Early events of protein folding at subzero temperatures
Zhijie Qin1*, Jinsong Li1, and Hiroshi Kihara1
address: Department of Chemistry and Biochemistry, UCSC,
We have applied
stopped-flow fluorescence, circular dichroism and x-ray scattering to the
elucidation of protein folding. The method is limited by the dead time of the
mixing of the stopped-flow, which did not allow us to investigate the early
events of protein folding faster than millisecond region. We, then, attempted
to slow down the folding process by decreasing temperatures lower than
We have investigated
apomyoglobin, bovine b-lactoglobulin,
ubiquitin, lambda repressor and src SH3 domain protein. Results can be
summarized as, (1) initial a-helical
foramation is too fast to be detected even at
How RNA and proteins recognize each other
Gabriele Varani1*, Yu Chen, Neil Dobson, Katherine S Godin
1Department of Biochemistry and Department of Chemistry,
RNA-binding proteins play many essential roles in the cell in the regulation of gene expression, but our knowledge of their structure and function has lagged behind DNA-binding proteins. The last few years have seen an explosion in the number of structures of RNA-binding proteins and their complexes: we now know the structures of most major RNA-binding protein domains and how they bind RNA. However, the molecular basis of specificity remains unclear even for the best-studied protein families, such as the RRM. If we were able to identify the interactions responsible for conveying specificity in RNA-binding proteins, we would understand their function much better. We would also be able to predict the specificity of an RNA-binding protein from its sequence and perhaps design RNA-binding proteins with new specificity and biological activity. Such proteins would provide valuable new probes of biological interactions and, potentially, new therapeutic agents. I will describe how two of the major RNA-binding protein families, the RRM (one of the largest protein families in all eukaryotic genomes) and dsRBD bind RNA. I will highlight what existing structures tell us about RNA recognition and specificity and discuss the role of RNA and protein dynamics in molecular recognition. I will discuss the development of a new atomic model of protein-nucleic acid interfaces based on the statistical analysis of high-resolution crystal structures of protein-DNA and protein-RNA complexes. The new potential is shown to predict the native amino acid sequence at the protein-RNA interface well and to discriminate native protein-RNA structures from incorrectly docked decoys. I will report on the early experimental tests of the predictive power of the model and associated design algorithms aimed at achieving specificity switches in model protein-RNA interfaces and highlight areas where experimental information remains to be gathered to overcome limitations of the model.
Automated molecular microscopy
B. Carragher*, A. Cheng, D. Fellmann, F. Guerra, J. Pulokas, J. Quispe, S. Stagg, C. Suloway,
C. Yoshida, Y. Zhu, and C. S. Potter
Department of Cell Biology, The Scripps Research Institute,
N. Torrey Pines Rd.,
The technique of molecular microscopy holds great promise for routinely and efficiently providing structural information at a resolution sufficient to resolve the secondary structure in proteins. It could thus be used in conjunction with high resolution x-ray structures of individual proteins to interpret very large complexes to near atomic resolution. The methods generally used are however both time consuming and labor intensive. This includes almost every aspect of the process; the preparation of suitable specimens, the acquisition of the required very large numbers of electron micrographs, and the supervision of the sometimes-complex software needed for analysis and reconstruction of the three dimensional electron density maps.
The challenge then is to transform EM structure determination into a high throughput methodology. Success in this endeavor will not only facilitate the process of molecular microscopy but has the potential to expand the scope of accessible problems and make possible investigations that are presently deemed too high risk because of the inordinate effort involved.
To this end we are focused on the development of technologies to address automation for specimen handling, image acquisition, data processing and data information integration. Several years ago we began to develop a system, called Leginon, which automatically collects electron micrographs of macromolecular structures under low dose conditions. This system has been integrated with automated particle selection algorithms and analysis and processing packages. We will discuss the current status of these efforts and our future plans for improving both the sustained throughput and the yield of automated data collection and analysis.
Thermodynamics and kinetics of protein folding: a mean field theory
K. K. Liang, M. Hayashi, Y. J. Shiu, Y. Mo, J. S. Shao, Y. J. Yan, and S. H. Lin
Institute of Atomic and Molecular Sciences, Academia Sinica.
The kinetic Ising model in the mean field approximation is applied to study the equilibrium and kinetic behaviors of protein folding-unfolding. In our model, we regard a protein as a topological collection of interacting peptide bonds (or other protein unites). According to this model, thermodynamics and kinetics of protein folding-unfolding are rated to the elementary process of folding←→unfolding of such interacting units. We shall show that even for the so-called two-state case of protein folding-unfolding, the kinetic behaviors are predicted to be in general non-exponential and that universal curves exist separately for the thermodynamic behaviors and kinetics behaviors of protein folding-unfolding. Our model can treat the effect of temperature and denaturant concentration on the thermodynamics and kinetics of protein folding-unfolding and provide the Chevron plot. It can also be used to calculate the force-extension curve in the atomic force microscopic studies of protein folding-unfolding. Satisfactory demonstrations are presented for treating experimental observations on the thermodynamical and kinetic responses of protein folding-unfolding to the changes in temperature and denaturant concentration and for exhibiting universal plots of protein.
Structural genomics and proteomics in Japanese University Community
Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto
606-8502, Japan and RIKEN Harima Institute / SPring-8, Koto
Japanese national project on structural
genomics and proteomics has established in fiscal year 2002 under the name of
"National Project on Protein Structural and Functional
Analyses." The aims of this project are to study structures
and functions of proteins and also to develop research techniques for
expression and structure analysis of proteins. The
project is divided to two programs; the "comprehensive analysis"
program and the "individual analysis" program. The former program is performed
by the structural genomics and proteomics initiative group of RIKEN. On the other hand, Japanese
university community mainly contributes to the latter program to perform
structural and functional analyses of a variety of individual target
proteins. In the
"individual analysis" program, eight consortiums (research groups)
were assigned for seven biological target fields. In our consortium of this
program (Kyoto University group), for example, we aim to perform structural
and functional studies of the proteins that are related to the higher-order
structure formation of proteins in order to display their intrinsic biological
functions, including molecular chaperons for protein folding, transfer of
proteins across the membrane, and the quality control of proteins by repair,
recycling and degradation. Our
consortium is composed of not only laboratories of
Enzyme as brownian motor, and the design of molecular and ion pumps
Tian Yow Tsong ( 鄭 天 佑 )
College of Biological Sciences,
Many popular biochemistry textbooks define enzyme as a biological catalyst which can enhance the rate but cannot shift the chemical equilibrium of a biochemical reaction. For a Michaelis-Menten enzyme working under the Briggs-Haldane Steady-State condition, in which the concentration of enzyme is much lower than that of the substrate and product, the above statement is for all practical purposes correct. However, in a living cell or a supra-molecular structure the concentrations of substrate, enzyme, and product are comparable and in dynamic fluxes. Coupling or cascading of different reactions is also common place. The presence of an enzyme may greatly alter the energy landscape of the catalyzed reaction. In this work I will redress Michaelis-Menten enzyme as a catalytic wheel and an energy transducer rather than a mere rate enhancer. With such premise I will present two models, the “conformational coupling model” and the “barrier surfing model” (the flashing ratchet), based on the formalism of the Brownian Motor, to explain some transport data on Na,K-ATPase. Our analysis has unraveled certain rules and these rules may be used as guide for the design of molecular and ion pumps. I propose that the catalytic wheel is a core mechanism in many nanometer scale bio-molecular motors, transporters, and locomotors. Muscle contraction and kinesin transport are examples. [References: Tsong TY, Chang CH, Assoc. Asia Pacific Phys. Soc. Bulletin, 2003 April Issue, Pp. 12-18; Chang CH, Tsong TY. Phys Rev E 69, 021914 (2004); Makhnovskii Yu A, et al. Phys Rev E 69, 021102 (2004); Rozenbaum VM et al. (2004) In Preparation.] email@example.com
Structure and mechanism of a high-fidelity and a low-fidelity
The structure and mechanism of a high fidelity DNA polymerase, mammalian Pol bb, as well as a low fidelity DNA polymerase, African Swine Fever Virus Pol X, will be presented. For Pol bb, we used stopped flow kinetics and substitution-inert metal complexes to dissect the reaction pathway, and used X-ray crystallography in collaboration with Michael Chan to determine the structure of the intermediate. The results led to a new mechanistic model. For Pol X, we used pre-steady state kinetic analysis to show that it has the lowest fidelity ever measured for a template directed DNA polymerase, and is the first polymerase reported to have no preference for a Watson-Crick base pair (G:C) over a “mismatched” base pair (G:G). NMR was used to solve the solution of Pol X in the free form as well as in the ternary complex (with DNA, dNTP, and magnesium). Further studies suggested that Pol X is likely to be part of a mutagenic base excision repair (BER) pathway, which could represent a novel mechanism by which a virus achieves hypervariability. The DNA ligase in this pathway has also been identified and shown to display extremely low fidelity.
Three-dimensional structural view of branch migration in
DNA homologous recombination
1Biomolecular Engineering Research Institute (BERI)
At the late stage of recombinational repair in prokaryotes, RuvA, RuvB, and RuvC proteins process the Holliday junction through formation of two types of complexes, which catalyze branch migration (RuvAB) and resolution (RuvABC resolvasome), respectively. We determined the three-dimensional structures of all three protein components by X-ray crystallography. The crystal structure of the RuvA-Holliday junction complex revealed that two base pairs near the crossover point are disrupted, suggesting the positive mechanistic role of RuvA in the branch migration. The crystal structure of the E.coli RuvC dimer indicated the catalytic centre of this resolvase, and allowed us to build a Holliday junction model bound to RuvC. The crystal structure of the Thermus thermophilus RuvB protomer revealed the RuvB architecture, classified into the AAA+ family, and the environments of the ATP or ADP binding site. The X-ray structure of the RuvA-RuvB complex, determined more recently, has revealed that two RuvA tetramers form the symmetric and closed octameric shell, where four RuvA domain IIIs spring out in the two opposite directions to be individually caught by a single RuvB. The binding of domain III deforms the protruding b-hairpin in the N-terminal domain of RuvB, and thereby appears to induce a functional and less symmetric RuvB hexameric ring structure. The fitting of this complex structure into the averaged electron microscopic images of the RuvA-RuvB-junction DNA ternary complex allows the model building, which implies that the functional scheme with a fixed RuvA-RuvB interaction may be preferable to that with their rotational interaction.