In my laboratory we employ a multifaceted approach to pursue our research. We determine the three-dimensional structures of proteins, 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 substitutions affect 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.
First recognized as a non-catalytic homology region in protein kinases related to Src oncogene, SH3 domains have now been identified in thousands of 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 it achieves by binding usually binding to PXXP-containing sequence motifs in target proteins. Our current work on SH3 domains is focused on understanding how these domains are able to specifically recognize biologically important targets within the cell. We are investigating several different SH3 domains from the yeast, S. cerevisiae . We have uncovered mechanisms by which these domains recognize very unusual target sequences and avoid non-specific interactions.
Bacteriophages (or phages) are viruses that infect bacteria. They are the most abundant biological entities on the planet, with a global population estimated at 10 31 particles. Tailed bacteriophages, which comprise 95% of phages, are complicated macromolecular assemblages composed of a DNA genome and well over 500 individual protein subunits encoded by at least 10 different genes. For tailed phage particles to assemble and mediate bacterial infection, myriad protein interactions must occur within precisely regulated assembly and disassembly pathways. In my lab, we employ a multifaceted approach aimed at elucidating the mechanisms regulating these tailed phage assembly pathways, which includes protein structure determination, bioinformatics, mutagenesis, and in vivo functional assays.
Phage, Pseudomonas aeruginosa , and Cystic Fibrosis
Pseudomonas aeruginosa (PA) is an environmentally ubiquitous Gram-negative bacteria, which is highly resistant to antibiotics. PA is the leading cause of ICU pneumonia, catheter-associated urinary tract infections, and burn wound infections that can lead to sepsis. Individuals with the genetic disease, Cystic Fibrosis (CF), are particularly prone to lung infections with PA, and this organism is the leading cause of death for this group. We are investigating the prevalence and effect of phages within the lungs of patients with CF in an effort to understand whether phages can modulate the frequency and severity of clinical exacerbations caused by PA. We are also carrying out investigations aimed at developing phage-based alternative therapies for PA.
Cardarelli, L., Pell, L.G., Neudecker, P., Pirani, N., Liu, A., Baker, L.A. , Rubinstein, J.L., Maxwell, K.L., Davidson, A.R. (2010). Phages have adapted the same protein fold to fulfill multiple functions in virion assembly. Proc Natl Acad Sci U S A 107, 14384-9.
Yu, Z., Reichheld, S.E., Savchenko, A., Parkinson, J., Davidson, A.R. (2010) A comprehensive analysis of structural and sequence conservation in the TetR family transcriptional regulators. J Mol Biol 400 , 847-864.
Cardarelli, L., Lam, R., Tuite, A., Baker, L. A., Sadowski, P. D., Radford, D. R., Rubinstein, J. L., Battaile, K. P., Chirgadze, N., Maxwell, K. L. & Davidson, A. R. (2010). The crystal structure of bacteriophage HK97 gp6: defining a large family of head-tail connector proteins. J Mol Biol 395 , 754-68.
Reichheld, S.E., Yu, Z., Davidson, A.R. (2009) The induction of folding cooperativity by ligand binding drives the allosteric response of tetracycline repressor. Proc Natl Acad Sci U S A. 106, 22263-22268.
Stollar, E.J., Garcia, B., Chong, P.A., Rath, A., Lin, H., Forman-Kay, J.D., Davidson, A.R. (2009) Structural, Functional, and Bioinformatic Studies Demonstrate the Crucial Role of an Extended Peptide Binding Site for the SH3 Domain of Yeast Abp1p. J Biol Chem 284 , 26918-26927.
Pell, L.G., Liu, A., Edmonds, L., Donaldson, L.W., Howell, P.L., Davidson, A.R. (2009) The X-Ray Crystal Structure of the Phage l Tail Terminator Protein Reveals the Biologically Relevant Hexameric Ring Structure and Demonstrates a Conserved Mechanism of Tail Termination Among Diverse Long-Tailed Phages. J Mol Biol. 389 , 938-951.
Pell, L.G., Kanelis, V., Donaldson, L.W., Howell, P.L., Davidson, A.R. (2009) The phage l major tail protein structure reveals a common evolution for long-tailed phages and the type VI bacterial secretion system. Proc Natl Acad Sci U S A 106, 4160-4165.
Zarrine-Afsar, A., Wallin, S., Neculai, A.M., Neudecker, P., Howell, P.L., Davidson, A.R.*, Chan, H.S.* (2008) Theoretical and experimental demonstration of the importance of specific nonnative interactions in protein folding. Proc Natl Acad Sci U S A 105 , 9999-10004. *Co-Corresponding Authors
Kim, J, Lee, C.D., Rath A., Davidson, A.R. (2008) Recognition of non-canonical peptides by the yeast Fus1p SH3 domain: elucidation of a common mechanism for diverse SH3 domain specificities. J Mol Biol. 377 , 889-901.
Reichheld, S. E. & Davidson, A. R. (2006). Two-way Interdomain Signal Transduction in Tetracycline Repressor. J Mol Biol 361 , 382-9.
Fraser, J. S., Yu, Z., Maxwell, K. L. & Davidson, A. R. (2006). Ig-like domains on bacteriophages: a tale of promiscuity and deceit. J Mol Biol 359 , 496-507.
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 , 813-23.
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 , 586-90.
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 U S A 101 , 7954-9.