Abstract
SecB is a homotetrameric, cytosolic chaperone that forms part of the protein translocation machinery in Escherichia coli. We have investigated the bound-state conformation of a model protein substrate of SecB, bovine pancreatic trypsin inhibitor (BPTI) as well as the conformation of SecB itself by using proximity relationships based on site-directed spin-labeling and pyrene fluorescence methods. BPTI is a 58-residue protein and contains three disulfide groups between residues 5 and 55, 14 and 38, as well as 30 and 51. Mutants of BPTI that contained only a single disulfide were reduced, and the free cysteines were labeled with either thiol-specific spin labels or pyrene maleimide. The relative proximity of the labeled residues was studied using either electron spin resonance spectroscopy or fluorescence spectroscopy. The data suggest that SecB binds a collapsed coil of reduced unfolded BPTI, which then undergoes a structural rearrangement to a more extended state upon binding to SecB. Binding occurs at multiple sites on the substrate, and the binding site on each SecB monomer accommodates less than 21 substrate residues. In addition, we have labeled four solvent-accessible cysteine residues in the SecB tetramer and have investigated their relative spatial arrangement in the presence and absence of the substrate protein. The electron spin resonance data suggest that these cysteine residues are in close proximity (15 A) when no substrate protein is bound but move away to a distance of greater than 20 A when SecB binds substrate. This is the first direct evidence of a conformational change in SecB upon binding of a substrate protein.
Highlights
Molecular chaperones are a class of proteins that prevent aggregation of newly synthesized or previously denatured polypeptides and proteins and mediate their folding into the native state [1]
We have investigated the binding of SecB to several bovine pancreatic trypsin inhibitor (BPTI) mutants that were labeled at two Cys residues with either pyrenes or spin labels
In a first set of experiments, we have incorporated into unfolded BPTI a pair of either spin labels or fluorescent labels at various positions along the primary sequence to investigate the proximity of the labels relative to each other in the free as well as the SecBbound state
Summary
Molecular chaperones are a class of proteins that prevent aggregation of newly synthesized or previously denatured polypeptides and proteins and mediate their folding into the native state [1]. Ular chaperones is the ability to recognize structural elements exposed in unfolded or partially denatured states such as hydrophobic surfaces They do not bind the native state of proteins and are capable of interacting with a variety of different polypeptide chains, often without apparent sequence preferences [1, 2]. 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]. BPTI, has been extensively used as a model protein for folding studies It consists of 58 residues and is stabilized by three disulfide bonds between the amino acids 5 and 55, 14 and 38, as well as 30 and 51. A similar approach has been employed recently to determine the proximity of helices and their relative movement upon photoactivation in bacteriorhodopsin [23, 24]
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