Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1998 May;7(5):1180–1185. doi: 10.1002/pro.5560070512

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

J E Bruce 1, V F Smith 1, C Liu 1, L L Randall 1, R D Smith 1
PMCID: PMC2144017  PMID: 9605322

Abstract

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 carried 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.

Full Text

The Full Text of this article is available as a PDF (1.5 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Diamond D. L., Randall L. L. Kinetic partitioning. Poising SecB to favor association with a rapidly folding ligand. J Biol Chem. 1997 Nov 14;272(46):28994–28998. doi: 10.1074/jbc.272.46.28994. [DOI] [PubMed] [Google Scholar]
  2. Goodlett D. R., Camp D. G., 2nd, Hardin C. C., Corregan M., Smith R. D. Direct observation of a DNA quadruplex by electrospray ionization mass spectrometry. Biol Mass Spectrom. 1993 Mar;22(3):181–183. doi: 10.1002/bms.1200220307. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Khisty V. J., Munske G. R., Randall L. L. Mapping of the binding frame for the chaperone SecB within a natural ligand, galactose-binding protein. J Biol Chem. 1995 Oct 27;270(43):25920–25927. doi: 10.1074/jbc.270.43.25920. [DOI] [PubMed] [Google Scholar]
  5. Konermann L., Collings B. A., Douglas D. J. Cytochrome c folding kinetics studied by time-resolved electrospray ionization mass spectrometry. Biochemistry. 1997 May 6;36(18):5554–5559. doi: 10.1021/bi970046d. [DOI] [PubMed] [Google Scholar]
  6. Light-Wahl K. J., Loo J. A., Edmonds C. G., Smith R. D., Witkowska H. E., Shackleton C. H., Wu C. S. Collisionally activated dissociation and tandem mass spectrometry of intact hemoglobin beta-chain variant proteins with electrospray ionization. Biol Mass Spectrom. 1993 Feb;22(2):112–120. doi: 10.1002/bms.1200220203. [DOI] [PubMed] [Google Scholar]
  7. Little D. P., Speir J. P., Senko M. W., O'Connor P. B., McLafferty F. W. Infrared multiphoton dissociation of large multiply charged ions for biomolecule sequencing. Anal Chem. 1994 Sep 15;66(18):2809–2815. doi: 10.1021/ac00090a004. [DOI] [PubMed] [Google Scholar]
  8. Little D. P., Thannhauser T. W., McLafferty F. W. Verification of 50- to 100-mer DNA and RNA sequences with high-resolution mass spectrometry. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2318–2322. doi: 10.1073/pnas.92.6.2318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Liu C., Wu Q., Harms A. C., Smith R. D. On-line microdialysis sample cleanup for electrospray ionization mass spectrometry of nucleic acid samples. Anal Chem. 1996 Sep 15;68(18):3295–3299. doi: 10.1021/ac960286j. [DOI] [PubMed] [Google Scholar]
  10. Liu Y. H., Bai J., Liang X., Lubman D. M., Venta P. J. Use of a nitrocellulose film substrate in matrix-assisted laser desorption/ionization mass spectrometry for DNA mapping and screening. Anal Chem. 1995 Oct 1;67(19):3482–3490. doi: 10.1021/ac00115a017. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Randall L. L., Topping T. B., Hardy S. J. No specific recognition of leader peptide by SecB, a chaperone involved in protein export. Science. 1990 May 18;248(4957):860–863. doi: 10.1126/science.2188362. [DOI] [PubMed] [Google Scholar]
  13. Randall L. L., Topping T. B., Hardy S. J., Pavlov M. Y., Freistroffer D. V., Ehrenberg M. Binding of SecB to ribosome-bound polypeptides has the same characteristics as binding to full-length, denatured proteins. Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):802–807. doi: 10.1073/pnas.94.3.802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Smith V. F., Hardy S. J., Randall L. L. Determination of the binding frame of the chaperone SecB within the physiological ligand oligopeptide-binding protein. Protein Sci. 1997 Aug;6(8):1746–1755. doi: 10.1002/pro.5560060815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Tame J. R., Murshudov G. N., Dodson E. J., Neil T. K., Dodson G. G., Higgins C. F., Wilkinson A. J. The structural basis of sequence-independent peptide binding by OppA protein. Science. 1994 Jun 10;264(5165):1578–1581. doi: 10.1126/science.8202710. [DOI] [PubMed] [Google Scholar]
  16. Tolić L. P., Bruce J. E., Lei Q. P., Anderson G. A., Smith R. D. In-trap cleanup of proteins from electrospray ionization using soft sustained off-resonance irradiation with fourier transform ion cyclotron resonance mass spectrometry. Anal Chem. 1998 Jan 15;70(2):405–408. doi: 10.1021/ac970828c. [DOI] [PubMed] [Google Scholar]
  17. Topping T. B., Randall L. L. Chaperone SecB from Escherichia coli mediates kinetic partitioning via a dynamic equilibrium with its ligands. J Biol Chem. 1997 Aug 1;272(31):19314–19318. doi: 10.1074/jbc.272.31.19314. [DOI] [PubMed] [Google Scholar]
  18. Verentchikov A. N., Ens W., Standing K. G. Reflecting time-of-flight mass spectrometer with an electrospray ion source and orthogonal extraction. Anal Chem. 1994 Jan 1;66(1):126–133. doi: 10.1021/ac00073a022. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES