Solvation and Crowding Effects on Unfolded Proteins

University Of Utah, Salt Lake City UT

Investigators

Abstract

Most protein molecules are able to exist in two dramatically different conformational states, a well-defined folded structure and an unfolded state that is composed of an immense number of rapidly interconverting conformations. Although biological function is usually associated with the folded state, unfolded polypeptides are now recognized to play important roles in both normal and aberrant cellular function, including protein localization and degradation and the formation of amyloid fibers. The physical properties of unfolded proteins remain poorly defined and the subject of considerable controversy. Most of the available information about these molecules comes from studies of dilute solutions in denaturants. This project is directed towards developing an improved understanding of unfolded proteins under more physiological conditions, using a combination of computational and experimental approaches. The physical properties of several model unfolded proteins will be examined using small-angle x-ray scattering, NMR-based diffusion measurements, and measurements of equilibrium constants for the formation of disulfide bonds between cysteine residues. These measurements will provide quantitative information about properties such as average dimensions, packing densities and the energetics of intramolecular interactions. In addition, small-angle neutron scattering will be used to study the unfolded proteins in the presence of high concentrations of other proteins. The experimental results will be directly compared to the predictions of computational simulations recently developed in this laboratory, so as to test the assumptions of the model and allow the experimental data to be interpreted in structural terms. Together, the computational and experimental results will provide unique information about the effects of solvent interactions and macromolecular crowding on unfolded proteins and will lead to an improved understanding of their behavior in the cellular environment. This research will have an immediate impact on scientific training through the participation of undergraduate and graduate students, who will be responsible for the majority of the work and will actively contribute to its intellectual progress. The project also includes an international collaboration, with Dr. Jill Trewhella of the University of Sydney (Australia), and exchange visits between the two laboratories will contribute to the broader education of members of each laboratory. Because the principal investigator is also actively engaged in the teaching of both undergraduate and graduate courses, the methods and ideas developed in this project are very likely to be incorporated into more formal educational activities, as exemplified by his previous development of a biochemistry laboratory course based on materials and methods from his research program. The computer programs and scripts written for this project will be made available to other investigators and educators, so as to allow further testing and refinement of the models, as well as their use as pedagogical tools.