个人简介

Ph.D. Case Western Reserve University Postdoctoral Associate, Cornell University 2000-2002 NSF Minority Postdoctoral Fellow, Cornell University 2002-2005 Robert C. and Sandra Connor Endowed Faculty Fellow, Fulbright College of Arts and Sciences 2007-2008

研究领域

biochemistry

Biophysical Characterization of Protein Structure and dynamics of Cdc42Hs forms related to its cell transforming capabilities A potent human oncoprotein, Dbl, decreases the affinity of the Cdc42Hs protein for GDP leading to cell transformation by increasing cycling between GTP/GDP-bound forms. In fact, Dbl has its greatest affinity for Cdc42Hs in the nucleotide-depleted form. However, the structural significance of this interaction in Cdc42Hs has not been explored. A single-point mutant of Cdc42Hs, Cdc42Hs(F28L), also shows decreased affinity for the nucleotide exhibited by an increase in the cycling rate between GTP and GDP bound forms of the protein. Research on the structure and dynamics of this mutant have shown that the nucleotide-binding site is greatly altered; however, no important protein-protein interacting regions seem to be affected (Adams, et al., 2004, 2006). The structure and dynamics of the nucleotide-depleted form of Cdc42Hs will be studied and the structural modifications in this construct will be compared to the Cdc42Hs-GDP complex. These studies will provide information on: 1) Conformation and flexibility at the nucleotide-binding site in the absence of the nucleotide, 2) Conformational changes in the nucleotide-binding site that may be caused by interactions with effectors (such as Dbl) that decrease the affinity of Cdc42Hs for GDP, and 3) Conformation and dynamics changes, in the absence of the nucleotide, in the region of Cdc42Hs that binds Dbl (Switch II) that may facilitate an optimal conformation for the Cdc42Hs-Dbl interaction. These studies will uncover structural mechanisms of cell cycle regulation by Dbl that affect cell signaling. Effectors of Cdc42Hs can initiate signaling cascades that regulate cell cycle progression, as well as, cause cellular transformation. Cdc42Hs(F28L), known to induce cellular transformation due to an increase in the rate of GTP-GDP cycling, has been used as a background to characterize transformation-deficient mutants. A 13-amino acid region of Cdc42Hs (designated the Rho insert region) is unique to the Rho subfamily of Ras proteins. When this insert region is deleted from the Cdc42Hs(F28L) mutant, rapid cycling between GTP and GDP remains, but the transformation ability of the protein is eradicated. Hence, the insert region plays a regulatory role in the function of Cdc42Hs, possibly by binding to some unknown effector that is key to cellular transformation. Presently, I am working on the 3-D structure and dynamics parameters of Cdc42Hs(?L8) (F28), which should answer the questions of whether or not the nucleotide-binding region is altered by the removal of the insert, as well as, if there are any significant changes in important effector-binding regions. The structure and dynamics of the double mutant, Cdc42Hs(F28L_?L8), will be studied in order to delineate structural ramifications of protein-protein interactions that mediate cell cycle progression and oncogenesis. This will include defining the binding surface of the unknown effector that diminishes the transformation capability of this protein, if applicable. Fluorescence Spectroscopy Studies of Conformation-Dependent Interactions with Cdc42Hs Correlation between conformational dynamics and protein-protein interactions is important to understanding protein function. Fluorescence spectroscopy will be used to characterize interactions of peptide derivatives of known effectors with Cdc42Hs constructs believed to be directly involved in effector/regulator interactions. The results from the fluorescence studies will be used to measure binding affinities, and extract thermodynamics parameters to characterize important protein interactions involving Cdc42Hs constructs that alter the protein’s function(s) in signal transduction. Studies of Cdc42Hs complexed with PBD, a peptide fragment of an important downstream effector of Cdc42Hs (PAK), have shown that, upon binding, the structure of the effector-binding Switch I and II regions exhibit less complex motions relative to free Cdc42Hs. Fluorescence studies characterizing the binding of PBD with Cdc42Hs(T35A) (Adams, et al, in preparation) have shown that this mutation in an important effector-binding region of Cdc42Hs (Switch I) facilitates a decreased binding affinity for PBD relative to Cdc42Hs wildtype, suggesting that a conformational change in the protein in this region modifies an important protein interaction that could affect proper cell function. It has been a widely debated topic as to how certain effectors specifically recognize and bind to Cdc42, as opposed to Rac or Rho (other GTP-binding proteins with similar sequences to Cdc42Hs), as well as, which effector controls what cellular events. These studies should provide an excellent way to monitor intricate molecular details on the mechanism of binding and activation of protein effectors/regulators that exploit even subtle changes in conformation that lead to aberrant cell behavior. Structural Studies of the Rheb Family of G Proteins The Rheb family of GTP-binding proteins is a relatively new class of proteins within the Ras superfamily that have been recently identified. These proteins play a role in cell growth regulation similar to other Ras proteins, and are also involved in tuberous sclerosis complex, a genetic disorder that can result in mental retardation, seizures and/or benign tumorigenesis (Roach, E. S., et al, Journal of Child Neurology, 2004 13, p. 624-628). Rheb proteins bind nucleotide in a similar manner as Cdc42Hs and other Ras proteins, however, the same mutations that convert Ras proteins (including Cdc42Hs) into active, transforming proteins, do not facilitate similar functions in Rheb (Tabancay, A.P., et al, Journal of Biological Chemistry, 2003, 278, p. 39921-39930). The structure of these Rheb mutant proteins, and the comparisons of the structure to the corresponding Cdc42Hs mutants, will undoubtedly uncover information on structural differences in similar regions between these two Ras proteins that account for Rheb protein mutants not exhibiting the same types of constitutively active state functions, even in the face of the same mutation(s). The study of Rheb mutant proteins will provide a link between the structure of Ras proteins and their particular biological roles in the face of certain types of mutations.

近期论文

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R. Chandrashekar, O. Salem, H. Krizova, R. McFeeters and P. D. Adams, “A switch 1 mutant of Ras protein Cdc42 exhibits decreased conformational freedom”, (2011) Biochemistry, DOI: 10.1021/bi2004284. K. Kathir, L. Gao, D. Rajalingam, A. Daily, S. Brixey, H. Liu, D. Davis, P.D. Adams, I. Prudovsky, and T.K.S. Kumar, “NMR Characterization of the Copper and Lipid Interactions of the C2B Domain of Synaptotagmin I-Relevance to the Non Classical Secretion of the Human Acidic Fibroblast Growth Factor (hFGF-1)”, Biochim. Biophys. Acta, (2009), vol 1798, #2, p. 297-302 D. Rajalingam, K. Kathir, K. Ananthamurthy, P.D. Adams, T.K.S. Kumar, "An Efficient Method to Prevent the Degradation of Recombinant Proteins by Thrombin," Analytical Biochemistry, (2008), 375, p.361-363 R.E. Oswald and P.D. Adams, "NMR Assignment of Cdc42(T35A), an Active Switch I Mutant of Cdc42," Biomolecular NMR Assignments, (2007), 1: p. 225-227 Adams, P.D., and Oswald, R.E. (2006). Solution structure of an oncogenic mutant of Cdc42Hs. Biochemistry 45, 2577-2583 Adams, P.D., Loh, A.P., and Oswald, R.E. (2004). Backbone dynamics of an oncogenic mutant of Cdc42Hs shows increased flexibility at the nucleotide-binding site. Biochemistry 43, 9968-9977 Adams, P.D., Chen, Y., Ma, K., Zagorski, M.G., Sonnichsen, F.D., McLaughlin, M.L., and Barkley, M.D. (2002). Intramolecular quenching of tryptophan fluorescence by the peptide bond in cyclic hexapeptides. J Am Chem Soc 124, 9278-9286 B. Liu, R.K. Thalji, P.D. Adams, F.R. Fronczek, M.L. McLaughlin, and M.D. Barkley. (2002) Fluorescence of cis-1-Amino-2-(3-indolyl)cyclohexane-1- carboxylic Acid: A Single Tryptophan ?1 Rotamer Model. J. Am. Chem. Soc. 124, 13329-13338