Project Summary An understanding of protein folding pathways is important in understanding protein function. Protein folding can be studied in vitro using full-length proteins in dilute solutions under conditions optimized for successful folding. However, in the complex environment of the cell, co- and post-translational folding of proteins has been observed. The vectorial nature of co-translational folding as well as interactions between the nascent chain, modifying enzymes, and molecular chaperones presumably inhibit unfavorable interactions such as aggregation and smooth the energy landscape, thereby making folding in the cell quite different from folding in the test-tube. To date, there is a dearth of information about conformations available to a nascent chain in cells and how its interacting partners affect these conformations. This gap in knowledge is mainly due to the experimental difficulty of observing the folding reaction in cells. To overcome this limitation, we aim to develop a new method for study protein folding. This method, entitled pulse-chase in-cell fast photochemical oxidation of proteins (pcIC- FPOP) couples traditional pulse-chase technology with a mass spectrometry-based in- cell footprinting method. pcIC-FPOP will provide higher resolution information than gel electrophoresis, which is the current analytical technique for analysis of pulse-chase data, as tandem mass spectrometry can provide information on the amino acid residue- level. The development of this method requires a redesign of the footprinting platform. We have designed a new platform for in-cell footprinting that includes a stage-top incubator and nanopositioning system. We will assemble and optimize the new platform to demonstrate its efficacy for pcIC-FPOP (specific aim 1). We will use alpha 1 antitrypsin (A1AT) as a model system to test the ability of the method to probe short- lived folding intermediates (specific aim 2). We will also study two mutants of A1AT, S and Z to determine whether pcIC-FPOP can detect protein misfolding (specific aim 3). The S mutant has a mild folding defect while the Z mutant has a more severe defect. The study of both proteins will determine the sensitivity of the method in detecting protein misfolding. The developed method would provide a new, higher resolution tool for studying protein folding in the native cellular environment.