Innovation of a Novel Pulse-Chase in Cell Footprinting Method for the Study of Protein Folding Phenomena

Abstract

Protein structure determines function for globular proteins, and the understanding of protein folding pathways aids in the understanding of their function. Traditionally, protein folding has been observed in vitro using full length protein sequences, but folding in the cell is quite different from folding studies in an isolated environment due to macromolecular crowding and specific interactions with the ribosome, chaperones and modifying enzymes. To overcome these limitations, we have begun development of a new method for protein folding studies. This method, Pulse Chase In-Cell Fast Photochemical Oxidation of Proteins (pcIC-FPOP), couples pulse-chase technology with mass spectrometry-based in cell footprinting which will allow for detailed characterization of short lived protein folding intermediates. FPOP coupled with mass spectrometry has become an invaluable tool used in structural proteomics to study surface accessibility in proteins. In our lab, FPOP has already been extended to protein labeling in live cells, allowing the study of protein conformations in the complex cell environment, and providing insight into ligand induced structural changes or conformational changes accompanying protein complex formation within the cellular context. pcIC FPOP will allow proteins synthesized during the pulse to be studied from birth to death. The novel pcIC-FPOP method required the design of a new platform for in-cell footprinting that includes a stage-top cell incubator and nanopositioning system automated to meet the high speed demands required to study protein folding intermediates. We are optimizing the new platform to demonstrate its efficacy for pcIC-FPOP. Preliminary benchmark data, will show the ability of this new method to provide comparable results to the already published method of in cell oxidative labeling, IC-FPOP. We believe this method will become powerful tool in probing protein folding and misfolding in the native cellular environment.