Thermodynamics of Substrate Binding to the Chaperone SecB

Panse, Vikram Govind; Swaminathan, Chittoor P.; Surolia, A.; Varadarajan, Raghavan

doi:10.1021/bi992065o

Cited by 20 publications

(31 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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.…”

Section: Thermodynamic Coupling Model Of B-chain Dissociation Bysupporting

confidence: 53%

“…The two results are not necessarily contradictory because of the very short length of the binding frame. Since SecB binds only small 10-residue regions of the substrate and the binding site on SecB is solvent accessible (10) it is possible for the bound fragment to be extended even though the remainder of the protein adopts a relatively compact conformation. Recent NMR studies of the ␣-lactalbumin molten globule have also shown that some regions of the molten globule adopt an unfolded conformation (33).…”

Section: Thermodynamic Coupling Model Of B-chain Dissociation Bymentioning

confidence: 99%

“…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). ITC studies with model protein substrates, which were either partially folded or unfolded, suggested that between 7 and 29 residues are buried upon substrate binding to SecB (10).…”

mentioning

confidence: 99%

“…SecB has also been shown to bind partially folded states of ␣-LA (10). ITC studies with model protein substrates, which were either partially folded or unfolded, suggested that between 7 and 29 residues are buried upon substrate binding to SecB (10). The exact nature of the translocation competent state of SecB's natural substrates is unknown, although it is believed to be a flexible molten globule state (6,11).…”

mentioning

confidence: 99%

See 3 more Smart Citations

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

Panse¹,

Vogel²,

Trommer³

et al. 2000

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Molecular chaperones prevent protein aggregation in vivo and in vitro. In a few cases, multichaperone systems are capable of dissociating aggregated state(s) of substrate proteins, although little is known of the mechanism of the process. SecB is a cytosolic chaperone, which forms part of the precursor protein translocation machinery in Escherichia coli. We have investigated the interaction of the B-chain of insulin with chaperone SecB by light scattering, pyrene excimer fluorescence, and electron spin resonance spectroscopy. We show that SecB prevents aggregation of the B-chain of insulin. We show that SecB is capable of dissociating soluble B-chain aggregates as monitored by pyrene fluorescence spectroscopy. The kinetics of dissociation of the B-chain aggregate by SecB has been investigated to understand the mechanism of dissociation. The data suggests that SecB does not act as a catalyst in dissociation of the aggregate to individual B-chains, rather it binds the small population of free B-chains with high affinity, thereby shifting the equilibrium from the ensemble of the aggregate toward the individual B-chains. Thus SecB can rescue aggregated, partially folded/misfolded states of target proteins by a thermodynamic coupling mechanism when the free energy of binding to SecB is greater than the stability of the aggregate. Pyrene excimer fluorescence and ESR methods have been used to gain insights on the bound state conformation of the B-chain to chaperone SecB. The data suggests that the B-chain is bound to SecB in a flexible extended state in a hydrophobic cleft on SecB and that the binding site accommodates approximately 10 residues of substrate.

show abstract

Section: Thermodynamic Coupling Model Of B-chain Dissociation Bysupporting

confidence: 53%

Section: Thermodynamic Coupling Model Of B-chain Dissociation Bymentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

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

Panse¹,

Vogel²,

Trommer³

et al. 2000

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…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).…”

mentioning

confidence: 99%

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

Panse¹,

Krishnan²,

Philipp³

et al. 2001

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

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 Å) when no substrate protein is bound but move away to a distance of greater than 20 Å when SecB binds substrate. This is the first direct evidence of a conformational change in SecB upon binding of a substrate protein.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). The common property shared by most molecular 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). SecB is a tetrameric chaperone in Escherichia coli that is involved in the translocation of polypeptide chains into the periplasmic space of the cell (3). In vivo, SecB binds to a subset of precursor proteins and maintains them in an unfolded, translocation-competent state, whereas in vitro it interacts with a variety of proteins in the non-native state (4 -7). The nature of the translocation-competent state of substrate proteins is unknown, although it is believed to be a flexible molten globule state (8). 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 lactal...

show abstract

Chaperone Function in Vitro

Büchner

Walter

2008

Protein Science Encyclopedia

View full text Add to dashboard Cite

Originally published in: Protein Folding Handbook. Part II. Edited by Johannes Buchner and Thomas Kiefhaber. Copyright © 2005 Wiley‐VCH Verlag GmbH & Co. KGaA Weinheim. Print ISBN: 3‐527‐30784‐2 The sections in this article are Introduction Basic Functional Principles of Molecular Chaperones Recognition of Nonnative Proteins Induction of Conformational Changes in the Substrate Energy Consumption and Regulation of Chaperone Function Limits and Extensions of the Chaperone Concept Co‐chaperones Specific Chaperones Working with Molecular Chaperones Natural versus Artificial Substrate Proteins Stability of Chaperones Acknowledgments

show abstract

Thermodynamics of Substrate Binding to the Chaperone SecB

Cited by 20 publications

References 51 publications

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

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

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

Chaperone Function in Vitro

Contact Info

Product

Resources

About