Influence of charge on the rate of amide proton exchange.

Nearest-neighbor inductive effects are known to influence amide proton exchange. Here we show that longrange electrostatic interactions also have a significant effect on the rate of amide proton exchange. We have measured the effects of salt (NaC1) on the exchange rates of the neutral polypeptide poly(DL-alanine) (PDLA) and of the positively charged polymer poly(DL-lysine) (PDLL). Our results show that, while exchange from PDLA is insensitive to ionic strength, exchange from PDLL is extremely sensitive to salt concentration. In the acid-catalyzed exchange of PDLL, the exchange rate increases approximately 3-fold between ionic strengths of 60 mM and 1 M. In base-catalyzed exchange, there is a decrease in exchange rate of almost 50-fold between ionic strengths of 2 mM and 1 M. As expected, salt produces a shift of the pH(the pH at which exchange is slowest) for PDLL but not for PDLA. Our results show a salt-dependent A m i d e proton exchange was introduced as a method of studying protein conformation by Hvidt & Linderstrram-Lang (1954). The method is useful for studying the conformation and conformational fluctuations of a protein since the rate of amide proton exchange is greatly reduced in native proteins (by as much as lo8) when the amide proton is hydrogen bonded or when it is otherwise protected from solvent. Recently, amide proton exchange has been used to (a) study conformational fluctuations in lysozyme (Knox & Rosenberg, 1980) and ribonuclease S (Rosa & Richards, 1979, 1981), (b) measure the dissociation constants of hemoglobin (Ide et al., 1976) and of ribonuclease S (Schreier & Baldwin, 1976,1977), (c) study ligand-induced conformational changes in hemoglobin (Englander et al., 1980), and (d) probe the structures of intermediates in the folding of ribonuclease A (Schmid & Baldwin, 1979; Kim & Baldwin, 1980). The NMR' assignment of the slowly exchanging amide protons in BPTI by Dubs et al. (1979) has made it possible to investigate the mechanisms of amide proton exchange in a protein by analyzing the exchange rates of individual amide protons in BPTI and its From the Department of Biochemistry, Stanford University Medical School, Stanford, California 94305. Receioed June 9, 2981. This research has been supported by grants to R.L. B. from the National Science Foundation (PCM 77-16834) and the National Institutes of Health (2 ROI GM-19988-21) and by a Medical Scientist Training Program grant to P.S. K. from the National Institutes of Health (GM07365). t Predoctoral fellow, Medical Scientist Training Program. January 5 , 1982 shift of 1.3 pH units in the pHmi, of PDLL between 0 and 2 M NaCl. These results unambiguously demonstrate a major effect of charge on the kinetics of amide proton exchange in unstructured polypeptides. We have also done some simple electrostatic calculations for the well studied protein basic pancreatic trypsin inhibitor (BPTI). Our calculations show some of the anomalous features of the pH dependence curves measured for individual amide protons of BPTI [Richarz, R. , Sehr, P., Wagner, G. , & Wiithrich, K. (1979) J . Mol. Biol. 130,1! 9-30], although the overall agreement is poor, indicating that additional factors (probably including the local electrostatic environment) affect the pH dependence curve. An obvious conclusion of this work is that long-range electrostatic effects, which alter the pH in the vicinity of a macromolecule, must be taken into account when interpreting proton exchange data. derivatives (Wiithrich et al., 1980, and references cited therein). In unstructured peptides, neighboring groups have a major effect on the kinetics of amide proton exchange. The importance of inductive effects was suggested by Leichtling & Klotz (1966), and an important advance was made when the inductive effects of the various amino acid side chains were quantitated by Molday et al. (1972). In general, electronwithdrawing neighbors, which make the amide a stronger acid, are found to enhance base-catalyzed exchange and decrease the acid-catalyzed exchange rate. In this paper we demonstrate that long-range electrostatic interactions also have a significant effect on amide proton exchange rates. This conclusion is based on a comparison of the effects of NaCl on the exchange rates of PDLA (a neutral polypeptide) and the positively charged polypeptide PDLL. We also show, using a smeared-charge model (LinderstrramLang, 1924; Tanford, 1962), that the effects of charge can be substantial in amide proton exchange from proteins. In particular, we have made calculations for BPTI and have Abbreviations: PDLA, poly(DL-alanine); PDLL, poly(DL-lySine); BPTI, basic pancreatic trypsin inhibitor; pH&, the pH at which proton exchange is slowest; NMR, nuclear magnetic resonance; pH,,,, the pH in the vicinity of a macromolecule; pH,,, the experimentally measured pH of the bulk solution; NMA, N-methylacetamide; Cw, concentration of condensed counterions surrounding a polyelectrolyte; DNA, deoxyribonucleic acid; Tris, tris(hydroxymethy1)aminomethane. 0006-2960/82/0421-0001$01.25/0