The major objective of this proposal is to develop and apply methods to elucidate the dynamic structure of proteins in solution. It is new generally recognized that proteins do not have static structures and that X-ray data must be supplemented by motional information about side-chain and backbone atoms if a more complete understanding of the nature of protein interactions is to be achieved. We propose to synthesize a variety of 13C- and 2H-labeled amino acids and incorporate them into HIV-1 protease, despentapeptideinsulin amide, and the 9th and 10th type III domains of fibronectin. These molecules will then be studied by high field (200 600 MHz) nuclear magnetic resonance (Nmr) spectroscopy. We are especially interested in studying side-chain motion in these proteins and therefore our primary purpose is to synthesize amino acids labeled at every other side-chain carbon with 13C. Then with site- specific T1, T2, and long-range NOE data in hand, we hope to be able to paint a detailed picture of the structure and dynamics at and around these labeled sites. Recent work suggests that in many proteins flexibility may play a crucial role in determining enzymatic activity. Thus, for example, in proteins such as HIV-1 Protease, flexibility in the conformationally mobile """"""""flap"""""""" region may control on-off access of the substrate and an understanding of this motion may provide information vital ta the rational design of protease inhibitors. To implement these projects, I am requesting support for two undergraduate students for four years. They will bear primary responsibility for the synthesis of all the labeled amino acids, peptides and insulin derivatives and they will routinely purify and analyze their products by chromatography and nmr. York's 200 MHz broadband FT-nmr, Sun Sparc5 workstation networked to an SGI with Biosym nmr processing/modeling software, and our solid phase peptide synthesizer will enable these students to participate fully in preparing, characterizing, and analyzing their 13C- and 2H- labeled products with the aid of forefront technology not generally available to undergraduates.

Project Start
1999-06-01
Project End
2000-05-31
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
24
Fiscal Year
1999
Total Cost
Indirect Cost
Name
York College
Department
Type
DUNS #
City
New York
State
NY
Country
United States
Zip Code
11451
Yuan, Yiping; Shen, Xiaotong; Pan, Wei (2012) Maximum Likelihood Estimation Over Directed Acyclic Gaussian Graphs. Stat Anal Data Min 5:
Knapp, John M; Zhu, Jie S; Tantillo, Dean J et al. (2012) Multicomponent assembly of highly substituted indoles by dual palladium-catalyzed coupling reactions. Angew Chem Int Ed Engl 51:10588-91
Fearnley, Stephen Philip; Thongsornkleeb, Charnsak (2010) Oxazolone cycloadducts as heterocyclic scaffolds for alkaloid construction: synthesis of (+/-)-2-epi-pumiliotoxin C. J Org Chem 75:933-6
Macneil, Margaret A; Purrier, Sheryl; Rushmore, R Jarrett (2009) The composition of the inner nuclear layer of the cat retina. Vis Neurosci 26:365-74
Desamero, Ruel Z B; Kang, Jeonghee; Dol, Chrystel et al. (2009) Spectroscopic characterization of the SH2- and active site-directed peptide sequences of a bivalent Src kinase inhibitor. Appl Spectrosc 63:767-74
Levinger, Louis; Hopkinson, Angela; Desetty, Rohini et al. (2009) Effect of changes in the flexible arm on tRNase Z processing kinetics. J Biol Chem 284:15685-91
Zhang, Chun-Yang; Johnson, Lawrence W (2009) Single quantum-dot-based aptameric nanosensor for cocaine. Anal Chem 81:3051-5
Juszczak, Laura J; Desamero, Ruel Z B (2009) Extension of the tryptophan chi2,1 dihedral angle-W3 band frequency relationship to a full rotation: correlations and caveats. Biochemistry 48:2777-87
MacNeil, Margaret A; Gaul, Paulette A (2008) Biocytin wide-field bipolar cells in rabbit retina selectively contact blue cones. J Comp Neurol 506:6-15
Arsov, I; Li, X; Matthews, G et al. (2008) BAC-mediated transgenic expression of fluorescent autophagic protein Beclin 1 reveals a role for Beclin 1 in lymphocyte development. Cell Death Differ 15:1385-95

Showing the most recent 10 out of 30 publications