Abstract
Nearly half a century ago, Hvidt and Linderstrøm-Lang established amide hydrogen (NH) isotope exchange as a fruitful probe of structure and dynamics in proteins (1–4). Rapid growth in the use of NH exchange over the last 15 yr has followed from advances in both technology (e.g., multidimensional nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS) and rapid-mixing methodologies) and in our understanding of how NH exchange in proteins is slowed relative to exchange in small peptides (5–11). Most applications of NH exchange involve one of the two major types of exchange experiments. These are distinguished by the population of native protein: the first and most popular type of experiment involves native protein alone, and the second type is used to follow exchange during the process of folding or unfolding. For practical reasons, almost all exchange experiments in proteins focus on the NHs of the peptide backbone. This chapter focuses on the exchange of these NHs from the native protein.
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References
Hvidt, A. and Linderstrøm-Lang, K. (1955) The pH-dependence of the deuterium exchange of insulin. Biochem. Biophys. Acta 18, 308.
Hvidt, A. (1955) Deuterium exchange between ribonuclease and water. Biochim. Biophys. Acta 18, 307–308.
Hvidt, A. and Nielsen, S. O. (1966) Hydrogen exchange in proteins. Adv. Protein Chem. 21, 287–386.
Hvidt, A. (1964) A discussion of the pH dependence of the hydrogen-deuterium exchange in proteins. CR Trav. Lab. Carlsberg 34, 299–317.
Udgaonkar, J. B. and Baldwin, R. L. (1988) NMR evidence for an early framework intermediate on the folding pathway of ribonuclease A. Nature 335, 694–699.
Roder, H., Elove, G. A., and Englander, S. W. (1988) Structural characterization of folding intermediates in cytochrome c by H-exchange labeling and proton NMR. Nature 335, 700–704.
Miranker, A., Robinson, C. L., Radford, S. E., Aplin, R. T., and Dobson, C. M. (1993) Detection of transient protein folding populations by mass spectrometry. Science 262, 896–900.
Kiefhaber, T. and Baldwin, R. L. (1995) Kinetics of hydrogen bond breakage in the process of unfolding of ribonuclease A measured by pulsed hydrogen exchange. Proc. Natl. Acad. Sci. USA 92, 2657–2661.
Gladwin, S. T. and Evans, P. A. (1996) Structure of very early protein folding intermediates: new insights through a variant of hydrogen exchange labeling. Folding and Design 1, 407–417.
Laurents, D. V., Bruix, M., Jamin, M., and Baldwin, R. L. (1998) A pulse-chase-competition experiment to determine if a folding intermediate is on or off-pathway: application to ribonuclease A. J. Mol. Biol. 283, 669–678.
Robertson, A. D. and Baldwin, R. L. (1991) Hydrogen exchange in thermally denatured ribonuclease A. Biochemistry 30, 9907–9914.
Woodward, C. K. and Hilton, B. D. (1979) Hydrogen exchange kinetics and internal motions in proteins and nucleic acids. Annu. Rev. Biophys. Bioeng. 8, 99–127.
Roder, H. (1986) Structural characterization of protein folding intermediates by protein magnetic resonance and hydrogen exchange. Meth. Enzymol. 176, 446–473.
Mayo, S. L. and Baldwin, R. L. (1993) Guanidinium chloride induction of partial unfolding in amide proton exchange in RNase A. Science 262, 873–876.
Bai, Y., Sosnick, T. R., Mayne, L., and Englander, S. W. (1995) Protein folding intermediates: native-state hydrogen exchange. Science 269, 192–197.
Swint-Kruse, L. and Robertson, A. D. (1996) Temperature and pH dependences of hydrogen exchange and global stability for ovomu-coid third domain. Biochemistry 35, 171–180.
Hosszu, L. L., Craven, C. J., Lorch, M., Spencer, J., Clarke, A. R., and Waltho, J. P. (1997) Structure of a kinetic protein folding intermediate by equilibrium amide exchange. Nat. Struct. Biol. 4, 801–804.
Xu, Y., Mayne, L., and Englander, S. W. (1998) Evidence for an unfolding and refolding pathway in cytochrome c. Nat. Struct. Biol. 5, 774–778.
Sivaraman, T., Kumar, T. K. S., and Yu, C. (1999) Investigation of the structural stability of cardiotoxin analogue III from the Taiwan cobra by hydrogen-deuterium exchange kinetics. Biochemistry 38, 9899–9905.
Chakshusmathi, G., Ratnaparkhi, G. S., Madhu, P. K., and Varadarajan, R. (1999) Native-state hydrogen-exchange studies of a fragment complex can provide structural information about the isolated fragments. Proc. Natl. Acad. Sci. USA 96, 7899–7904.
Neira, J. L., Selvilla, P., Menendez, M., Bruix, M., and Rico, M. (1999) Hydrogen exchange in ribonuclease A and ribonuclease S: evidence for residual structure in the unfolded state under native conditions. J. Mol. Biol. 285, 627–643.
Woodward, C. (1993) Is the slow-exchange core the protein folding core? Trends Biochem. Sci. 18, 359–360.
Li, R. and Woodward, C. (1999) The hydrogen exchange core and protein folding. Protein Sci. 8, 1571–1591.
Clarke, J., Itzhaki, L. S., and Fersht, A. R. (1997) Hydrogen exchange at equilibrium: a short cut for analyzing protein-folding pathways? Trends Biochem. Sci. 22, 284–287.
Englander, S. W. (1998) Native-state HX. Trends Biochem. Sci. 23, 378.
Woodward, C. and Li, R. (1998) The slow-exchange core and protein folding. Trends Biochem. Sci. 23, 379.
Clarke, J., Itzhaki, L. S., and Fersht, A. R. (1998) A reply to Englander and Woodward. Trends Biochem. Sci. 23, 379–381.
Roder, H., Wagner, G., and Wuthrich, K. (1985) Amide proton exchange in proteins by EX1 kinetics: studies of the basic pancreatic trypsin inhibitor at variable p2H and temperature. Biochemistry 24, 7396–7407.
Pedersen, T. G., Thomsen, N. K., Andersen, K. V., Madsen, J. C., and Poulsen, F. M. (1993) Determination of the rate constants k1 and k2 of the Linderstrøm-lang model for protein amide hydrogen exchange: a study of the individual amides in hen egg-white lysozyme. J. Mol. Biol. 230, 651–660.
Arrington, C. B. and Robertson, A. D. (1997) Microsecond protein folding kinetics from native-state hydrogen exchange. Biochemistry 36, 8686–8691.
Arrington, C. B., Teesch, L. M., and Robertson, A. D. (1999) Defining protein ensembles with native-state NH exchange: kinetics of interconversion and co-operative units from combined NMR and MS analysis. J. Mol. Biol. 285, 1265–1275.
Arrington, C. B. and Robertson, A. D. (2000) Kinetics and thermody namics of conformational equilibria in native proteins by hydrogen exchange. Methods Enzymol. 323, 104–124.
Arrington, C. B. and Robertson, A. D. (2000) Microsecond to minute dynamics revealed by EX 1-type hydrogen exchange at nearly every backbone hydrogen bond in a native protein. J. Mol. Biol. 296, 1311–1321.
Bai, Y., Milne, J. S., Mayne, L., and Englander, S. W. (1993) Primary structure effects on peptide group hydrogen exchange. Proteins 17, 75–86.
Huyghes-Despointes, B. M. P., Scholtz, J. M., and Pace, C. N. (1999) Protein conformational stabilities can be determined from hydrogen exchange rates. Nature Struct. Biol. 6, 910–912.
Bai, Y., Sosnick, T. R., Mayne, L., and Englander, S. W. (1995) Protein folding intermediates: native-state hydrogen exchange. Science 269, 192–197.
Chamberlain, A. K., Handel, T. M., and Marqusee, S. (1996) Detection of rare partially folded molecules in equilibrium with the native conformation of Rnase H. Nat. Struct. Biol. 3, 782–787.
Jackson, S. E. (1998) How do small single-domain proteins fold? Folding and Design 3, R81–R91.
Swint-Kruse, L. and Robertson, A. D. (1995) Hydrogen bonds and the pH dependence of ovomucoid third domain stability. Biochemistry 34, 4724–732.
Bai, Y., Milne, J. S., Mayne, L., and Englander, S. W. (1994) Protein stability parameters measured by hydrogen exchange. Proteins Struct. Funct. Genet. 20, 4–14.
Fersht, A. (1999) Structure and Mechanism in Protein Science, W. H. Freeman and Co., New York, NY.
Davis, J. H. (1995) Refocusing revisited: an optimized, gradient-enhanced refocused HSQC and its applications in 2D and 3D NMR and in deuterium exchange experiments. J. Biomol. NMR 5, 433–437.
Hwang, T. L., van Zijl, P. C. M., and Mori, S. (1998) Accurate quantitation of water-amide proton exchange rates using the phase-modulated CLEAN chemical Exchange (CLEANEX-PM) approach with a fast-HSQC (FHSQC) detection scheme. J. Biomol. NMR 11, 221–226.
Baxter, N. J. and Williamson, M. P. (1997) Temperature dependence of 1H chemical shifts in proteins. J. Biomol. NMR 9, 359–369.
Forsyth, W. R. and Robertson, A. D. (1996) Intramolecular electrostatic interactions accelerate hydrogen exchange in diketopiperazine relative to 2-piperidone. J. Am. Chem. Soc. 118, 2694–2698.
Evans, J. N. S. (1995) Biomolecular NMR spectroscopy, Oxford University Press, Oxford, U.K.
Westcott, C. C. (1978) Electrodes, in pH Measurements, 1st ed., Academic Press, Inc., Orlando, FL, pp. 41–78.
Bates, R. (1973) pH standards, in Determination ofpH: Theory and Practice, 2nd Ed., John Wiley and Sons, New York, NY, pp. 59–104.
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Sivaraman, T., Robertson, A.D. (2001). Kinetics of Conformational Fluctuations by EX1 Hydrogen Exchange in Native Proteins. In: Murphy, K.P. (eds) Protein Structure, Stability, and Folding. Methods in Molecular Biology™, vol 168. Humana Press. https://doi.org/10.1385/1-59259-193-0:193
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DOI: https://doi.org/10.1385/1-59259-193-0:193
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