Intellectual Merit. The pathways by which proteins attain their folded structure are poorly understood in the context of the cellular environment. The goal of this project is to address the fundamental principles governing protein folding in a biologically relevant context, as nascent polypeptides exit from the ribosomal tunnel during translation. The investigations are based on the working hypothesis that long-range folding interactions cannot be established among portions of the chain that have not been synthesized yet. Therefore, in vitro folding pathways of full length proteins are likely to be different from the corresponding pathways of cotranslationally elongating chains. Using methodological advances developed under prior NSF support, specific structural aspects of cotranslational protein folding as a function of chain elongation will be elucidated. The project will focus on two single-domain proteins (apo-myoglobin and thymidylate kinase) with predominantly alpha-helical structure and a large number of long-range native contacts. Nascent polypeptides will be analyzed by hydrogen/deuterium exchange followed by time-of-flight MALDI mass spectrometry. This approach unveils the degree of chain compaction and backbone hydrogen bonding as a function of chain elongation. High resolution information will be gained by in situ limited proteolysis under quenching conditions. The above studies are carried out in a cell-free system either containing or lacking the gene for the ribosome-associated Trigger Factor chaperone.
Broader Impacts. The Cavagnero group is strongly committed to undergraduate participation to research and to the active involvement of underrepresented groups in academia. Undergraduate students contribute significantly to the research and regularly appear as coauthors of research publications. Several past and current undergraduate students have received prestigious campus-wide undergraduate research awards. The principal investigator is involved in educational curriculum development in collaboration with the NSF-sponsored Center for the Integration of Research, Teaching and Learning (CIRTL) at UW-Madison and its campus program, Delta. This includes an initiative to develop connections between general chemistry principles and everyday life-science-related applications.
