Won et al. (2002) J. Biol. Chem. 277, 11450-11455.

H.-S. Won et al. (2000) Biochemistry 39, 13953-13962.

H.-S. Won et al. (2000) J. Biomol. NMR 16, 79-80.




Compressibility-structure-function relationship of proteins

Kunihiko Gekko

Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University,

Higashi-Hiroshima 739-8526, Japan


To address the structure-fluctuation-function relationship of proteins, we have measured the adiabatic compressibility (bs) and partial specific volume (v) of many globular proteins by means of the precise sound velocity and density measurements. Most globular proteins show positive bs due to the positive contribution of internal cavity overcoming the negative contribution of hydration. The bs increases with increasing the cavity, hydrophobicity, and helix content but disulfide bonds contribute to depress the fluctuation. Binding of cofactor and substrate to E. coli dihydrofolate reductase (DHFR) brings about large changes in v and bs, indicating the modified fluctuation of DHFR during the enzyme reaction. The bs value of DHFR is significantly influenced by single amino acid substitutions at three sites, Gly67, Gly121, and Ala145, which are located at the centers of three flexible loops. The mutant with a large bs value shows high enzyme activity. These results demonstrate that a small alteration in local atomic packing dramatically influences an overall protein dynamics and that the structural fluctuation plays important role to enhance the enzyme function.




Biophysical characteristics of cytotoxic ribonucleases from Rana catesbeiana

Yuan-Chao Lou1, Chun-Hua Hsu2, Yun-Ru Pan1, Yi-Hsuan Ho1, Chung-De Chen3, You-Di Liao1, Chinpan Chen1*

1Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan; 2Institute of Biological Chemistry,

Academia Sinica, Taipei, Taiwan; 3Department of Physics, Fu-Jen University, Taipei, Taiwan

No. 128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan


Bullfrog ribonucleases from Rana catesbeiana are found to exhibit cytotoxicities toward tumor cells in addition to possessing different base specificities and ribonucleolytic activities. Therefore, understanding the structure/function relationships of these enzymes would be helpful toward investigating whether they have potential for use as agents in tumor therapy. We have expressed and purified several frog ribonucleases as well as mutant proteins. With good stability and well-dispersed NMR data, these proteins are superb systems for NMR structural studies as well as for other biophysical studies. CD and NMR data clearly reveal that these frog ribonucleases possess highly similar secondary structures. Based on the comparison between native and recombinant protein structures, we concluded that the reduction in catalytic and cytotoxic activities for the recombinant protein, which contains an extra Met residue and has a Gln instead of pyroglutamate at the N-terminus, is primarily due to the loss of two H-bonds in the N-terminal Gln residue. Mutational studies of RC-RNase 4 and RC-RNase 2 clearly indicated that Trp15 plays an important role in high thermostability and causes a unique characteristic of CD data with an ellipticity minimum at 228 nm for RC-RNase 4. NMR chemical shift perturbations between free- and complex- structures identified the substrate-bound related residues in the base specificity for the frog ribonucleases. In addition, the complex structure of RC-RNase L1 with d(ApCpGpA) substrate analog with also be discussed.




Altered sarcoplasmic reticular Ca2+-uptake function of H9c2 cells cultured

in high glucose medium

Eun Hee Lee, Young-Hoon Kim, Hye Kyung Lee, and Hae Won Kim*

Department of Pharmacology, University of Ulsan College of Medicine, Seoul 138-736, Korea


Altered intracellular Ca2+ homeostasis is presumably the primary mechanism of the diastolic impairment in diabetic cardiomyopathy. Previously, using in vivo model, the underlying mechanisms for these functional derrangements were investigated with respect to SRCa2+-ATPase(SERCA), phospholamban(PLB), Ca2+-release channel(ryanodine receptor, RYR), and plasma membrane Ca2+ channel(dihydropyridine receptor) at the transcriptional and translational levels. Reduction of the SERCA level would contribute to decreased rates of SR Ca2+ uptake and that this function is further impaired by the enhanced inhibition by PLB due to its increased expression in the diabetic heart. The Ca2+ release through the RyR appears to be impaired but this was only functional without involving the transcriptional or translational steps. However, causal relations of numerous environmental changes observed in the diabetic heart have been left unresolved. In the present study, we sought to establish an in vitro model of diabetic cardiomyopathy using H9c2 cardiac myocyte cell line. Confluent H9c2 cells cultured for 2 weeks showed morphology of differentiated cardiomyocytes. High glucose (25 mM) did not affect the proliferation or differentiation during this period. We developed a method to measure SR Ca2+ uptake in digitonin-permeabilized cells and analysed the SR Ca2+ uptake parameters in cell groups exposed to normal and high glucose medium. Cells exposed to high glucose showed markedly suppressed SR Ca2+ uptake rate (40 % decrease in Vmax by exposure to high glucose for 2 weeks), and the suppression of SR Ca2+ uptake was dependent on the duration of exposure to high glucose medium. The suppression was reversible by normalizing the medium glucose concentration for 5 days. Protein expression level of SERCA and PLB was analysed, indicating increased expression of PLB without changes in SERCA expression in high glucose group. Increased expression of PLB was also reversible by lowering the medium glucose concentration. It could be concluded that the changes in SR Ca2+ uptake function of H9c2 cells by high glucose was compatible with those observed in diabetic animal models. In addition, it was revealed that high glucose alone can induce SR dysfunction, and decreased SR Ca2+ uptake function was, at least in part, attributable to increased expression of PLB.




Structural and mechanistic studies of photosynthetic water oxidation by light-induced FTIR difference spectroscopy

Hsiu-An Chu*

Institute of Botany, Academia Sinica, Taipei, Taiwan, 11529

No.128 Academia Road Section 2, Nan-Kang, Taipei, 11529, Taiwan


Photosystem II reaction centers utilize the light energy to catalyze the conversion of water to molecular oxygen.  The catalytic site for water oxidation in PSII contains a (Mn)4-Ca cluster that interacts closely with a redox-active tyrosine residue know as YZ.  The (Mn)4 cluster accumulates oxidizing equivalents in response to photo-induced electron transfer reactions within PSII.  There are over a dozen structural models for photosynthetic water oxidation in the literatures to be tested.  Our ability to test these proposals, however, is limited by a lack of appropriately incisive experimental tool.  During my postdoctoral work at Michigan State University, my colleagues and I have developed low-frequency FTIR difference techniques so that metal-ligand vibration can be detected in systems, like (Mn)4-Ca site, in PSII.  We have successfully identified Mn-O-Mn cluster vibration modes of the oxygen-evolving complex in PSII.  Our work is also the first report of a low-frequency, metal-ligand vibrational mode in a protein that has been identified by FTIR.  In this presentation, we will present our new progress on the structural and mechanistic studies of photosynthetic water oxidation.




Spinal glycine receptors are the targets of the general anesthetics

Tian-Le Xu, Hui Lü, Xian-Ping Dong, Zheng-Xiong Zhang

Laboratory of Receptor Pharmacology, Department of Neurobiology and Biophysics, University of Science and Technology of China, Hefei 230027, People’s Republic of China


Although many general anesthetics have been found to produce anesthetic and analgesic effects by augmenting GABAA receptor (GABAAR) function, the role of glycine receptor (GlyR) in this process is not fully understood at the neuronal level in the spinal cord. We investigated the effects of a barbiturate general anesthetic, pentobarbital (PB), on the glycinergic miniature inhibitory postsynaptic currents (mIPSCs) and the responses to exogenously applied glycine (Gly), or taurine, a low-affinity GlyR agonist, by using the whole-cell patch-clamp technique in the rat spinal dorsal horn neurons isolated using a novel mechanical method. Bath application of 30 mM PB significantly prolonged the decay time constant of the spontaneous glycinergic mIPSC without changing its amplitude and frequency. Co-application of 0.3 mM PB reduced the peak amplitude, affected the macroscopic desensitization and deactivation of the response to externally applied Gly in a concentration-dependent manner. In addition, the recovery of Gly response from desensitization was also prolonged by PB.  However, PB did not change the desensitization and deactivation kinetics of the taurine-induced response. The GABAAR antagonist bicuculline (BMI, 10 M) did not affect the effect of PB on the Gly response. Thus, PB prolonged the spinal glycinergic mIPSCs by slowing desensitization and deactivation of GlyR. Two other structurally different intravenous anesthetics, i.e. propofol (10 M) and etomidate (ET, 3 M), prolonged the duration of the glycinergic mIPSC in the rat spinal dorsal horn neurons. In conclusion, on GlyR-Cl- channel complexes there may exist action site(s) of intravenous general anesthetics. GlyR and glycinergic neurotransmission may play an important role in the modulation of general anesthesia in the mammalian spinal cord.




Role of non-planar amino groups in DNA structural rigidity

Rabi Majumdar1*, Anjan K. Dasgupta2 and Dhananjay Bhattacharyya3

1Department of Biophysics, Molecular Biology and Genetics, University of Calcutta, 92 APC Road, Calcutta-700009, India, 2 Department of Biochemistry, University of Calcutta, 35 Ballygunge Circular Road, Calcutta-700019, India and 3 Biophysics Division, Saha Institute of Nuclear Physics, 37 Belgachia Road, Calcutta-700037, India


It was shown for the first time by our group that the sequence specific structural rigidity of DNA arises due to the possibility of formation of a series of bifurcated cross-strand hydrogen bonds across the successive base pairs. We have further demonstrated, on the basis of x-ray diffraction data for DNA crystal structures and molecular modeling, that the lengths of these ‘unusual’ bifurcated hydrogen bonds are rather large and the angles subtended at the hydrogen atoms are small. As a result of this unfavorable geometry, the unusual bifurcated hydrogen bonds appear to be weak in comparison with the usual base pairing hydrogen bonds. However, we have demonstrated from more realistic quantum chemical computations that the right amount of non-planar pyramidal character of the base amino groups leads to a more favorable geometry, where the strength of the bifurcated cross-strand hydrogen bonds improve substantially. As a result, these hydrogen bonds seem to be in a better position to regulate the sequence specific structural rigidity of DNA, which may control its chromosomal packaging or gene expression. Inspired by these results, we have further analyzed protein crystal structures, on the basis of x-ray as well as neutron diffraction data, to assess the amount of pyramidal character of the chemically similar amide groups in proteins. Our investigations reveal significant pyramidal character in these cases as well. Though it is not yet clear how nature exploits this non-planarity in proteins, we are tempted to believe that this property can perhaps be useful in studying the mechanism of enzyme action or DNA-protein interactions.




The coiled-coil region of gp41: Its role in membrane fusion and the application for a new target for anti-HIV agent

Yeon Gyu Yu

Division of Life Sciences, Korea Institute of Science and Technology

PO Box 131 Cheongryang, Seoul, Korea, 136-650


The membrane fusion protein of HIV, gp41, contains two helical regions in the ectodomain. The first amphiphatic helical region, N-helix, has a tendency to form coiled coil and to interact with the second helical region of gp41, C-helix. In this study, the peptide or chimera protein representing the N-helix were shown to insert into the hydrophobic phase of lipid bilayer and disrupt the integrity of membrane. Interestingly, some of fusion defective mutations in the N-helix significantly reduced the membrane binding property of the N-helix, without affecting the ability to form coiled coil and interact with the C-helix. When the N-helix binds to the C-helix, it no longer interacts with membrane. Furthermore, the C-helix interacted with the membrane-bound N-helix and detached it from membrane. These observations suggested that the binding of the N-helix to membrane is important for the membrane fusion process, and such interaction of the N-helix to membrane can be regulated by the C-helix of gp41.




Crystal Structure of the BAFF and BAFF-R Complex: Implications for the Receptor Activiation


Jie-Oh Lee

Department of Chemistry, Korea Institute of Science and Technology, Daejeon, Korea

373-1 Kusong-dong, Yusong-gu, Daejeon, Korea


B-cell activating factor (BAFF) is a key regulator of B-lymphocyte development. Its biological role is mediated by the specific receptors BCMA, TACI, and BAFF-R. We have determined the crystal structure of BAFF-R extracellular domain bound to BAFF at a resolution of 3.3 Å. The cysteine-rich domain (CRD) of the BAFF-R extracellular domain adopts a b-hairpin structure and binds to the virus-like BAFF cage in a 1:1 molar ratio. The conserved DxL motif of BAFF-R is located on the tip of the b-turn, and plays an indispensable role in the binding of BAFF. The crystal structure shows that a unique dimeric contact occurs between the BAFF-R monomers in the virus-like cage complex. Both of the CRDs of TACI contain the DxL motifs and simultaneously interact with the BAFF dimer in the virus-like cage.




Fluorescence Resonance Energy Transfer (FRET) Study on Interaction of

Heat Shock Protein hsp27 with p38 MAP kinase in Single Living Cell


Chunlei Zheng1, Ziyang Lin2, Yajun Yang1, Hanben Niu2 and Xun Shen1*

1Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China and

Institute of Optoelectronics, Shenzhen University, Shenzhen 518060, China


The stress response small heat shock protein 27 (hsp27) helps the cell in repair processes after environmental stress such as heat, UV-irradiation and oxidative stress. MAP kinase p38 is often activated by stress and various cytokines. It has been suggested that the activated p38 can phosphorylate MAPK-activated protein kinase 2 (MK2), then, the later phosphorylates hsp27. However, it has not been clear if there is any direct interaction between hsp27 and p38. In this study, we demonstrate a direct interaction between these two proteins by using fluorescence resonance energy transfer (FRET) technology in single living cell.

Two vectors expressing CFP (cyan fluorescence protein)-hsp27 fusion protein and YFP (yellow fluorescence protein)-p38 fusion protein were co-transfected into L929 cells. All FRET microscopic observations were performed on a Leica confocal laser scanning microscope system at 37°C 12h after transfection. Time-correlated single photon counting fluorescence lifetime imaging (FLIM) was also performed to obtain the donor CFP fluorescence lifetime images in the cells in the presence and absence of YFP-p38 respectively.

It was found that the fluorescence intensity of donor CFP significantly decreased in the presence of acceptor YFP, while the fluorescence of the YFP-p38 fusion protein induced by energy transfer from CFP was markedly enhanced by the presence of the CFP-hsp27 fusion protein. Photobleaching of the acceptor YFP led to about 50 % increase of the fluorescence of the CFP-hsp27 fusion protein, indicating at least 35 % excited energy of CFP in the fusion protein was transferred to YFP in the YFP-p38 chimera. The FLIM studies revealed that the lifetime of the CFP-hsp27 fusion protein was shortened from 2.27 ns in the absence of the YFP-p38 to 1.17 ns in the presence of the YFP-p38 chimera. The results suggest that the protein hsp27 and p38 are so close that the resonance energy transfer occurs between the two proteins. The effect of the p38 activation on the interaction of hsp27 with p38 was also investigated through stimulating the transfected cells with either UV-irradiation or hydrogen peroxide. It was interesting to notice that after UV-irradiation or H2O2-stimulation the fluorescence lifetime of the hsp27-fused CFP went up, indicating the weakening of the interaction between hsp27 and p38. The results imply that the FRET from CFP-hsp27 to YFP-p38 in living cells decreased when p38 was activated. Although it was reported that hsp27 exists in a signaling complex containing p38, MK2 and Akt, and the phosphorylation of hsp27 leads to its dissociation from the complex, it may need further investigation on the p38 activation-caused disruption of the interaction between hsp27 and p38.




Molecular and Brownian dynamics simulations of ion channels

Serdar Kuyucak

School of Physics, University of Sydney, NSW 2006, Australia


Ion channels are formed by proteins embedded in cell membranes and provide pathways for fast and controlled flow of selected ions, which is crucial for normal physiological function of cells. In recent breakthroughs, molecular structures of several types of ion channels have been determined, which has opened the way for detailed studies of structure-function relationships in these channels.  In this talk, I will give a brief review of the basic structural and functional properties of ion channels and discuss various models that attempt to relate their function to structure.  A combined Brownian/molecular dynamics approach is argued to be the most appropriate for this purpose, and its application to potassium, calcium and gramicidin channels is presented. 




Sensitization of regulated exocytosis by protein Kinase C

Tao Xu

Institute of Biophysics and Biochemistry, School of Life Science and Technology, Huazhong University of Science

and Technology, Wuhan 430074, P.R.China


Neurons and endocrine cells release neurotransmitters and hormones by highly regulated exocytosis of secretory vesicles or granules. Intracellular Ca2+ is the principal regulator of stimulus-secretion coupling, however, the exact mechanism underlying the Ca2+ sensing of exocytosis and whether the so-called Ca2+-sensor of exocytotic machinery could be regulated and how remain to be established. In this study, we determined the calcium dependence of secretion and the size of the readily releasable pool (RRP) of secretory granules in pituitary gonadotropes by photorelease of caged-calcium. The calcium affinity for exocytosis was roughly doubled by activation of protein kinase C (PKC) by a phorbol ester, whereas the size of the RRP was not greatly enhanced. The effect was due to activation of PKC since it was blocked by a PKC inhibitor and was not mimicked by an inactive phorbol ester analogue. This finding provides direct evidence for regulation of secretion by enhancement of Ca2+-sensing steps. Since exocytosis depends on the third- to fourth-power of [Ca2+]i, this mechanism ensures a powerful up-regulation of hormone release and may explain how PKC can stimulate exocytosis without an increase of Ca2+ in certain cell types. The sensitization of Ca2+ sensitivity of RRP would also enable exocytosis at sites relatively distant from Ca2+ channels at intermediate or resting [Ca2+]i level and it may constitute an ubiquitous pathway for secretion control.




From optical trapping to single-particle spectroscopy & optical force microscopy for Bio- and Nano- applications

Arthur Chiou

Institute of Biophotonics Engineering,  National Yang-Ming University

No.155, Sec. 2, Linong St., Taipei City 112, Taiwan (R.O.C.)


Optical trapping and manipulation of micro-particles have been investigated for a wide variety of applications since the first experimental observation reported by Ashkin in 1970.  This subject has received increasing attention in recent years due to their unique potential applications in biological, biomedical, and nano- sciences and technologies. An important biological application of optical trapping is the measurement of forces (on the order of pico-Newton) between biological objects such as DNA, RNA, protein, cell, and tissues. For such applications, optical forces have to be measured and calibrated. To date the most popular method for the measurement of the optical transverse gradient force on micro-particles is by dragging against the viscous force.  In this approach a sample cell containing micro-particles (usually suspended in de-ionized water or in other transparent liquid) is dragged across the beam in a direction perpendicular to the beam axis when a particle is trapped in the beam. For a fixed dragging speed, the constant viscous force (given by the Stokes Law, Fvis = 6phrv, where, h is viscosity of the fluid, r the radius of the particle, and v the dragging speed) is balanced against the transverse optical gradient force. This approach is, however, quite time-consuming, and the result is subjected to a significant amount of unavoidable statistical fluctuation.

In this talk, I will give a brief historical overview of optical trappings and discuss some of their potential applications including optical force transduction, optical patterning, single-particle spectroscopy, and optical force microscopy.  Several novel approaches for the measurement of optical forces (on the order of tens of fN to tens of pN) and optical force constants (on the order of pico-m) will be presented, and a few selected examples of bio- and nano- applications will be highlighted.




Protein-structure annotation database (GTOP) and its application to genome wide comparison of proteins from bacteria living in extreme environments

Satoshi Fukuchi

Center for Information Biology and DNA Data Bank of Japan,

 National Institute of Genetics, Mishima 411-8540, Japan


The proteins of bacteria living in extreme environments, such as high temperatures or high salts, have been known to possess biased amino acid compositions. Recently the complete genome sequences of many of these bacteria have been determined, making it possible to perform systematic analysis of the encoded proteins. We have been systematically comparing proteins from organisms living in extreme environments with other proteins using a protein-structure annotation database called GTOP (http://spock.genes.nig.ac.jp/~genome/gtop.html) in which the results of genome-wide PSI-BLAST searches against the PDB and SCOP databases have been compiled for various completely sequenced organisms. Utilizing structures stored in the GTOP database, we have found that the compositional bias in the proteins of thermophilic bacteria, namely enrichment of charged residues and depletion of polar ones, becomes much more pronounced when the residues exposed to the protein surface are used instead of those in the interior, and this trend is independent of bacterial lineages. The proteins of halophilic bacteria show the same tendency: acidic residues are clearly over-represented not in the interior of proteins, but on the surface. These results suggest that alternations of the surface may be a simple, commonly employed strategy to adapt proteins to various environments.


Kawabata et al. GTOP, a database of protein structures predicted from genome sequences. Nucl. Acids Res. 30, 294-298 (2002)

Fukuchi and Nishikawa. Protein surface amino-acid composition distinctively differ between thermophilic and mesophilic bacteria.  J. Mol. Biol., 309, 835-843 (2001).

Fukuchi et al. Unique amino acid composition of proteins in halophilic bacteria. J. Mol. Biol, 327, 347-357 (2003).




Away from the edge: SAD phasing from the sulfur anomalous signal measured in-house with chromium radiation

C. Yang*, J. W. Pflugrath, D. A. Courville, C. N. Stence and J. D. Ferrara

Rigaku/MSC, Inc., 9009 New Trails Drive, The Woodlands, TX 77381 USA


Anomalous scattering with soft X-ray radiation opens new possibilities in phasing for macromolecular crystallography.  Anomalous scattering from sulfur atoms collected on an in-house chromium radiation source (λ = 2.29 Å) was used to phase the X-ray diffraction data from thaumatin (22 kDa), trypsin (24 kDa) and glucose isomerase (44 kDa) crystals.  The contribution to the anomalous term, Δƒ″=1.14 e-, from sulfur for CrKa radiation is doubled compared to that for CuKa radiation, Δƒ″=0.56 e-. For thaumatin and trypsin, the direct methods programs RANTAN or SHELXD successfully found sulfur positions using data sets with resolution limited to 3.5 Å.  The statistical phasing program SHARP was able to produce interpretable electron density maps using the sulfur anomalous signal alone at a low resolution (~3.0 – 3.5 Å).   Much less data, that is lower redundancy, is required for this sulfur SAD phasing procedure compared to the highly redundant data reported in the sulfur SAD phasing procedure with CuKa radiation [1,2].  Glucose isomerase has a lower sulfur-amino acid ratio than that of most of proteins. The Bijvoet ratio (<ΔF>/<F>) is only 0.6% for CuKa radiation, making S-SAD phasing very difficult.  However, using CrKa radiation, the Bijvoet ratio is doubled, to ~1.2%.  An automatically interpretable electron density map can be obtained at low resolution (3.0 Å) with only 180º of data using SOLVE/RESOLVE using CrKa radiation. Furthermore, CrKa radiation can also improve the strength of the anomalous scattering of many other elements in macromolecules like selenium, calcium, zinc, and phosphorus because of increased Δƒ″.  This experimental study shows using CrKa radiation from an in-house rotating anode X-ray generator can provide sufficient phasing power from sulfur anomalous signals for routinely phasing protein diffraction data.  In addition, the longer wavelength of CrKa radiation can greatly increase the in-house ability of resolving long unit cell and enhance the diffraction power for small crystals.  These indicate CrKa radiation may be a good alternative to CuKa radiation for an in-house source.   

1. Yang, C. & Pflugrath, J.W. (2001) Acta Cryst. D57, 1480-1490

2. Dauter, Z.,  Dauter, M., de La Fortelle, E., Bricogne, G. & Sheldrick, G. M. (1999) J. Mol. Biol., 289, 83-92.




NMR Studies of intact Spermatozoa

Girjesh Govil

Department of Chemical Sciences,

Tata Institute of Fundamental Research, Mumbai 400 005


A somewhat less explored frontier of NMR involves studies on the chemistry of living cells, body fluids and tissues (metabonomics). One generally uses high field spectrometers meant for molecular studies and designs strategies to keep the cells viable while they are in the spectrometer. We have used NMR to study chemistry of intact spermatozoa during its various phases of maturation in epididymis tract, during capacitation and during acrosomal reactions. Changes in the kinetics of the metabolic processes under the influence of activators or drugs have been followed. The information has been used to detect: (a) Presence of several unusual and unexpected molecules. (b) Changes in the levels of compounds during various phases of spermatozoa. (c) Detection of metabolites during cell activity and monitoring of unusual metabolism. (d) Effect of environmental factors and storage on the health of spermatozoa.





Conformation of the aM1 segment of the nicotinic acetylcholine receptor

in a membrane environment

M.R.R. de Planque1, D.T.S. Rijkers2 and F. Separovic1*

1School of Chemistry, The University of Melbourne, VIC 3010, Australia and 2Department of Medicinal Chemistry, Utrecht University, The Netherlands

1School of Chemistry, The University of Melbourne, VIC 3010, Australia


The molecular mode of action of general and local anesthetics probably involves a conformational change in the transmembrane segments that line the narrow membrane pore of ion channels such as the nicotinic acetylcholine receptor (nAChR). The extensive transmembrane domain of the nAChR is predominantly, but not exclusively, a-helical; three of the four different transmembrane nAChR segments (M2, M3, M4) appear to be completely a-helical, while the remaining segment (M1) may contain non-helical structural elements. M1 has a Pro residue in the middle of its sequence, which might give rise to a helix-kink-helix structure.

Using solid-state NMR methods, we have studied the conformation of a synthetic nAChR-aM1 peptide from Torpedo californica to ascertain whether aM1 has an inherent tendency to form a distorted a-helix in model membranes. Our results indicate that aM1 strongly interacts with lipid bilayers and that aM1 is indeed not entirely a-helical near the Pro residue, with the extent of non-helicity dependent on the lipid environment. Such a conformation is expected to be more flexible than an undistorted helix, and hence anesthetics might act by inducing a structural change in M1 that results in closure of the ion channel pore.




Crystal structure of human tryptophanyl-tRNA synthetase at 3.0 Å resolution

Jianping Ding1,2*, Yadong Yu1, Yunqin Liu1, Xiang Xu1, Zhen Xu1, Feng Xu1, Jie Jia1, Youxin Jin1, and Edward Arnold2

1 Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China

2 Center for Advanced Biotechnology and Medicine (CABM) and Rutgers University Chemistry Department, 679 Hoes Lane, Piscataway, NJ 08854-5638, USA


Tryptophanyl-tRNA synthetase (TrpRS) catalyzes tryptophan activation by ATP and its subsequent aminoacylation to tRNA(Trp).  TrpRS belongs to the class I aminoacyl-tRNA synthetase (AaRS) family, whose members contain highly conserved motifs HIGH and KMSKS at the catalytic site.  So far, all known structures of aminoacyl-tRNA synthetases, including TrpRS, come from bacteria or yeast.  The only structure of AaRS from mammalian is the recently reported structure of the N-terminal domain of human tyrosyl-tRNA synthetase (TyrRS) (Yang et al., PNAS, 99, 15369-15374 (2002)).  We report here the crystal structure of human tryptophanyl-tRNA synthetase (hTrpRS) at 3.0 Å resolution which was solved using the multi-wavelength anomalous diffraction (MAD) method.  Different from the structure of TrpRS from Bacillus stearothermophilus (bTrpRS), hTrpRS contains an extra N-terminal domain which consists of 140 amino acid residues.  Though the catalytic domains of hTrpRS and bTrpRS share a very low sequence homology of 22%, their overall structure and the secondary structure topology are similar.  Detailed structure and function analyses of hTrpRS will be discussed in the meeting.


Please address correspondence to Dr. Jianping Ding (Phone: 86-21-64331914, Fax: 86-21-64338357

E-mail: ding@sunm.shcnc.ac.cn)





Ca2+-Activated K+ Channels in Mammalian Central Nervous System: Their Biophysical Characteristics and Molecular Mechanisms


Chul-Seung Park, Ph.D.

Department of Life Science, Kwangju Institute of Science and Technology (K-JIST), Gwangju 500-712, Korea


Small conductance Ca2+-activated K+ channels (or SKCa channels) are a group of K+-selective ion channels activated by sub-micromolar concentrations of intracellular Ca2+ independent of membrane voltages.  We expressed a cloned SKCa channel, rSK2, in Xenopus oocytes and investigated the effects of intracellular divalent cations on the current-voltage (I-V) relationship of the channels.  Both Mg2+ and Ca2+ reduced the rSK2 channel currents in voltage-dependent manners from intracellular side and thus rectified the I-V relation at physiological concentration ranges.  The apparent affinity of Mg2+ was changed as a function of both transmembrane voltage and intracellular Ca2+ concentration.  Extracellular K+ altered the voltage-dependence as well as the apparent affinities of Mg2+ binding from intracellular side.  Thus, the inwardly rectifying I-V relationship of SKCa channels is likely due to the voltage-dependent blockade of intracellular divalent cations and that the binding site is located within the ion-conducting pathway. From site-directed substitution of hydrophilic residues in K+-conducting pathway and subsequent functional analysis of mutations, we identified an amino acid residue, Ser359, in the pore-forming region of rSK2 critical for the strong rectification of I-V relationship.  This residue interacts directly with intracellular divalent cations and determines the ionic selectivity.  Therefore, we confirmed our proposition by localizing the divalent cation-binding site within the conduction pathway of SKCa channel.  Since the Ser residue unique for the subfamily of SKCa channels is likely to locate closely to the selectivity filter of the channels, it may also contribute to other permeation characteristics of SKCa channels.




Molecular mechanisms of acquisition and extinction of memory

Po-Wu Gean

Department of Pharmacology, College of Medicine, National Cheng-Kung University, Tainan, Taiwan 70101


Animals learn to express a fear response when encountering a predictive or conditioned stimulus (CS) that was previously paired with a noxious or unconditioned stimulus (US). This fear conditioning is widely used as a leading behavioral paradigm for studying the neural mechanisms through which emotional memory is formed and stored. Conversely, if animals are exposed only to the CS without pairing with the US, previously acquired fear responses will gradually decrease. This fear inhibition known as fear extinction receives increasing attention because it could become an effective intervention for the treatment of fear-related disorders. Converging evidence indicate that enhancement of synaptic strength in the lateral (LA) and basolateral amygdala (BLA) are thought to underlie the encoding of fear memory. We identified the first messenger (glutamate acts on NMDA receptors), the second messengers (Ca++, cAMP and the activation of protein kinases) and the third messengers (transcription factors, CREB) responsible for the consolidation of fear memory. We have further shown that fear training-induced phosphorylation of specific substrates in the rat amygdala is reduced following extinction trials and is accompanied by an increase in the protein level and enzymatic activity of calcineurin. Calcineurin inhibitors prevented extinction-induced protein dephosphorylation as well as extinguishment of fear memory. Administration of NMDA receptor, PI-3 kinase, MAPK, calcineurin or protein synthesis inhibitors blocked extinction of memory. Thus, extinction not only shares some common mechanisms with acquisition of memory but also initiates a calcineurin signal to weaken the original memory.





Azimuth-tuning Characteristics of Auditory Cortical Neurons

Jun-Xian SHEN

Center for Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences

No. 15 Datun Road, Chaoyang District, Beijing 100101,China


Auditory scene analysis involves identifying the content (what) and the location (where) of sounds in the environment. Some studies in mammals asserted that the forebrain, specifically the primary auditory cortex, is essential for sound localization, while others claimed that it is not. The basis for it is that many of the ‘high directional’ neurons in the primary auditory cortex of cat and monkey respond strongly to sounds presented across areas as large as half the sound field. Moreover, physiological studies of the auditory cortex have failed to demonstrate any evidence of a space map containing sharply tuned neurons.

Azimuth-tuning characteristics of the neurons in the primary auditory cortex (AC) were studied. New results are: 1) The auditory neurons recorded from AC show the diversity of the azimuth-tuning functions and are classified into four categories based on azimuth sensitivity curve, best azimuth location and preferred azimuth range (PAR). 2) Cortical EE neurons are directionally sensitive with best azimuth located at contralateral about 20°- 40°, and ipsilateral 30°, and a narrower PAR at higher sound levels. 3) Cortical EI neurons are directionally sensitive with azimuth-tuning functions of broader PAR. 4) Spatial receptive fields of cortical neurons are antagonistically organized with a center-surround pattern. The receptive field size is dependent on individual neuron. 5) Cortical neurons isolated within an orthogonally penetrated electrode have about the same best azimuth location, suggesting a columnar organization.




Protein Folds, Functions and Evolution

Yuh-Fan Liu1 and Ueng-Cheng Yang 1,2*

1Institute of Biochemistry and 2Institute of Bioinformatics, National Yang-Ming University,
No. 155, Sec. 2, Li-Noun St., Taipei 11221, Taiwan.


Many protein folds are re-usable modules, which is independently folded. In other words, these folds still maintain its structure and function even though they are relocated to other part of the genome in evolution. An ancestral gene may go through speciation, duplication, or exon shuffling events. The products of speciation and gene duplication are orthologous genes and paralogous genes, respectively. Exon shuffling may create novel genes that have different functions. In a given genome, only the paralogous genes and the products of exon shuffling will be observed. Unless an exon shuffling takes place after speciation, all the proteins that have the same folds evolve with the given protein fold in them. We thus argue that the functional relation of proteins might be represented by the phylogenetic relation of the given protein fold.

To explore this hypothesis, we have created a tool to perform phylogenetic analysis of protein domains. This tool automate the analysis by connecting the following 3 parts into a pipeline. The first part collects all the proteins that have a given protein fold from Internet. The second part performs multiple sequence alignment and phylogentic analysis. The last part present the functional annotation and a tree to display the relation of different proteins. We have used this tool to analyze the functional classification of death-domain containing proteins, p10 and p20 domain of caspases, and the ligand binding and the zinc finger domains of steroid hormone receptors. The phylogentic analysis of protein domain alone appears to correlate with the functional classification of these proteins quite well. In the case of Unc5 family, the domain classification even correlates with the gene expression pattern. Taken together, domain classification analysis may help biologists to discover the functional difference among those proteins that have a given protein domain.




A trial of genome function prediction through gene positional information in the case of DNA repair proteins

Kei Yura*, Hidetoshi Kono, Nobuhiro Go

Quantum Bioinformatics Group, Japan Atomic Energy Research Institute, Japan

8-1, Umemidai, Kizu, Souraku, Kyoto, 619-0215, Japan


One of the greatest challenges in post genome-sequencing era is to retrieve information out of genome sequences. We have obtained genome sequences of more than 100 species, but we have not fully been able to obtain biological information out of them. The amino acid sequences of the proteins encoded in those genomes are predicted, yet biological functions of half of those proteins remain to be discovered. Conventional methods of function prediction are based on amino acid sequence similarity between ones predicted from genome sequences and ones of sequences in databases. We are in a trial to utilize information other than coding regions for gene function predictions.

Proteins that function in a concerted manner are expected to be transcribed and be translated at the same. The seemingly most effective system for co-transcription is an operon system found in prokaryotic genomes. In the operon system, a single promoter controls consecutive genes on the genome and hence the genes are transcribed at the same time. We, therefore, analyzed the functional correlations of proteins encoded in the same operons and found out that there were cases that functionally related proteins were encoded by genes within the same operon, though strength of the relationships varies on species. Based on this observation, we developed a method to predict biological functions of proteins encoded in the same operons. The method is applied to the prediction of new proteins related to DNA repair systems.




Amplifying collective motions in computer simulations of

proteins and peptides

Haiyan Liu*, Zhiyong Zhang, Jianbin He, Yunyu Shi

School of Life Sciences, University of Science and Technology of China, Hefei, 230026, China


We present a novel method that uses collective modes obtained with a coarse-grained model to guide atomic-level simulations. Based on this model, local collective modes can be calculated according to a single configuration in the conformational space of the protein. In the molecular dynamics simulations, the motions along the slowest few modes are coupled to a higher temperature by the weak coupling method, in order to amplify the collective motions. The amplified-collective-motion (ACM) method allows for extensive sampling in conformational space while still restricts the sampled configurations within low energy areas. The method can be applied in both explicit and implicit solvent simulations, and may be further applied to important biological problems, such as long time-scale functional motions, protein folding/unfolding and structure prediction. We further describe a method for efficient sampling of the energy landscape of a protein in atomic molecular dynamics simulations. A simulation is divided into alternative relaxation phases and excitation phases. In the relaxation phase (conventional simulation), we use a frequently updated reference structure and deviations from this reference structure to mark whether the system has been trapped in a local minimum. In that case, the simulation enters the excitation phase, during which the ACM method is employed to assist minimum-escaping (ACM-AME).  After the system has escaped from the minimum the simulation reenters the relaxation phase. The ACM method is applied to two test systems. One is a S-peptide analogue. We realized the refolding of the denatured peptide in 8 simulations out of 10 using the method. The other system is Bacteriophage T4 lysozyme (T4L). Much more extensive domain motions between the N-terminal and C-terminal domain of T4L are observed in the ACM simulation compared to a conventional simulation. The ACM-AME (amplified collective motion-assisted minimum escaping) sampling is compared with conventional simulations as well as an alternative scheme that elevates the temperature of all degrees of freedom during the excitation phase. In terms of sampling low energy conformations and continuously sampling new conformations throughout a simulation, the ACM-AME scheme demonstrates good performance.