University of Toronto Biochemistry

	Alan Davidson Professor Ph.D., University of Toronto, 1991 Post-Doctoral Fellowship, Massachusetts Institute of Technology, 1991-1995
	Medical Sciences Building, Room 4285 416-978-0332 alan.davidson@utoronto.ca

In Vitro and In Vivo Studies of Protein Folding and Protein-Protein Interactions

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Member of the following Training Program of interest to students and PDF's:
CIHR Training Program in Protein Folding: Principles and Diseases

Research Synopsis

Research Approach
In my laboratory we employ a multifaceted approach to research. We study the three-dimensional structures of proteins in detail and use this information to construct hypotheses about interactions that may be crucial for a protein's stability or function. We then test these hypotheses by using molecular biology techniques to create mutant proteins with amino acid substitutions at positions we believe to be important. The in vitro properties of these mutant proteins are assessed using biophysical methods such as circular dichroism spectroscopy, tryptophan fluorescence spectroscopy, and nuclear magnetic resonance spectroscopy. In this way, can determine exactly how particular amino acid substitutionsaffect the protein's activity and thermodynamic stability. We also assess the ability of these mutant proteins to carry out their normal functions inside the cell. All of this research is aided by our studies in the area of bioinformatics. These investigations involve the construction and careful analysis of large and comprehensive sequence alignments. We are developing new methods to extract useful information from this rich source of data, and we have designed a number of successful experiments based on conclusions drawn from our bioinformatics studies.

Model Systems
First recognized as a non-catalytic homology region in protein kinases related to Src oncogene, SH3 domains have now been identified in more than 350 different proteins in organisms ranging from yeast to humans. SH3 domains are found in kinases, lipases, GTPases, adaptor proteins, structural proteins and others, and these proteins act in diverse processes including signal transduction, cell cycle regulation, and actin organization. The function of the SH3 domain is to mediate specific protein-protein interactions, which itachieves by binding to PXXP-containing sequence motifs in target proteins. SH3 domains generally bind their targets with affinities between 1 and 50 microM and individual domains display distinct specificities. The above figure shows the three dimensional structure of the SH3 domain from the Fyn tyrosine kinase. The yellow amino acid side chains comprise the hydrophobic core of this domain and are critical for its stability. The red (aromatic) and blue (negatively charged) side chains lie on the surface of the domain and are crucial for peptide-binding. SH3 domains, which are only 60 residues long, are among the smallest folded monomeric proteins and they are amenable to a variety of in vitro functional and thermodynamic analyses. Hundreds of SH3 domain sequences are available for analysis and, very importantly, these sequences are very diverse, providing a broad sampling of sequences that are consistent with the SH3 domain fold. In addition, forty-four structures of nineteen different SH3 domains are available in the structural database.

Areas of Research
In the past few years the genomes of C. elegans, S. cerevisiae and a number of bacterial species have been sequenced and in the near future the human genome will be completed. The crucial challenge for biologists in the future will be to make sense of this information, much of which will consist of protein sequences. To fully understand the function of proteins in living cells, detailed knowledge of their structures will be required. Since the experimental determination of protein structures can be a difficult and laborious task, the development of methods to accurately predict protein structures from their amino acid sequences alone is of crucial importance. Some of my research is aimed at understanding how the amino acid sequence of a protein specifies its three dimensional structure. This "protein folding problem" is a fundamental question in biology and its solution will lead to accurate protein structure prediction and will also aid in the design of novel proteins with unique properties useful for medical or industrial purposes.

Studies in my laboratory are also focused on the question of how proteins specifically recognize and bind to eachother. Since specific protein-protein interactions form the basis of most processes within cells such as signal transduction, gene regulation, and morphogenesis, knowledge in this area is essential. The figure above shows an SH3 domain and an overlay of the structures of 7 different peptides as they appear when bound to SH3 domains. The red and blue side chains belong to the amino acid residues that contact target peptide in most structures. The red residues, which are highly conserved in the SH3 domain sequence alignment are seen to contact the structurally conserved portion of the target peptide conformation. The target peptides are seen to adopt a much wider variety of conformations where they contact the blue residues, which are less conserved positions in the sequence alignment. The blue residues are likely to play an important role in determining binding specificity.

Recent Publications

Zarrine-Afsar, A., Larson, S.M., Davidson, A.R. (2005) The family feud: do proteins with similar structures fold via the same pathway? Curr. Opin. Struct. Biol. 15 , 42-49.

Maxwell, K.L., Wildes, D., Zarrine-Afsar, A., De Los Rios, M.A., Brown, A.G., Friel, C.T., Hedberg, L., Horng, J.C., Bona, D., Miller, E.J., Vallee-Belisle, A., Main, E.R., Bemporad, F., Qiu, L., Teilum, K., Vu, N.D., Edwards, A.M., Ruczinski, I., Poulsen, F.M., Kragelund, B.B., Michnick, S.W., Chiti, F., Bai, Y., Hagen, S.J., Serrano, L., Oliveberg, M., Raleigh, D.P., Wittung-Stafshede, P., Radford, S.E., Jackson, S.E., Sosnick, T.R., Marqusee, S., Davidson, A.R., Plaxco, K.W. (2005) Protein folding: Defining a "standard" set of experimental conditions and a preliminary kinetic data set of two-state proteins. Protein Sci .14, 602-616.

Korzhnev, D. M., Salvatella, X., Vendruscolo, M., Di Nardo, A. A., Davidson, A. R., Dobson, C. M. & Kay, L. E. (2004). Low-populated folding intermediates of Fyn SH3 characterized by relaxation dispersion NMR. Nature 430 , 586-590.

Yu, B., Paroutis, P., Davidson, A. R. & Howell, P. L. (2004). Disruption of a salt bridge dramatically accelerates subunit exchange in duck delta 2 crystallin. J. Biol. Chem. 279 , 40972-40979 .

Marles, J. A., Dahesh, S., Haynes, J., Andrews, B. J. & Davidson, A. R. (2004). Protein-protein interaction affinity plays a crucial role in controlling the Sho1p-mediated signal transduction pathway in yeast. Mol. Cell 14 , 813-823.

Di Nardo, A. A., Korzhnev, D. M., Stogios, P. J., Zarrine-Afsar, A., Kay, L. E. & Davidson, A. R. (2004). Dramatic acceleration of protein folding by stabilization of a nonnative backbone conformation. Proc. Natl. Acad. Sci. USA 101 , 7954-7959.

Di Nardo, A.A., Larson, S.M., and Davidson, A.R. (2003) The relationship between conservation, thermodynamic stability, and function in the SH3 domain hydrophobic core. J. Mol. Biol. 333 , 641-655.

Mittermaier, A.K., Davidson, A.R., and Kay, L.E. (2003) Correlation between 2H side-chain parameters and sequence conservation in globular proteins. J. Am. Chem. Soc. 125 , 9004-9005.

Sheng, Y, Ip, H., Liu, J, Davidson, A.R., and Bognar, A.L. (2003) Binding of ATP as Well as Tetrahydrofolate Induces Conformational Changes in Lactobacillus casei Folylpolyglutamate Synthetase in Solution. Biochemistry 42 , 1537-1543.

Zarrine-Afsar, A. and Davidson, A.R. (2003) The analysis of protein folding kinetic data produced in protein engineering experiments. Methods 34 , 41-50.

Leung, G.C., Hudson , J.W., Kozarova, A., Davidson, A.R., Dennis, J.W., and Sicheri, F. (2002) The Sak polo-box comprises a structural domain sufficient for mitotic subcellular localization. Nature Struct. Biol. 9 , 719-724.

Northey, J.G.B., Maxwell, K.L. & Davidson, A.R. (2002) Protein folding kinetics beyond the F value: using multiple amino acid substitutions to investigate the structure of the SH3 domain folding transition state. J. Mol. Biol. 320 , 389-402.

Maxwell, K.L., Yee, A.A., Arrowsmith, C.H., Gold, M., and Davidson, A.R. (2002) The solution structure of the bacteriophage lambda head-tail joining protein, gpFII. J. Mol. Biol. 318 , 1395-1404.

Northey, J.G.B., Di Nardo, A.A., and Davidson, A.R. (2002) Hydrophobic core packing in the SH3 domain folding transition state. Nature Struct. Biol. 9 , 126-30.

Larson, S.M., Ruczinski, I., Davidson, A.R., Baker, D., and Plaxco, K.W. (2002) Residues participating tn the folding nucleus do not exhibit preferential evolutionary conservation. J . Mol. Biol. 316 , 225-233.

Yu, B., Thompson, G.D., Yip, P., Howell, P.L., and Davidson, A.R. (2001) Mechanisms for intragenic complementation at the human argininosuccinate lyase locus. Biochemistry 40 , 15581-15590.

Maxwell, K.L., Yee, A.A., Booth, V., Arrowsmith, C.H., Gold, M., and Davidson, A.R. (2001) The solution structure of bacteriophage lambda protein W, a small morphogenetic protein possessing a novel fold. J. Mol. Biol. 308 , 9-14.

Last Modified: 21 August 2008, 12:35 PM