This project will investigate the mechanisms and principles that drive protein folding. Protein folding is fundamental to all living organisms. The pathway of intermediate structures that carry proteins from their initially synthesized unstructured condition to their biologically functional structure is arguably the most centrally important reaction in all of biology. Further, it is key to understanding many normal processes and to the effort to interdict challenging misfolding processes. The folding problem has proven extremely difficult. All available methodologies have been directed at the problem but they commonly cannot describe detailed structure formation during the kinetic folding process. This laboratory has developed and recently demonstrated a new approach, named hydrogen exchange analyzed by mass spectrometry (HX-MS), that shows great promise for delving into and perhaps solving this long-lasting enigma. The understanding of the folding process may provide the knowledge necessary to interfere in a constructive way with misfolding processes. The investigator has a superb record of training graduate students and postdocs and will continue these training activities. In addition, the proposed method development and software tools are likely to benefit the scientific community at large.
Recent work in this laboratory has developed the pulse labeling HX MS method and shown it can be used to define, for the first time, the structure of the increasingly folded intermediate forms that together construct the protein folding pathway. Results so far obtained for three proteins (ribonuclease H, cytochrome c, and maltose binding protein) clearly display the timed development of structures that carry the initially unfolded proteins (U) through intermediate forms (Ii) to their final native state (N). Further the results suggest three principles that appear to govern the folding process. The plan going forward is to test for the reality and generality of these results. Initial efforts will be directed at the alpha-subunit of tryptophan synthase, a moderate-sized protein with 265 residues in a TIM barrel structure, which represents the most common protein fold, accounting for ~10% of all known proteins. The investigation will then be extended to the small all-beta protein ubiquitin (76 residues) and then ultimately to the immense Hsp104 protomer (908 residues).
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
