Cotranslational Events and Folding Pathways of an Alpha-Helical Model Protein

University Of Wisconsin-Madison, Madison WI

Investigators

Abstract

Protein folding is a multifaceted poorly understood phenomenon of great biological significance. Most of the published literature on the subject deals with the in vitro refolding of purified full-length denatured polypeptides. In contrast, this project addresses cotranslational protein folding and misfolding, as it occurs within the ribosomal machinery. The main goal of this research is to develop a novel methodology to study the mechanistic and conformational aspects of protein folding within the prokaryotic translation apparatus. The folding kinetics of nascent polypeptide chains will be investigated in the context of the Escherichia coli translation machinery. These studies will be performed by hydrogen/deuterium exchange pulse labeling in cell-free systems. Liquid chromatography-coupled time-of-flight electrospray mass spectrometry and time-of-flight MALDI mass spectrometry will be employed for detection. Apomyoglobin (apoMb) serves as a good model system for these studies since it is a well-characterized cytoplasmic protein with single domain containing all alpha-helical segments. An apoMb gene with E. coli-optimized codon usage will be employed. Control experiments will also be performed in parallel on chloramphenicol acetyltransferase. The methodology developed in this project will set the stage to enable future investigations addressing important aspects of biologically relevant protein folding questions. These include (a) studying whether cotranslational folding takes place during expression of small single domain proteins; and (b) mapping nascent polypeptide conformation, and identifying which specific amino acids become folded or misfolded at different stages of chain elongation. Very little is known about the structural aspects of how proteins fold or misfold in the cell. This project aims at devising initial steps towards filling this gap.