Abstract
Methods are presented for monitoring solvent accessibility of protein-ligand and protein-protein interfaces. The kinetics of solvent accessibility at the protein-protein interface is monitored by amide hydrogen/deuterium (H/2H) exchange detected by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). A straightforward theoretical analysis is presented for determining the concentration of a weakly binding ligand that is required for achieving a situation in which the receptor is essentially 100% bound, and this is verified by control experiments. We show that when the receptor is essentially 100% bound it is possible to distinguish amide exchange as a result of solvent accessibility at the interface from amide exchange caused by complex dissociation. Methods are also presented for the measurement of tightly bound complexes of large interactions such as antibody-antigen complexes. Quantitation of the number of amides sequestered at the interface can be related to the number of H2O molecules excluded from the interface.
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References
Mandell J. G., Falick A. M., and Komives E. A. (1998) Identification of protein-protein interfaces by decreased amide proton solvent accessibility. Proc. Nat. Acad. Sci. USA 95, 14,705–14,710
Mandell J. G., Baerga-Ortiz A., Akashi S., Takio K., and Komives E. A. (2001) Solvent accessibility of the thrombin-thrombomodulin interface. J. Mol. Biol. 306, 575–589.
Baerga-Ortiz A., Hughes C. A., Mandell J. G., and Komives E. A. (2002) Epitope mapping of a monoclonal antibody against human thrombin by H/D-exchange mass spectrometry reveals selection of a diverse sequence in a highly conserved protein. Protein Sci. 11, 1300–1308.
Anand G. S., Law D., Mandell J. G., Snead A. N., Tsigelny I., Taylor S. S., Ten Eyck L., and Komives E. A. (2003) Identification of the Protein Kinase A Regulatory RI-Catalytic Subunit Interface by Amide H/2H Exchange and Protein Docking. Proc. Nat. Acad. Sci. USA 100, 13,264–13,269.
Hughes C. A., Mandell J. G., Anand G. S., Stock A. M., and Komives E. A. (2001) Phosphorylation causes subtle changes in solvent accessibility at the interdomain interface of methylesterase CheB. J. Mol. Biol. 307, 967–976.
Englander S., Mayne L., Bai Y., and Sosnick T. (1997) Hydrogen exchange: the modern legacy of Linderstrøm-Lang. Protein Science 6, 1101–1109.
Bai Y., Milne J. S., Mayne L., and Englander S. W. (1993) Primary structure effects on peptide group hydrogen exchange. Proteins 17, 75–86.
Dharmasiri K. and Smith D. L. (1996) Mass spectrometric determination of isotopic exchange rates of amide hydrogens located on the surfaces of proteins. Analytical Chemistry 68, 2340–2344.
Baerga-Ortiz A., Bergqvist S. P., Mandell J. G., and Komives E. A. (2004) Two different proteins that compete for binding to thrombin have opposite kinetic and thermodynamic profiles. Protein Sci. 13, 166–176.
Zhang Z. and Smith D. L. (1993) Determination of amide hydrogen exchange by mass spectrometry∶ a new tool for protein structure elucidation. Protein Sci. 2, 522–531.
Mandell J. G., Falick A. M., and Komives E. A. (1998) Measurement of amide hydrogen exchange by MALDI-TOF mass spectrometry. Analytical Chemistry 70, 3987–3995.
Gemmecker G., Jahnke W., and H. K. (1993) Measurement of fast proton exchange rates in isotopically labeled compounds. J. Am. Chem. Soc. 115, 11,620–11,621.
Petrey D. and Honig B. (2003) GRAPZ∶ visualization, surface properties, and electrostatics of macromolecular structures and sequences. Methods Enzymol. 374, 492–509.
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© 2005 Humana Press Inc.Totowa, NJ
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Mandell, J.G., Baerga-Ortiz, A., Falick, A.M., Komives, E.A. (2005). Measurement of Solvent Accessibility at Protein-Protein Interfaces. In: Ulrich Nienhaus, G. (eds) Protein-Ligand Interactions. Methods in Molecular Biology, vol 305. Humana, Totowa, NJ. https://doi.org/10.1385/1-59259-912-5:065
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DOI: https://doi.org/10.1385/1-59259-912-5:065
Publisher Name: Humana, Totowa, NJ
Print ISBN: 978-1-58829-372-5
Online ISBN: 978-1-59259-912-7
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