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David Goldenberg

professor of biology

A.B. Whitman College

Ph.D. Massachusetts Institute of Technology

Research

References

 

David Goldenberg's Lab Page

Research

The long-range goal of research in my laboratory is to understand the mechanisms by which polypeptide chains fold into stable structures and assemble into functional complexes. We are particularly interested in understanding the role of protein flexibility in determining the stabilities of protein structures, the specificity of their formation and their functional properties.

At present, the majority of our efforts are directed towards studying the interactions between a protease, bovine trypsin, and a natural inhibitor of this enzyme, bovine pancreatic trypsin inhibitor (BPTI).   BPTI is a member of a large class of natural protease inhibitors that act by binding to the active sites of their targets, much as a substrate would, but are resistant to hydrolysis.   By blocking the access of potential substrates to the active sites, these inhibitors play critical biological roles in preventing unregulated proteolysis.   The interaction between BPTI and trypsin is remarkable for both its very high stability (with a dissociation constant of about 10 -12 M) and a very slow rate of hydrolysis of the inhibitor (with a half-time measured in years).   Over the past several years, we have found that amino acid replacements at specific sites in BPTI can, at the same time, increase the flexibility of the free inhibitor, decrease the stability of the complex it forms with trypsin and increase its likelihood of being hydrolyzed by the enzyme.   Together, these observations suggest that flexibility, or the lack thereof, is a critical factor in the function of this and other protease inhibitors.

To study protein flexibility, we use high resolution NMR spectroscopy, which provides unique information about the motions of individual atoms or bonds in proteins on a wide range of time scales.   We have used this approach to study the effects of several amino acid replacements on the flexibility of the free form of BPTI, and we are now beginning to investigate the dynamics of BPTI variants when bound to the enzyme. From these studies, we expect to learn much more about the factors that determine flexibility in the context of an enzyme-substrate complex and about the nature of the motions that are necessary for catalytic function. The NMR studies are being complemented by x-ray crystallography of the enzyme-inhibitor complexes, in collaboration with our colleague Martin Horvath. We are also studying the thermodynamics of formation of these complexes in order to gain further insights into the energetic factors that determine binding and inhibition.

Close up view of the interaction between trypsin and BPTI. (Huber et al. (1974) J. Mol. Biol. 89 , 73)

References