Abstract
Electrospray ionization mass spectrometry was used to investigate the structure of the Escherichia coli chaperone protein SecB. It was determined that the N-terminal methionine of SecB has been removed and that more than half of all SecB monomers are additionally modified, most likely by acetylation of the N-terminus or a lysine. The use of gentle mass spectrometer interface conditions showed that the predominant, oligomeric form of SecB is a tetramer that is stable over a range of solution pH conditions and mass spectrometer interface heating (i.e., inlet capillary temperatures). At very high pH, SecB dimers are observed. SecB contains a region that is hypersensitive to cleavage by proteinase K and is thought to be involved in conformational changes that are crucial to the function of SecB. We identified the primary site of cleavage to be between Leu 141 and Gln 142. Fourteen amino acids are removed, but the truncated form remains a tetramer with stability similar to that of the intact form.
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- Braig K., Otwinowski Z., Hegde R., Boisvert D. C., Joachimiak A., Horwich A. L., Sigler P. B. The crystal structure of the bacterial chaperonin GroEL at 2.8 A. Nature. 1994 Oct 13;371(6498):578–586. doi: 10.1038/371578a0. [DOI] [PubMed] [Google Scholar]
- Breukink E., Kusters R., De Kruijff B. In-vitro studies on the folding characteristics of the Escherichia coli precursor protein prePhoE. Evidence that SecB prevents the precursor from aggregating by forming a functional complex. Eur J Biochem. 1992 Sep 1;208(2):419–425. doi: 10.1111/j.1432-1033.1992.tb17203.x. [DOI] [PubMed] [Google Scholar]
- Fasman G. D., Park K., Randall L. L. Chaperone SecB: conformational changes demonstrated by circular dichroism. J Protein Chem. 1995 Oct;14(7):595–600. doi: 10.1007/BF01886885. [DOI] [PubMed] [Google Scholar]
- Hardy S. J., Randall L. L. A kinetic partitioning model of selective binding of nonnative proteins by the bacterial chaperone SecB. Science. 1991 Jan 25;251(4992):439–443. doi: 10.1126/science.1989077. [DOI] [PubMed] [Google Scholar]
- Holmgren A., Bränden C. I. Crystal structure of chaperone protein PapD reveals an immunoglobulin fold. Nature. 1989 Nov 16;342(6247):248–251. doi: 10.1038/342248a0. [DOI] [PubMed] [Google Scholar]
- Kraus E., Kiltz H. H., Femfert U. F. The specificity of proteinase K against oxidized insulin B chain. Hoppe Seylers Z Physiol Chem. 1976 Feb;357(2):233–237. [PubMed] [Google Scholar]
- Kumamoto C. A., Chen L., Fandl J., Tai P. C. Purification of the Escherichia coli secB gene product and demonstration of its activity in an in vitro protein translocation system. J Biol Chem. 1989 Feb 5;264(4):2242–2249. [PubMed] [Google Scholar]
- Kumamoto C. A., Nault A. K. Characterization of the Escherichia coli protein-export gene secB. Gene. 1989 Jan 30;75(1):167–175. doi: 10.1016/0378-1119(89)90393-4. [DOI] [PubMed] [Google Scholar]
- Morshauser R. C., Wang H., Flynn G. C., Zuiderweg E. R. The peptide-binding domain of the chaperone protein Hsc70 has an unusual secondary structure topology. Biochemistry. 1995 May 16;34(19):6261–6266. doi: 10.1021/bi00019a001. [DOI] [PubMed] [Google Scholar]
- Randall L. L. Peptide binding by chaperone SecB: implications for recognition of nonnative structure. Science. 1992 Jul 10;257(5067):241–245. doi: 10.1126/science.1631545. [DOI] [PubMed] [Google Scholar]
- Smith R. D., Loo J. A., Edmonds C. G., Barinaga C. J., Udseth H. R. New developments in biochemical mass spectrometry: electrospray ionization. Anal Chem. 1990 May 1;62(9):882–899. doi: 10.1021/ac00208a002. [DOI] [PubMed] [Google Scholar]
- Szyperski T., Pellecchia M., Wall D., Georgopoulos C., Wüthrich K. NMR structure determination of the Escherichia coli DnaJ molecular chaperone: secondary structure and backbone fold of the N-terminal region (residues 2-108) containing the highly conserved J domain. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11343–11347. doi: 10.1073/pnas.91.24.11343. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Topping T. B., Randall L. L. Determination of the binding frame within a physiological ligand for the chaperone SecB. Protein Sci. 1994 May;3(5):730–736. doi: 10.1002/pro.5560030502. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vrielink A., Beamer L., Le T., Eisenberg D. Crystallization of the chaperone protein SecB. Protein Sci. 1995 Aug;4(8):1651–1653. doi: 10.1002/pro.5560040824. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Watanabe M., Blobel G. Cytosolic factor purified from Escherichia coli is necessary and sufficient for the export of a preprotein and is a homotetramer of SecB. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2728–2732. doi: 10.1073/pnas.86.8.2728. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weiss J. B., Ray P. H., Bassford P. J., Jr Purified secB protein of Escherichia coli retards folding and promotes membrane translocation of the maltose-binding protein in vitro. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8978–8982. doi: 10.1073/pnas.85.23.8978. [DOI] [PMC free article] [PubMed] [Google Scholar]