Interaction of SecB with intermediates along the folding pathway of maltose‐binding protein

Diamond, Deborah L.; Strobel, Sharon; Chun, Sang-Yearn; Randall, Linda L.

doi:10.1002/pro.5560040610

Cited by 23 publications

(21 citation statements)

References 34 publications

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“…3A), and a portion of the sample was warmed to 25°C. The difference in temperature affects only the folding rate of maltosebinding protein and not the ability of the SecB to bind ligand as has been demonstrated previously by the formation of complexes between SecB and mutationally altered species of maltose-binding protein that fold slowly (9,19). As was the case for galactose-binding protein, when the folding rate of the maltosebinding protein ligand increased the complex was disrupted (Fig.…”

Section: Resultssupporting

confidence: 54%

Chaperone SecB from Escherichia coli Mediates Kinetic Partitioning via a Dynamic Equilibrium with Its Ligands

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Randall

1997

Journal of Biological Chemistry

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We have shown that the complexes between SecB, a chaperone from Escherichia coli, and two physiological ligands, galactose-binding protein and maltose-binding protein, are in rapid, dynamic equilibrium between the bound and free states. Binding to SecB is readily reversible, and each time the ligand is released it undergoes a kinetic partitioning between folding to its native state and re-binding to SecB. Binding requires that the polypeptide be devoid of tertiary structure; once the protein has folded, it is no longer a ligand. Conditions were established in which folding of the polypeptides was sufficiently slow so that at each cycle of dissociation rebinding was favored over folding and a kinetically stable complex between SecB and each polypeptide ligand was observed. Evidence that the ligand is continually released to the bulk solution and rebound was obtained by altering the conditions to increase the rate of folding of each ligand so that folding of the ligand was faster than reassociation with SecB thereby allowing the system to partition to free SecB and folded polypeptide ligand. We conclude that complexes between the chaperone SecB and ligands are in dynamic, rapid equilibrium with the free states. This mode of binding is simpler than that documented for chaperones that function to facilitate folding such as the Hsp70s and Hsp60s, where hydrolysis of ATP is coupled to the binding and release of ligands. This difference may reflect the fact that SecB does not mediate folding but is specialized to facilitate protein export. Without a requirement for exogenous energy it efficiently performs its sole duty: to keep proteins in a nonnative conformation and thus competent for export.SecB is a cytosolic chaperone from Escherichia coli that binds a subset of polypeptides destined to be exported to the periplasmic space or to the outer membrane. SecB keeps these proteins in a nonnative conformation, which is competent for export, while delivering them to SecA, the next component in the export pathway. There is no specific sequence of amino acids that distinguishes a polypeptide as a ligand for SecB. The only requirement for binding is that the ligand be devoid of tertiary structure. The binding frames for three physiological ligands, maltose-binding protein, galactose-binding protein, and the oligopeptide-binding protein, have been determined (1, 2).1 In all three cases it was shown that the sequence in contact with SecB is large, minimally comprising 150 amino acids in a continuous stretch. There is no similarity in sequence among the binding frames nor is it obvious that a structural element provides recognition. The large area of contact between SecB and its ligands allows for interaction at multiple subsites with each site having low affinity yet together giving tight binding; the dissociation constant for ligands of SecB is in the range of 10 Ϫ8 M (3-5). Even though the affinity is high there are several studies that indicate that the complex of SecB and its ligand is in rapid equilibrium with the unbound ...

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Section: Resultssupporting

confidence: 54%

Chaperone SecB from Escherichia coli Mediates Kinetic Partitioning via a Dynamic Equilibrium with Its Ligands

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Randall

1997

Journal of Biological Chemistry

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“…The spectra obtained under acidic conditions lead to a molecular weight measurement of 58,809.3 * 0.6 Da (95% confidence limits, n = 10 most abundant charge states) in excellent agreement with the predicted MW of 58,808 kDa. Previous studies of SecB in complex with maltose-binding protein (Diamond et al, 1995;Diamond & Randall, 1997). galactosebinding protein (Khisty et al, 1995;, and oligopeptide-binding protein (Smith et al, 1997).…”

Section: Resultsmentioning

confidence: 99%

The observation of chaperone‐ligand noncovalent complexes with electrospray ionization mass spectrometry

et al. 1998

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Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) was applied for the study of noncovalent chaperone SecB-ligand complexes produced in solution and examined in the gas phase with the aid of electrospray ionization (ESI). Since chaperone proteins are believed to recognize and bind only with ligands with nonnative tertiary structure, this work required careful unfolding of the ligand and subsequent reaction with the intact chaperone (the noncovalent tetrameric protein, SecB). A high denaturant concentration was employed to produce nonnative structures of the OppA, and microdialysis of the resulting solutions containing the chaperone-ligand complexes was camed out to rapidly remove the denaturant prior to analysis. Multistage mass spectrometry was essential to the successful study of these complexes since the initial mass spectra indicated extensive adduction that precluded mass measurements, even after microdialysis. However, low energy collisional activation of the ions in the FTICR trap proved useful for adduct removal, and careful control of excitation level preserved the intact complexes of interest, revealing a 1 : 1 SecB:OppA stoichiometry. To our knowledge, these results present the first direct observation of chaperone-ligand noncovalent complexes and the highest molecular weight heterogeneous noncovalent complex observed to date by mass spectrometry. Furthermore, these results highlight the capabilities of FTICR for the study of such complex systems, and the development of a greater understanding of chaperone interactions in protein export.Keywords: dissociation; Fourier transform ion cyclotron resonance; MS/MS; OppA; protein export; SecB; stoichiometry Mass spectrometry has evolved to contribute to biological research on many frontiers (Ganem et al

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“…Protein-The details of this assay have been reported previously (12,15,19,21). Fluorescence measurements were made using a Shimadzu RF-540 fluorescence spectrophotometer.…”

Section: Fluorescence Assay Of Interaction Between Secb and Maltose-bmentioning

confidence: 99%

“…For example, at 25°C rapid folding of wild-type mature maltosebinding protein precludes formation of a detectable complex with SecB; however, if the folding reaction is slowed by decreasing the temperature from 25 to 5°C, SecB binds the mature protein (12,19). Furthermore, three species of maltose-binding protein have been shown to contain single aminoacyl substitutions in the mature portion of the protein (MalE Y283D, MalE A276G, and MalE W10A) that slow folding sufficiently so that SecB can bind even when the temperature of the folding reaction is 20 -25°C (12,19,21). It is clear from these examples that kinetic partitioning can be poised to favor association with SecB by changing the rate constant for folding of the polypeptide (k f ) (refer to Fig.…”

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confidence: 99%

Kinetic Partitioning

Diamond

Randall

1997

Journal of Biological Chemistry

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Chaperones are a class of proteins that possess the remarkable ability to selectively bind polypeptides that are in a nonnative state. The selectivity of SecB, a molecular chaperone in Escherichia coli, for its ligands can be explained in part by a kinetic partitioning between folding of the polypeptide and association with SecB. It has clearly been established that kinetic partitioning can be poised to favor association with SecB by changing the rate constant for folding of the ligand. We now demonstrate that binding to SecB can be given a kinetic advantage over the pathway for folding by modulating the properties of the chaperone. By poising SecB to expose a hydrophobic patch, we were able to detect a complex between SecB and maltose-binding protein under conditions in which rapid folding of the polypeptide otherwise precludes formation of a kinetically stable complex. The data presented here are interpreted within the framework of a kinetic partitioning between binding to SecB and folding of the polypeptide. We propose that exposure of a hydrophobic patch on SecB increases the surface area for binding and thereby increases the rate constant for association. In this way association of SecB with the polypeptide ligand has a kinetic advantage over the pathway for folding.

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Interaction of SecB with intermediates along the folding pathway of maltose‐binding protein

Cited by 23 publications

References 34 publications

Chaperone SecB from Escherichia coli Mediates Kinetic Partitioning via a Dynamic Equilibrium with Its Ligands

Chaperone SecB from Escherichia coli Mediates Kinetic Partitioning via a Dynamic Equilibrium with Its Ligands

The observation of chaperone‐ligand noncovalent complexes with electrospray ionization mass spectrometry

Kinetic Partitioning

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