PL1

Light and Life

Lubert Stryer

Department of Neurobiology, Stanford University School of Medicine,

Stanford, California 94305 USA

 

It is a pleasure and privilege for me to give this lecture on Light and Life in remembrance of Felicia Wu, a fine scientist and a wonderful person. The interplay of light and life is evident in processes such as vision, bioluminescence, and photosynthesis. Light is also a powerful means of revealing the structure, dynamics, and interactions of biomolecules and cells. Moreover, light can be used to direct spatially-ordered chemical syntheses on the micrometer scale. I will discuss three areas of my research over a span of many decades that illustrate these themes: (1) fluorescence resonance energy transfer as a spectroscopic ruler; (2) the molecular mechanism of visual excitation; and (3) the use of photolithography to generate DNA arrays, powerful tools for probing gene expression and genetic diversity.

 

 

PL2

Modeling the structure of proteins and macromolecular assemblies

Andrej Sali

Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, and California Institute for

Quantitative Biomedical Research

Mission Bay Genentech Hall, 600 16thStreet N472D

University of California, San Francisco, San Francisco, CA 94143-2240

 

The structures of most protein domains will eventually be characterized by structural genomics, which aims to determine most protein folds by experiment, allowing the remaining protein sequences to be modeled with useful accuracy by computational methods. In the case of assemblies, however, the structure is usually obtained by a number of experimental methods of varying accuracy and resolution (eg, X-ray crystallography of the subunits, low-resolution electron microscopy of the assembly, and chemical cross-linking). Therefore, there is a need for a computational framework that can take into account all available information about the structure of an assembly and calculate at the appropriate resolution all models that are consistent with the given input. To this end, it is useful to express structure determination as an optimization problem. The three components of this approach are (i) representation of an assembly; (ii) a scoring function consisting of individual spatial restraints; and (iii) optimization of the scoring function to obtain the models. This approach will be illustrated by the modeling of the yeast nuclear pore complex.

 

 

PL3

Experimental molecular evolutions with and without cellular interaction

Tetsuya Yomo

Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University

2-1 Yamadaoka, Suita, Osaka 565-0871 Japan

 

We conducted two experimental evolutions accelerated by random mutagenesis to address the following questions: 1) How much variety in the sequences is needed to prompt the evolution of protein functions from random polypeptides? 2) How does cellular interaction affect the dynamics of molecular evolution to allow genetic diversity in population?    

The first experimental evolution was carried out on a small library of phage-displayed polypeptides with random sequences of about 140 amino acid residues.  With less than twenty cycles of random mutagenesis and some different functional selections, the polypeptides evolved with different protein functions, such as esterase activity, DNA binding activity, and so forth.  The experimental results basically mean that the evolution of protein functions can be prompted from a small sequence variety, even from a single arbitrarily chosen random sequence.

To know the role of cellular interaction in molecular evolution, we conducted the second experimental evolution, three serial cycles of random mutagenesis of the glutamine synthetase gene and chemostat culture of the transformed Escherichia coli cells containing the mutated genes.  The molecular phylogeny and population dynamics of the experimental evolution show the coexistence of some mutant cells having different level of glutamine synthetase activity at each cycle.  The coexistence was proven to be stable and require the cellular interaction via the medium.  In addition, the mutant gene once extinct at the earlier generation was found to coexist with the population of the final generation.  These results show that cellular interaction brought about the change of the fitness of each mutant, giving a chance to increase and maintain genetic diversity even in a spatially-unbiased environment. 

 

 

PL4

Structure and Function of Nickel-containing Superoxide Dismutase

 

Sa-Ouk Kang

Laboratory of Biophysics, School of Biological Sciences, and Institute of Microbiology, Seoul National University,

Seoul 151-742, Korea

 

Nickel-containing superoxide dismutase (NiSOD) was prepared to homogeneity from Streptomyces seoulensis and was used for X-ray absorption spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, electron-nuclear double resonance spectroscopy and X-ray crystallography. The molecular mass of NiSOD subunit was determined to be 13.2 kDa by matrix assisted laser desorption ionization - time of flight mass spectrometry. And the molecular mass of the native enzyme was determined to be 73 kDa by gel filtration chromatography and to be 84.9 kDa by sedimentation equilibrium using analytical ultracentrifuge. When the sodN gene of Streptomyces seoulensis was expressed in Streptomyces lividans TK24 or Streptomyces coeolicolor A3(2), a total of seven kind hybrid SOD bands appeared. This result, together with the gel filtration and sedimentation equilibrium data indicated that the quaternary structure of NiSOD is a homohexamer, not the previously reported homotetramer, which is novel among SODs.

The EPR spectrum of NiSOD as isolated exhibited resonances typical of a rhombically distorted S = 1/2 Ni(III) signal with unique g values (gxyz = 2.306, 2.232 and 2.016) at 100 K. The large anisotropy and the average g value of 2.18 of NiSOD EPR spectrum indicate that the unpaired electron mainly resides in a metal-based orbital. The fact that gx and gy are larger than gz and that gz is nearly 2.00, together with the observation of a superhyperfine splitting of the gz component, indicates a (dz2 )1 electron configuration for Ni(III). The EPR spectrum could be simulated as an effective S = 1/2 system, using a Gaussian line-shape function and the following parameters; gxyz = 2.306, 2.232, 2.016, Axyz = 16.2, 17.7, 24.6 G and lxyz = 28, 17, 7.8 G. At pH 4.0, the EPR spectrum showed g-values (gxyz = 2.282, 2.24, 2.0155) which is more axial than that at pH 7.4, indicating that the active site structure has changed. In addition, the EPR spectrum taken at pH 5 and above pH 8 showed clear superhyperfine splitting in the gy region.

The crystal structure of NiSOD demonstrated that NiSOD is a hexameric enzyme consisting of four-helix-bundle subunits. The hexamer exhibited a three-fold symmetry axis with three two-fold axes perpendicular to the three-fold axis. The subunit structure which comprises 117 residues in the mature enzyme revealed a four-helix bundle in the canonical all-antiparallel topology. The crystal structures of the resting NiSOD revealed that each Ni(III) ion is coordinated by the amino group of His1, the amide group of Cys2, and two thiolate groups, Cys2 and Cys6. Upon reduction, a systematic increase in bond lengths was observed. The water molecule closest to the Ni(III) in the resting form of the enzyme was absent in the reduced crystal form, the second water molecule still being present at a slightly shortened average distance of 4.0 Å.

 

 

PL5

Protein systems in excitation-contraction coupling

Do Han Kim

Department of Life Science, Kwangju Institute of Science and Technology (K-JIST), 1 Oryong-dong, Buk-gu,

 Gwangju, 500-712, Korea

 

For excitation-contraction (E-C) coupling in striated muscles, dihydropyridine receptor (DHPR) senses depolarization of the transverse (T)-tubules, and transmits signals to trigger Ca2+ release from the sarcoplasmic reticulum (SR) for muscle contraction. The major protein responsible for Ca2+ release from the SR is the ryanodine receptor (RyR)/Ca2+ release channel. The native RyR (565 kDa) complex exists as a homo-tetramer, which is associated with FKBP in 1:1 molar ratio. The N-terminal region of FKBP could bind the RyR. The RyR in the junctional SR forms a quaternary protein complex with calsequestrin (CSQ), triadin and junctin. CSQ is a high capacity Ca2+ binding protein, and is physically anchored to the RyR by triadin and junctin. The aspartate-rich region of CSQ, the KEKE motif of triadin and the intralumenal loop II of RyR could participate in the protein-protein interactions in Ca2+-dependent manner. Perturbation of the quaternary protein complex by overexpression of junctin or junctate could lead to an abnormal Ca2+ handling in cardiac cells. The overall protein systems involved in the E-C coupling will be discussed.

 

 

PL6

Decision Making in Drosophila Facing Competing Visual Cues

Aike Guo1,2, Shiming Tang2, Jianzeng Guo1, Ke Zhang1, Yueqing Peng1 and Wang Xi

1Institute of Neuroscience, Shanghai Institutes for Biological Sciences, CAS, Shanghai 200031;

2Institute of Biophysics,CAS,Beijing 100101,PR CHINA

 

A choice is required when an organism is confronted with alternatives for which an action is necessary to acquire or avoid one or more of alternatives because of a desire, goal or preference. Human and most animals can make a rapid and rational decision/choice among competing alternatives by assessing the advantages and disadvantages based on knowledge and experience. We have explored a simple choice-making behavior in Drosophila facing competing visual cues at flight simulator. Individual flies were conditioned to choose a flight direction in accordance to the color and shape cues in a flight simulator and tested with contradictory cues following the conditioning. It was found that wild type flies could make a discrete and firm choice in their flight orientation between two competing alternatives as the relative salience of color and shape cues gradually changes. We have also revealed that the mushroom bodies in Drosophila are required to the decision -making. The decision ability was greatly diminished in mutant(mbm1)flies with miniature mushroom bodies or with hydroxyurea ablation of mushroom bodies. Control experiments showed that these mutant flies exhibited normal color vision and visual learning and that the discrete choice in flight behavior was not due to overshadowing by or selective attention to one visual cue. The molecular genetic manipulations, coupled with behavior studies were used for elucidating the neural basis of decision-making in Drosophila. Although our decision-making paradigms developed for Drosophila are rudimentary tasks compared with human decision-making under more complex conditions, but the information gained from these studies will provide a foundation for future experiments that investigate more complex decision behavior.

 

(1)     Tang & Guo (2001) Choice behavior of Drosophila facing contradictory visual cues, Science, Vol 294:1543

(2)   Jeffrey D.Schall(2001) Neural basis of Deciding, Choosing and Acting, Nature Review/Neuroscience,2:33

 

PL7

Is water essential for life?

An approach through hydration structure analysis of proteins

 

Masayoshi Nakasako

Department of Physics, Faculty of Science and Technology, Keio University3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan.

 

Water is a complex fluid having unusual physical properties, such as high boiling and melting temperatures and strong surface tension. Networks of hydrogen bonds running through water are the main cause for the properties, and the dynamical reorientation of water molecules induces the reorganization of networks ensuring the fluidity of water. From the viewpoint of biology, water is a natural medium of life. Living cells are occupied by water more than 60% of their volume, and biological molecules composing cells are constantly bathed in an aqueous environment. Any protein molecule does not fold correctly without water environment, and the association of water molecules with proteins is essential for the stability and the functions of proteins. Therefore, structures and interaction modes at the interface between water and proteins are the subject of much discussion to understand how proteins fold and work in aqueous environment. Recent progresses in cryogenic X-ray crystallography and molecular dynamics simulation have made it possible to characterize protein hydration. Firstly, we show our recent studies on the static pictures of hydration structures obtained through crystallographic and computational methods. Secondly, we report an experimental result as to dynamical coupling of hydration structure changes and domain motion of an enzyme. Finally, we would like to discuss the influences of hydration on protein folding and dynamics toward understanding the dynamics of ¡¥molecular machines¡¦ in aqueous environment.

 

 

PL8

The Chinese Hybrid Rice Genome Project

Jun Wang

Beijing Genomics Institute

Beijing Airport Industrial Zone B6, Beijing 101300, P.R. China

 

The rice genomes are organized primarily as a ¡§ship-in-cannel¡¨ model where the ¡§ship¡¨ or genes (often described as gene islands or blocks in lower copy numbers) are intervened by repeats (often described as repeat clusters in higher copy numbers) of retrotransposons that along with many other types of repetitive sequences form a rather elastic ¡§cannel¡¨ along a dozen chromosomes. To catch all the ¡§ship¡¨ in action and to size up the ¡§cannel¡¨, we have simultaneously produced two sequence maps of the rice genome from whole-short-gun derived data for the major cultivated rice subspecies, indica and japonica, which are gene-centric, positioned on the chromosomes, and with a gene coverage of 97% and accuracy of 99.9%. Domain-based protein analysis has yielded 50,000 protein-coding transcripts that are potentially important candidates for functional studies for rice biology. The gene maps together with identified genes provide rice biologists and geneticists a guidebook for identifying and mapping genes that are of biologically and agronomically importance. It also paves the way for the genome analysis of other Gramineae crop species whose genomes are a few folds or even magnitudes larger than that of the rice.

 

 

 

 

PL9

New NMR Approaches to Protein-Protein and Protein-Ligand Interactions

James H. Prestegard

Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602

 

The determination of structures of protein and their complexes by Nuclear Magnetic Resonance (NMR) methods normally involves the measurement of Nuclear Overhauser Effects (NOEs) and the interpretation of these effects in terms of inter-proton distance constraints.  While these constraints are extremely valuable, their short-range character limits applicability in situations where the relationship of more remote parts of a system needs to be defined.  Such situations occur in defining the relationship of one protein to another in a multi-protein complex and in defining the relationship of a protein active site to a bound ligand.  Residual dipolar couplings, which are capable of returning orientational constraints as opposed to distance constraints, provide a valuable alternative in these situations.  Methods for acquiring and analyzing these new types of data will be described, and applications to proteins involved in carbohydrate binding and synthesis will be used to illustrate the methods.