SecB Binds Only to a Late Native-like Intermediate in the Folding Pathway of Barstar and Not to the Unfolded State

Abstract

SecB is a cytosolic, tetrameric chaperone of Escherichia coli which maintains precursor proteins in a translocation competent state. We have investigated the effect of SecB on the refolding kinetics of the small protein barstar in 1 M guanidine hydrochloride at pH 7.0 and 25 degreesC using fluorescence spectroscopy. We show that SecB does not bind either the native or the unfolded states of barstar but binds to a late near-native intermediate along the folding pathway. For barstar, polypeptide collapse and formation of a hydrophobic surface are required for binding to SecB. SecB does not change the apparent rate constant of barstar refolding. The kinetic data for SecB binding to barstar are not consistent with simple kinetic partitioning models.

A Thermodynamic Coupling Mechanism for the Disaggregation of a Model Peptide Substrate by Chaperone SecB

“…Thus, the data is consistent with a model that the B-chain is bound on the surface of the chaperone SecB in a flexible extended state. In certain cases it has been shown that SecB binds to partially folded conformations of proteins such as ␣-lactalbumin and barstar (8,10). In the present work we suggest that the bound conformation is an extended conformation.…”

“…SecB purification was done as reported previously (8). The purified protein was estimated to be 99% pure by SDS-polyacrylamide gel electrophoresis as detected by silver staining (16) and analytical gel filtration high Performance liquid chromatography.…”

“…In vivo, SecB binds to a subset of precursor proteins and maintains them in an unfolded, translocation competent state, while in vitro it interacts with a variety of proteins in the non-native state (6 -9). Studies on a model protein substrate of SecB, barstar, revealed that SecB did not bind the folded or unfolded state, but trapped a near native-like molten globule state (8). SecB has also been shown to bind partially folded states of ␣-LA (10).…”

Electron Spin Resonance and Fluorescence Studies of the Bound-state Conformation of a Model Protein Substrate to the Chaperone SecB

“…SecB purification was done as reported previously (6). The purified protein was estimated to be 99% pure by SDS-polyacrylamide gel electrophoresis as detected by silver staining (28) and analytical gel filtration high performance liquid chromatography.…”

“…For some proteins the translocationcompetent state contains significant secondary and tertiary structure (4). Studies on the model protein substrate, barstar, revealed that SecB does not bind the folded or unfolded state but traps a near native-like molten globule state (6). SecB has also been shown to bind partially folded states of lactalbumin (9).…”