The long term goal of this project is to elucidate the role of protein flexibility in determining the energetic and functional aspects of protein-protein interactions, particularly in the context of serine proteases and their natural protein inhibitors. The serine proteases play numerous metabolic and regulatory roles in humans and other organisms, and an important mechanism of their own regulation involves proteins that bind to their active sites with very high affinities, apparently mimicking a substrate, but without undergoing proteolysis at a significant rate. This study will focus on one member of this large class of inhibitors, bovine pancreatic trypsin inhibitor (BPTI). Although aspects of this protein have been extensively studied by biophysical and mutational methods, it is not yet known why it (or other members of the class) are so dramatically resistant to hydrolysis. Various destabilizing chemical and mutational modifications can increase the rate of cleavage, however, suggesting that the stability or rigidity of the structure when bound to the enzyme somehow prevents motions that are necessary for catalysis. The rigidity of the structure, which undergoes very little change upon binding to trypsin, is also likely to contribute to the very high stability of the enzyme-inhibitor complex. To test these hypotheses, the dynamics of several BPTI variants, both free and when bound to trypsin, will be studied by NMR relaxation methods. In addition, the thermodynamics of the interactions will be studied by calorimetric and fluorescence methods, and x-ray crystal structures will be determined for the BPTI-trypsin complexes. The combination of data from these three approaches will lead to an improved understanding of how protein flexibility, or the lack thereof, influences the regulation of a medically important class of enzymatic reactions. More generally, this work will contribute to the fundamental knowledge necessary to predict and understand the interactions among biological macromolecules, an area of rapidly increasing biomedical importance.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM042494-17
Application #
7197318
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Wehrle, Janna P
Project Start
1989-09-01
Project End
2010-03-31
Budget Start
2007-04-01
Budget End
2010-03-31
Support Year
17
Fiscal Year
2007
Total Cost
$265,494
Indirect Cost
Name
University of Utah
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Goldenberg, David P (2010) The Product Operator Formalism: A Physical and Graphical Interpretation. Concepts Magn Reson Part A Bridg Educ Res 36A:49-83
Zakharova, Elena; Horvath, Martin P; Goldenberg, David P (2009) Structure of a serine protease poised to resynthesize a peptide bond. Proc Natl Acad Sci U S A 106:11034-9
Zakharova, Elena; Horvath, Martin P; Goldenberg, David P (2008) Functional and structural roles of the Cys14-Cys38 disulfide of bovine pancreatic trypsin inhibitor. J Mol Biol 382:998-1013
Wang, Yuanyuan; Trewhella, Jill; Goldenberg, David P (2008) Small-angle X-ray scattering of reduced ribonuclease A: effects of solution conditions and comparisons with a computational model of unfolded proteins. J Mol Biol 377:1576-92
Hanson, W Miachel; Domek, Gretchen J; Horvath, Martin P et al. (2007) Rigidification of a flexible protease inhibitor variant upon binding to trypsin. J Mol Biol 366:230-43
Hanson, W Miachel; Beeser, Scott A; Oas, Terrence G et al. (2003) Identification of a residue critical for maintaining the functional conformation of BPTI. J Mol Biol 333:425-41
DeLa Cruz, R; Whitby, F G; Buczek, O et al. (2003) Detergent-assisted oxidative folding of delta-conotoxins. J Pept Res 61:202-12
Bulaj, G; Goldenberg, D P (1999) Early events in the disulfide-coupled folding of BPTI. Protein Sci 8:1825-42
Beeser, S A; Oas, T G; Goldenberg, D P (1998) Determinants of backbone dynamics in native BPTI: cooperative influence of the 14-38 disulfide and the Tyr35 side-chain. J Mol Biol 284:1581-96
Bulaj, G; Kortemme, T; Goldenberg, D P (1998) Ionization-reactivity relationships for cysteine thiols in polypeptides. Biochemistry 37:8965-72

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