Summary
Nuclear magnetic resonance (NMR) is a very powerful tool for determining the boundaries of peptide epitopes recognized by antibodies. NMR can be used to study antibodies in complexes that exhibit a wide range of binding affinities from very weak and transient to very tight. Choice of the specific method depends upon the dissociation constant, especially the ligand off-rate.
Epitope mapping by NMR is based on the difference in mobility between the amino acid residues of a peptide antigen that interact tightly with the antibody and residues outside the epitope that do not interact with the antibody. The interacting peptide residues become considerably immobilized upon binding. Their mobility will resemble that of the antibody’s residues. Several NMR methods were developed based on these characteristics. In this chapter we discuss some of these methods, including dynamic filtering, comparison of 1H-15N HSQC peaks’ intensities, transverse relaxation time, measurements of 1H-15N nuclear Overhauser effect (NOE) values, and measurements of T 1ρ relaxation time.
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References
Williams, D. C., Jr., Rule, G. S., Poljak, R. J., and Benjamin, D. C. (1997) Reduction in the amide hydrogen exchange rates of an antilysozyme Fv fragment due to formation of the Fv–lysozyme complex. J. Mol. Biol. 270, 751–762.
Tugarinov, V., Zvi, A., Levy, A., Hayek, Y., Matsushita, S., and Anglister, J. (2000) NMR structure of an anti-gp120 antibody complex with a V3 peptide reveals a surface important for co-receptor binding. Struct. Fold. Des. 8, 385–395.
Rosen, O., Chill, J., Sharon, M., Kessler, N., Mester, B., Zolla-Pazner, S., and Anglister, J. (2005) Induced fit in HIV-neutralizing antibody complexes: evidence for alternative conformations of the gp120 V3 loop and the molecular basis for broad neutralization. Biochemistry 44, 7250–7258.
Rosen, O., Sharon, M., Quadt-Akabayov, S. R., and Anglister, J. (2006) Molecular switch for alternative conformations of the HIV-1 V3 region: implications for phenotype conversion. Proc. Natl. Acad. Sci. USA 103, 13950–13955.
Sharon, M., Kessler, N., Levy, R., Zolla-Pazner, S., Gorlach, M. and Anglister, J. (2003) Alternative conformations of HIV-1 V3 loops mimic beta hairpins in chemokines, suggesting a mechanism for coreceptor selectivity. Structure 11, 225–236.
Sharon, M., Rosen, O., and Anglister, J. (2005) NMR studies of V3 peptide complexes with antibodies suggest a mechanism for HIV-1 co-receptor selectivity. Curr. Opin. Drug Discov. Dev. 8, 601–612.
Scherf, T., Hiller, R., Naider, F., Levitt, M., and Anglister, J. (1992) Induced peptide conformations in different antibody complexes: molecular modeling of the three-dimensional structure of peptide–antibody complexes using NMR-derived distance restraints. Biochemistry 31, 6884–6897.
Zilber, B., Scherf, T., Levitt, M., and Anglister, J. (1990) NMR-derived model for a peptide–antibody complex. Biochemistry 29, 10032–10041.
Zvi, A., Feigelson, D. J., Hayek, Y., and Anglister, J. (1997) Conformation of the principal neutralizing determinant of human immunodeficiency virus type 1 in complex with an anti-gp120 virus neutralizing antibody studied by two-dimensional nuclear magnetic resonance difference spectroscopy. Biochemistry 36, 8619–8627.
Zvi, A., Kustanovich, I., Feigelson, D., Levy, R., Eisenstein, M., Matsushita, S., Richalet Secordel, P., Regenmortel, M. H., and Anglister, J. (1995) NMR mapping of the antigenic determinant recognized by an anti-gp120, human immunodeficiency virus neutralizing antibody. Eur. J. Biochem. 229, 178–187.
Zvi, A., Tugarinov, V., Faiman, G. A., Horovitz, A., and Anglister, J. (2000) A model of a gp120 V3 peptide in complex with an HIV-neutralizing antibody based on NMR and mutant cycle-derived constraints. Eur. J. Biochem. 267, 767–779.
Scherf, T., and Anglister, J. (1993) A T1 rho-filtered two-dimensional transferred NOE spectrum for studying antibody interactions with peptide antigens. Biophys. J. 64, 754–761.
Samson, A. O., Chill, J. H., Rodriguez, E., Scherf, T., and Anglister, J. (2001) NMR mapping and secondary structure determination of the major acetylcholine receptor alpha-subunit determinant interacting with alpha-bungarotoxin. Biochemistry 40, 5464–5473.
Sattler, M., Schleucher, J., and Griesinger, C. (1999) Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog. Nucl. Magn. Reson. Spectrosc. 34, 93–158.
Kay, L. E., Torchia, D. A., and Bax, A. (1989) Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease. Biochemistry 28, 8972–8979.
Ishima, R., Wingfield, P. T., Stahl, S. J., Kaufman, J. D., and Torchia, D. A. (1998) Using amide 1H and 15N transverse relaxation to detect millisecond time-scale motions in perdeuterated proteins: application to HIV-1 protease. J. Am. Chem. Soc. 120, 10534–10542.
Samson, A. O., Chill, J. H., and Anglister, J. (2005) Two-dimensional measurement of proton T1rho relaxation in unlabeled proteins: mobility changes in alpha-bungarotoxin upon binding of an acetylcholine receptor peptide. Biochemistry 44, 10926–10934.
Gross, E. (1967) The cyanogen bromide reaction, in Methods in Enzymology (Hirs, C. H. W.), Academic, New York, NY, pp. 238–255.
Kaiser, R., and Metzka, L. (1999) Enhancement of cyanogen bromide cleavage yields for methionyl-serine and methionyl-threonine peptide bonds. Anal. Biochem. 266, 1–8.
Wuthrich, K. (ed.) (1986) NMR of proteins and nucleic acids. Wiley, New York, NY, pp. 130–161.
Braunschweiler, L., and Ernst, R. R. (1983) Coherence transfer of isotropic mixing: application to proton correlation spectroscopy. J. Magn. Reson. 53, 521–528.
Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293.
Shaka, A. J., Keeler, J., and Freeman, R. (1983) Evaluation of a new broadband decoupling sequence: WALTZ-16. J. Magn. Reson. 53, 313–340.
Piotto, M., Saudek, V., and Sklenar, V. (1992) Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J. Biomol. NMR 2, 661–665.
Acknowledgments
We thank Drs. Avraham Samson, Anat Zvi, Irina Kustanovic, Michal Sharon, and Naama Kessler, who did some of the studies described in this chapter. We gratefully acknowledge help from Dr. Tali Scherf in maintaining the NMR spectrometers and setting up some of the experiments. We thank Dr. Sandy Livnat for editorial assistance. This study was supported by the National Institute of Health Grant GM 53329 to Jacob Anglister who is the Dr. Joseph and Ruth Owades Professor of Chemistry.
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Rosen, O., Anglister, J. (2009). Epitope Mapping of Antibody–Antigen Complexes by Nuclear Magnetic Resonance Spectroscopy. In: Schutkowski, M., Reineke, U. (eds) Epitope Mapping Protocols. Methods in Molecular Biology™, vol 524. Humana Press. https://doi.org/10.1007/978-1-59745-450-6_3
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DOI: https://doi.org/10.1007/978-1-59745-450-6_3
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