Research Project
3296307
The goal of this project is to introduce new methods for characterization of flexible partially ordered structures of peptides and proteins, based on nonradiative energy transfer measurements between site specifically attached donor and acceptor probes. The folding of bovine pancreatic trypsin inhibitor (BPTI) was chosen as a first case to be studied using the new methods since it has a well defined native structure and its pathway of folding comprising preferentially populated disulfide intermediates has been elucidated (Creighton 1978). Despite great efforts, the search for the conformational forces which direct the nonsequential pathway of disulfide bond formations is hampered mainly by lack of appropriate methods for characterizing the partially ordered states. A method which can yield distributions of intramolecular distances at very low concentrations is required. This can be achieved with the proposed method. In the proposed study I plan to proceed in two parallel ways: (1) I will develop analytical methods and install new equipment based on a synchronously pumped dye laser system and new software capable of determining equilibrium interprobe distance distributions even in the presence of Brownian motion. (2) I will introduce new probes and methods of labeling designed to increase sensitivity and minimize conformational effects. The structure of reduced denatured BPTI (R-BPTI) will be investigated, searching for clues for the kinetic dominance of two single disulfide bond intermediates, (5-30) and (30-51). A hypothesis that formation of secondary structures (beta and alpha) exclude cys 55 from the major early disulfide bond intermediates will be tested. The formation of the alpha-helix (residues 50-55) will be monitored in all intermediate states by labeling close to its ends (residues 46 and 58). Time permitting we shall study the conformation of the dominant single disulfide intermediate (30- 51) testing the hypothesis that flexibility of the N-terminal segment and a loop (residues 35-40) direct the formation of the obligatory nonnative second disulfides 5-14 and 5-38. The methods developed here should prove useful for investigation of many systems involving partially ordered states such as flexible hormone peptides and their interactions with other macromolecules or flexible multidomain enzyme molecules in solution and hopefully also in more complex environments.
