About Our Research
Protein Folding
Over the years, the main activity of this lab has been the study of the mechanisms by which proteins form their three-dimensional structures. In particular, we have used a combination of biochemical, biophysical and mutational methods to study the folding of proteins that are stabilized by disulfide bonds, especially bovine pancreatic trypsin inhibitor (BPTI) and an omega-conotoxin. By taking advantage of the ability to physically isolate and characterize the disulfide-bonded intermediates in the folding of these proteins, we have been able to study in great detail the effects of amino acid replacements on the folding mechanism. These studies are directed towards the long term goal of understanding the relationship between a protein's amino acid sequence and it's three-dimensional structure. In addition, these studies have important implications for understanding the effects of mutations on protein structure and function, including mutations that give rise to a variety of human diseases.
Protein Dynamics and Protease Inhibitor Function
More recently, we have been using NMR spectroscopy to study the effects of mutations on the dynamics of BPTI in its folded and partially folded states. These studies have already revealed that specific amino acid residues play particularly important roles in maintaining the relatively fixed conformation of the native state, particularly in the region of the protein that binds to and inhibits serine proteases. Although BPTI normally binds to serine proteases (such as trypsin) just as a substrate would, catalytic turnover is extremely slow. Some of the mutations that increase the flexibility of the trypsin binding region also increase it's susceptibility to cleavage by the enzyme.
We are now beginning to direct more of our efforts towards studying the interaction between BPTI and trypsin. We plan to investigate the backbone dynamics of BPTI variants when bound to the enzyme, again using NMR methods. From these studies, we hope 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.
Computational Chain Statistics
Another relatively new area for us is computational studies of protein conformation. Although, many investigators have applied theoretical and computational methods to the simulation of protein folding and the problem of predicting a protein's structure from its sequence, there have been relatively few simulations of the unfolded state. The unfolded state is important both because it serves as the starting point for folding reactions and because there is growing evidence that some proteins, or portions of proteins, are largely unfolded in vivo. Our simulations are intended to address questions concerning the relative importance of local and non-local interactions on the distribution of conformations making up the unfolded state and may provide new insights into the early events in the folding process.