Skip to main content
SpringerLink
Log in

Alterations in chemical shifts and exchange broadening upon peptide boronic acid inhibitor binding to α-lytic protease

  • Published:
Journal of Biomolecular NMR Aims and scope Submit manuscript

    We’re sorry, something doesn't seem to be working properly.

    Please try refreshing the page. If that doesn't work, please contact support so we can address the problem.

Abstract

α-Lytic protease, a bacterial serine protease of 198 aminoacids (19800 Da), has been used as a model system for studies of catalyticmechanism, structure–function relationships, and more recently forstudies of pro region-assisted protein folding. We have assigned thebackbones of the enzyme alone, and of its complex with the tetrahedraltransition state mimic N-tert-butyloxycarbonyl-Ala-Pro-boroVal, usingdouble- and triple-resonance 3D NMR spectroscopy on uniformly15N- and 13C/15N-labeled protein.Changes in backbone chemical shifts between the uncomplexed and inhibitedform of the protein are correlated with distance from the inhibitor, thedisplacement of backbone nitrogens, and change in hydrogen bond strengthupon inhibitor binding (derived from previously solved crystal structures).A comparison of the solution secondary structure of the uninhibited enzymewith that of the X-ray structure reveals no significant differences.Significant line broadening, indicating intermediate chemical exchange, wasobserved in many of the active site amides (including three broadened toinvisibility), and in a majority of cases the broadening was reversed uponaddition of the inhibitor. Implications and possible mechanisms of this linebroadening are discussed.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Baker, D., Sohl, J.L. and Agard, D.A. (1992) Nature, 356, 263–265.

    Article  ADS  Google Scholar 

  • Bax, A. and Ikura, M. (1991) J. Biomol. NMR, 1, 99–104.

    Article  Google Scholar 

  • Bone, R., Shenvi, A.B., Kettner, C.A. and Agard, D.A. (1987) Biochemistry, 26, 7609–7614.

    Article  Google Scholar 

  • Bone, R., Frank, D., Kettner, C.A. and Agard, D.A. (1989a) Biochemistry, 28, 7600–7609.

    Article  Google Scholar 

  • Bone, R., Silen, J.L. and Agard, D.A. (1989b) Nature, 339, 191–195.

    Article  ADS  Google Scholar 

  • Bone, R., Fujishige, A., Kettner, C.A. and Agard, D.A. (1991a) Biochemistry, 30, 10388–10398.

    Article  Google Scholar 

  • Bone, R., Sampson, N.S., Bartlett, P.A. and Agard, D.A. (1991b) Biochemistry, 30, 2263–2272.

    Article  Google Scholar 

  • Boucher, W., Laue, E.D., Campbell-Burk, S. and Domaille, P.J. (1992a) J. Am. Chem. Soc., 114, 2262–2264.

    Article  Google Scholar 

  • Boucher, W., Laue, E.D., Campbell-Burk, S. and Domaille, P.J. (1992b) J. Biomol. NMR, 2, 631–637.

    Article  Google Scholar 

  • Brayer, G.D., Delbaere, L.T.J. and James, M.N.G. (1979) J. Mol. Biol., 131, 743–775.

    Article  Google Scholar 

  • Clubb, R.T., Thanabal, V. and Wagner, G. (1992) J. Biomol. NMR, 2, 203–210.

    Article  Google Scholar 

  • Davis, J.H. (1995) J. Biomol. NMR, 5, 433–437.

    Article  Google Scholar 

  • Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J. and Bax, A. (1995) J. Biomol. NMR, 6, 277–293.

    Article  Google Scholar 

  • Dolgikh, D.A., Gilmanshin, R.I., Brazhnikov, E.V., Bychkova, V.E., Semisotnov, G.V., Venyaminov, S.Yu. and Ptitsyn, O.B. (1981) FEBS Lett., 136, 311–315.

    Article  Google Scholar 

  • Fujinaga, M., Delbaere, L.T.J., Brayer, G.D. and James, M.N.G. (1985) J. Mol. Biol., 184, 479–502.

    Article  Google Scholar 

  • Grzesiek, S. and Bax, A. (1992) J. Am. Chem. Soc., 114, 6291–6293.

    Article  Google Scholar 

  • Haggett, K.D., Graham, L.D. and Whitaker, R.G. (1994) Biotechnol. Techniques, 8, 203–208.

    Article  Google Scholar 

  • Hunkapiller, M.W., Smallcombe, S.H., Whitaker, D.R. and Richards, J.H. (1973) Biochemistry, 12, 4732–4743.

    Article  Google Scholar 

  • Ikura, M., Kay, L.E. and Bax, A. (1990) Biochemistry, 29, 4659–4667.

    Article  Google Scholar 

  • Kabsch, W. and Sander, C. (1983) Biopolymers, 12, 2577–2637.

    Article  Google Scholar 

  • Kay, L.E., Ikura, M., Tschudin, R. and Bax, A. (1990) J. Magn. Reson., 89, 496–514.

    Google Scholar 

  • Kettner, C.A. and Shenvi, A.B. (1984) J. Biol. Chem., 259, 15106–15114.

    Google Scholar 

  • Kettner, C.A., Bone, R., Agard, D.A. and Bachovchin, W.W. (1988) Biochemistry, 27, 7682–7688.

    Article  Google Scholar 

  • Kraulis, P.J. (1989) J. Magn. Reson., 84, 627–633.

    Google Scholar 

  • Kraulis, P.J., Domaille, P.J., Campbell-Burk, S.L., Vanaken, T. and Laue, E.D. (1994) Biochemistry, 33, 3515–3531.

    Article  Google Scholar 

  • Levy, G.C. and Lichter, R.L. (1979) Nitrogen-15 Nuclear Magnetic Resonance Spectroscopy, Wiley, New York, NY, U.S.A.

    Google Scholar 

  • Mace, J.E. and Agard, D.A. (1995) J. Mol. Biol., 254, 720–736.

    Article  Google Scholar 

  • Mace, J.E., Wilk, B.J. and Agard, D.A. (1995) J. Mol. Biol., 251, 116–134.

    Article  Google Scholar 

  • Marion, D., Driscoll, P.D., Kay, L.E., Wingfield, P.T., Bax, A., Gronenborn, A.M. and Clore, G.M. (1989a) Biochemistry, 28, 6150–6156.

    Article  Google Scholar 

  • Marion, D., Kay, L.E., Sparks, S.W., Torchia, D.A. and Bax, A. (1989b) J. Am. Chem. Soc., 111, 1515–1517.

    Article  Google Scholar 

  • Muhandiram, D.R. and Kay, L.E. (1994) J. Magn. Reson., B103, 203–216.

    Google Scholar 

  • Silen, J.L., McGrath, C.N., Smith, K.R. and Agard, D.A. (1988) Gene, 69, 237–244.

    Article  Google Scholar 

  • Silen, J.L., Frank, D., Fujishige, A., Bone, R. and Agard, D.A. (1989) J. Bacteriol., 171, 1320–1325.

    Google Scholar 

  • Wishart, D.S., Sykes, B.D. and Richards, F.M. (1991) J. Mol. Biol., 222, 311–333.

    Article  Google Scholar 

  • Wittekind, M., Metzler, W.J. and Müller, L. (1993) J. Magn. Reson., B101, 214–217.

    Google Scholar 

  • Wittekind, M. and Müller, L. (1993) J. Magn. Reson., B101, 201–205.

    Google Scholar 

  • Zuiderweg, E.R.P. and Fesik, S.W. (1989) Biochemistry, 28, 2387–2391.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Graduate Group in Biophysics, University of California, San Francisco, CA, 94143-0448, U.S.A

    Jonathan H. Davis & David A. Agard

  2. Department of Biochemistry and Biophysics, University of California, San Francisco, CA, 94143-0448, U.S.A

    David A. Agard

  3. Howard Hughes Medical Institute, University of California, San Francisco, CA, 94143-0724, U.S.A

    David A. Agard

  4. Department of Molecular and Cellular Biology, University of California, Berkeley, CA, 94720, U.S.A

    Tracy M. Handel

  5. Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, 94143-0446, U.S.A

    Vladimir J. Basus

Authors
  1. Jonathan H. Davis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David A. Agard
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tracy M. Handel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Vladimir J. Basus
    View author publications

    You can also search for this author in PubMed Google Scholar

Electronic Supplementary Material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Davis, J.H., Agard, D.A., Handel, T.M. et al. Alterations in chemical shifts and exchange broadening upon peptide boronic acid inhibitor binding to α-lytic protease. J Biomol NMR 10, 21–27 (1997). https://doi.org/10.1023/A:1018314808361

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1018314808361

Search

Navigation