Integrated Process for Functional Site Feature Analysis

Wake Forest University, Winston Salem NC

Investigators

Abstract

Sequence and structural genomics projects have identified and predicted molecular functions in proteins, yet researchers still cannot determine biological mechanisms of, for example, catalysis or substrate specificity or inhibitor binding, without detailed biochemical and biophysical analysis of a single protein. While structural genomics projects are providing the necessary data, they are not being used to reveal the general principles underlying biological mechanism. Sequence, structure, bioinformatics, and biophysical methods will be used to characterize the molecular function sites of 6 protein superfamilies, focusing on the following objectives: 1) characterizing the sequence and structure of functional-site features and using the results to develop methods for clustering the peroxiredoxin family; 2) analyzing the electrostatics, including ionizable residue pKas, residues affecting these pKas, and electrostatic potential, at peroxiredoxin functional sites and testing them experimentally; 3) integrating the electrostatic, sequence and structural information to create a robust profiling method that can identify peroxiredoxin subfamilies; and 4) using it to create active-site signatures and profiles for a well-studied and important set of protein superfamilies. Crossing the gap from molecular function to biological mechanism requires integrating sequence, structure, and physical-chemical data. The peroxiredoxin superfamily makes a good test case, because of the emerging body of interesting sequence, structure, and function information, much of it generated by the Poole group. Combined strengths of the computational and experimental groups involved in the project will allow iterative improvements to knowledge in both areas. The detailed functional site analysis of 5 other superfamilies will yield insights into biological mechanisms, leading to hypotheses that can be experimentally tested. In the long term, the resulting methods will enable more accurate functional site identification from sequence. The development of general concepts for identifying and classifying molecular functional site features of proteins will improve the ability to predict molecular function, to design enzymes with novel functions and mechanisms, and to determine what molecules bind to proteins of unknown structure and function. Wake Forest University is a leading liberal arts institution, where professors have regular and intense intellectual interaction with undergraduate and graduate students. The institution fosters the Teacher/Scholar ideal: professors are active researchers, engaging students both in the classroom and the laboratory. Students involved in this project will gain leading-edge, cross-disciplinary molecular biophysics training that will fuel their success in productive scientific careers. In addition, two of the investigators teach an interdisciplinary molecular biophysics course in which students are introduced to computational methods and work to interpret data in terms of protein structure. This past fall, graduate and undergraduate students from physics, biochemistry, chemistry, and biology, as well as researchers from a local biopharmaceutical company, studied the peroxiredoxin family in this course and were introduced to the ideas and communication skills necessary in an interdisciplinary research project.