Energetics of substrate unfolding by ClpA

Abstract

Caseinolytic protease A (ClpA), a member of the Hsp 100 family of heat shock proteins, is the regulatory subunit of the E. coli protease ClpAP. As such, it recognizes proteins targeted for degradation by the cell and uses the energy of ATP hydrolysis to unfold substrates and translocate them to ClpP for proteolysis. ClpA's in vivo contribution to cellular maintenance is small, but it serves as an ideal model for energy-dependent molecular machines. The goal of this work is to describe the energetics of ClpA's interactions with both soluble and membrane-bound protein substrates. Green fluorescent protein modified with an 11-amino acid tag that facilitates ClpA recognition (GFP-ssrA) is used as a model soluble substrate, and ssrA-modified acetylcholine receptor (AChR-ssrA) is used as a model membrane-bound substrate.The structural and spectroscopic properties of GFP have been previously described. Here, in order to determine the minimum amount of energy needed to unfold this protein, we present two estimates of the free energy of unfolding (AGunfolding) for GFP-ssrA derived from thermal and chemical denaturation data. We find that GFP-ssrA is highly kinetically stable (tequil > 600 hrs.) and that AGunfolding = 0-2 kcal/mol. In addition, we show that ClpA retains its enzymatic activity under conditions which support single-molecule patch-clamp electrophysiology. These observations made it possible to carry out pilot studies for a novel electrophysiological unfoldase assay. ClpA may be able to actively extract protein substrates from the lipid bilayer. We are currently testing this hypothesis, and we are exploring ways to observe the process of substrate removal in real-time. The proposed electrophysiological unfoldase assay, once optimized, could be used to elucidate the mechanistic details of ClpA's interaction with membrane-bound substrates.

(cont.) We are specifically interested in determining the timescale of substrate removal and determining whether or not the substrate removal process occurs in one high-energy step or several low-energy steps.