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

Bruce, James E.; Smith, Virginia F.; Liu, Chuanliang; Randall, Linda L.; Smith, Richard

doi:10.1002/pro.5560070512

Cited by 36 publications

(34 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Unfolded polypeptides bind to SecB with a stoichiometry of one polypeptide per tetramer. 18 The portion of the ligand in contact with SecB has been estimated to be approximately 150 aminoacyl residues. [5][6][7] It seems unlikely that this stretch of extended polypeptide could simultaneously make contact with all four symmetrically related residues.…”

Section: Resultsmentioning

confidence: 99%

Sites of Interaction of a Precursor Polypeptide on the Export Chaperone SecB Mapped by Site-directed Spin Labeling

Crane

Suo

Lilly

et al. 2006

Journal of Molecular Biology

Self Cite

View full text Add to dashboard Cite

SummaryExport of protein into the periplasm of Escherichia coli via the general secretory system requires that the transported polypeptides be devoid of stably folded tertiary structure. Capture of the precursor polypeptides before they fold is achieved by the promiscuous binding to the chaperone SecB. SecB delivers its ligand to export sites through its specific binding to SecA, a peripheral component of the membrane translocon. At the translocon the ligand is passed from SecB to SecA and subsequently through the SecYEG channel. We have previously used site-directed spin labeling and electron paramagnetic resonance spectroscopy to establish a docking model between SecB and SecA. Here we report use of the same strategy to map the pathway of a physiologic ligand, the unfolded form of precursor galactose-binding protein, on SecB. Our set of SecB variants each containing a single cysteine, which was used in the previous study, has been expanded to forty-eight residues which cover 49% of the surface of SecB. The residues on SecB involved in contacts were identified as those that, upon addition of the unfolded polypeptide ligand, showed changes in spectral line shape consistent with restricted motion of the nitroxide. We conclude that the bound precursor makes contact with a large portion of the surface of the small chaperone. The sites on SecB that interact with the ligand are compared with the previously identified sites that interact with SecA and a model for transfer of the ligand is discussed.

show abstract

Section: Resultsmentioning

confidence: 99%

Sites of Interaction of a Precursor Polypeptide on the Export Chaperone SecB Mapped by Site-directed Spin Labeling

Crane

Suo

Lilly

et al. 2006

Journal of Molecular Biology

Self Cite

View full text Add to dashboard Cite

show abstract

“…Electrospray ionization coupled to time-of-flight mass spectrometry (MS) provides a unique insight into defining the subunit composition of noncovalent complexes (14-16) including sHSPs (17,18) and other chaperones (19)(20)(21)). It has not been possible, however, to interpret data from polydisperse proteins such as ␣B-crystallin.…”

mentioning

confidence: 99%

Polydispersity of a mammalian chaperone: Mass spectrometry reveals the population of oligomers in αB-crystallin

Aquilina

Benesch

Bateman

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

238

284

View full text Add to dashboard Cite

The quaternary structure of the polydisperse mammalian chaperone ␣B-crystallin, a member of the small heat-shock protein family, has been investigated by using electrospray mass spectrometry. The intact assemblies give rise to mass spectra that are complicated by the overlapping of charge states from the different constituent oligomers. Therefore, to determine which oligomers are formed by this protein, tandem mass spectrometry experiments were performed. The spectra reveal a distribution, primarily of oligomers containing 24 -33 subunits, the relative populations of which were quantified, to reveal a dominant species being composed of 28 subunits. Additionally, low levels of oligomers as small as 10-mers and as large as 40-mers were observed. Interpretation of the tandem mass spectral data was confirmed by simulating and summing spectra arising from the major individual oligomers. The ability of mass spectrometry to quantify the relative populations of particular oligomeric states also revealed that, contrary to the dimeric associations observed in other small heat-shock proteins, there is no evidence for any stable substructures of bovine ␣〉-crystallin isolated from the lens. T he small heat-shock proteins (sHSPs) constitute a family of molecular chaperones involved in protein stabilization under conditions of stress (1). The mammalian sHSP ␣〉-crystallin is systemically expressed (2) and, along with ␣A-crystallin, is also a major structural protein of the eye lens. ␣〉-crystallin has been demonstrated to display molecular chaperone activity by suppressing the aggregation of unfolded protein (3). Overexpression of ␣B-crystallin outside the lens is associated with a number of aberrant protein disease states including Alzheimer's, Alexander's, Parkinson's, and Creutzfeldt-Jakob diseases (4-7). Despite increasing amounts of structural information for prokaryotic (8) and plant sHSPs (9), the structures of mammalian members of this family, including the ␣-crystallins, HSP27 and HSP20, have yet to be elucidated. These sHSPs are isolated from tissue as polydisperse multimers, and it is this heterogeneity that has confounded the efforts of structural biologists (10, 11).␣-Crystallin, in particular, has long been the subject of conflicting results regarding its molecular mass distribution (12); however, it is accepted that from the lens, ␣-crystallin is isolated as a multimeric assembly ranging in molecular mass from 300 kDa to Ͼ1 MDa (10). Recently, a combination of size-exclusion chromatography and multiangle laser light scattering was used to determine the mass range of human recombinant ␣B-crystallin as 530-684 kDa, with a population of 585 kDa at the peak of the elution profile (13). This represents the most accurate assessment of the polydispersity inherent in the ␣-crystallins to date; however, it is confined to the major species in the elution profile and provides little insight into the relative populations of oligomers within the distribution.Electrospray ionization coupled to time-of-flight mass spectrometry (...

show abstract

“…Molecules with masses of over 2 MDa have been mass-measured in this way (32). We and others (33)(34)(35)(36)(37) have previously used mass spectrometry to characterize and investigate the properties of different molecular chaperones including GroEL (33), HSP90 (34), SecB (35,36), and MtGimC/prefoldin (37). In the case of MtGimC, the subunit stoichiometry and interactions were elucidated by MS by following the assembly of the complex in real-time.…”

mentioning

confidence: 99%

Subunit Exchange of Multimeric Protein Complexes

Sobott¹,

Benesch²,

Vierling³

et al. 2002

Journal of Biological Chemistry

182

View full text Add to dashboard Cite

The subunit exchange of the small heat shock proteins (sHSPs) PsHSP18.1 from pea and TaHSP16.9 from wheat has been monitored in real-time using nanoelectrospray mass spectrometry. By preserving the noncovalent interactions between subunits in the mass spectrometer, we show that these proteins are dodecameric. After mixing PsHSP18.1 and TaHSP16.9, a distribution of heterododecamers is formed. A comparison with spectra obtained from statistical modeling demonstrates that after equilibration the distribution of these heterocomplexes is governed by the starting ratio of the two components rather than an inherent preference for certain stoichiometries. This finding suggests that the two different sHSP subunits interact in a very similar manner. Following the kinetics of this reaction by mass spectrometry reveals that exchange proceeds via sequential incorporation of subunits with dimeric species being the principal units of exchange. Therefore, we conclude that sHSP complexes are in rapid dissociation/reassociation equilibria with suboligomeric forms. More generally, these experiments illustrate a powerful approach for the real-time analysis of the evolution of transient species and their relative populations during the subunit exchange of multimeric protein complexes.The small heat shock protein (sHSP) 1 family is both large and diverse with members found in virtually all organisms. The sHSPs are induced under a variety of stresses and act to increase cellular tolerance to these conditions. Additionally, they have been implicated in cancer, cataract, neurodegenerative diseases, and myopathy (1-4). It is not clearly understood how the sHSPs become involved in such diseases, but current models propose that they act primarily as molecular chaperones, preventing improper protein associations (5). This is evidenced by their ability to bind nonnative proteins under different stress conditions, thereby preventing their irreversible aggregation and consequent damage to the cell (6 -11). These bound substrates can subsequently be refolded in an ATPdependent manner upon interaction with the HSP70 system or DnaK (in eukaryotic and prokaryotic cells, respectively) and co-chaperones (8 -10, 12, 13).The sHSPs are characterized by a conserved C-terminal "␣-crystallin" domain (14, 15) and typically have subunit masses between 15 and 40 kDa. The proteins assemble into large oligomers containing 9 to Ͼ24 monomers of varying heterogeneity. Some sHSPs such as those from plants form assemblies with well defined stoichiometries, whereas other sHSPs including the mammalian proteins form a range of oligomeric sizes (16). This polydispersity has limited the amount of structural information available, and consequently, only two x-ray structures have been reported thus far for intact sHSPs, those of Methanococcus jannaschii HSP16.5 (17) and Triticum aestivum HSP16.9 (18). Compared with the wealth of such information available for other molecular chaperones, the sHSP family remains relatively poorly understood. Fig. 1 shows the structure of Ta...

show abstract

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

Cited by 36 publications

References 39 publications

Sites of Interaction of a Precursor Polypeptide on the Export Chaperone SecB Mapped by Site-directed Spin Labeling

Sites of Interaction of a Precursor Polypeptide on the Export Chaperone SecB Mapped by Site-directed Spin Labeling

Polydispersity of a mammalian chaperone: Mass spectrometry reveals the population of oligomers in αB-crystallin

Subunit Exchange of Multimeric Protein Complexes

Contact Info

Product

Resources

About