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15NH/D-SOLEXSY experiment for accurate measurement of amide solvent exchange rates: application to denatured drkN SH3

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Abstract

Amide solvent exchange rates are regarded as a valuable source of information on structure/dynamics of unfolded (disordered) proteins. Proton-based saturation transfer experiments, normally used to measure solvent exchange, are known to meet some serious difficulties. The problems mainly arise from the need to (1) manipulate water magnetization and (2) discriminate between multiple magnetization transfer pathways that occur within the proton pool. Some of these issues are specific to unfolded proteins. For example, the compensation scheme used to cancel the Overhauser effect in the popular CLEANEX experiment is not designed for use with unfolded proteins. In this report we describe an alternative experimental strategy, where amide 15N is used as a probe of solvent exchange. The experiment is performed in 50% H2O–50% D2O solvent and is based on the (HACACO)NH pulse sequence. The resulting spectral map is fully equivalent to the conventional HSQC. To fulfill its purpose, the experiment monitors the conversion of deuterated species, 15ND, into protonated species, 15NH, as effected by the solvent exchange. Conceptually, this experiment is similar to EXSY which prompted the name of 15NH/D-SOLEXSY (SOLvent EXchange SpectroscopY). Of note, our experimental scheme, which relies on nitrogen rather than proton to monitor solvent exchange, is free of the complications described above. The developed pulse sequence was used to measure solvent exchange rates in the chemically denatured state of the drkN SH3 domain. The results were found to correlate well with the CLEANEX-PM data, r = 0.97, thus providing a measure of validation for both techniques. When the experimentally measured exchange rates are converted into protection factors, most of the values fall in the range 0.5–2, consistent with random-coil behavior. However, elevated values, ca. 5, are obtained for residues R38 and A39, as well as the side-chain indole of W36. This is surprising, given that high protection factors imply hydrogen bonding or hydrophobic burial not expected to occur in a chemically denatured state of a protein. We, therefore, hypothesized that elevated protection factors are an artefact arising from the calculation of the reference (random-coil) exchange rates. To confirm this hypothesis, we prepared samples of several short peptides derived from the sequence of the drkN SH3 domain; these samples were used to directly measure the reference exchange rates. The revised protection factors obtained in this manner proved to be close to 1.0. These results also have implications for the more compact unfolded state of drkN SH3, which appears to be fully permeable to water as well, with no manifestations of hydrophobic burial.

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Notes

  1. Generally speaking, our experiment can be described as an EXSY-style counterpart to the lineshape analyses by Hawkes et al. and spin echo experiments by Kateb et al., extending the observation window toward the slower exchange rates. At the same time, there is clearly a parallel with 15N zz-exchange experiment by Kay and co-workers (Farrow et al. 1994).

  2. Our scheme is, therefore, well suited for measurements at or near physiological pH, but cannot be used with acid-denatured samples at pH ~2 where exchange rates are too slow.

  3. The convergence of the fitting procedure generally presents no problem; for several residues it is recommendable to first fit the data with \( (0 , N_{z}^{D} (0)) \) initial conditions and then use the results as a starting point for the optimization involving \( (\varepsilon_{H} , N_{z}^{D} (0)) \).

  4. In principle, such an experiment providing a full complement of two ‘axial peaks’ and two ‘cross-peaks’ can be designed using a HA(CACO)N-type sequence, although the detection of 1H resonances near the residual water line presents some practical difficulty.

  5. There is another slowly exchanging residue, I27, which yields an exchange rate 0.34 ± 0.20 s−1 in our experiment, but cannot be detected in the CLEANEX spectra.

  6. It can be pointed out that the e-PHOGSY scheme does not protect against the Overhauser transfer. This, however, is also true for CLEANEX outside the macromolecular limit. Given our successful validation of CLEANEX in this work, this does not appear to be a significant problem.

  7. In addition, we have determined the protection factor for the W36 indole in the salt-stabilized F exch state, PF = 1.9. This result is in line with expectations since the tryptophan side chain is fully exposed to solvent in the folded protein. It is also consistent with the previous observations that W36 has the same (high) degree of solvent exposure in the F exch and U exch states (Evanics et al. 2006 Biochemistry 45:14120–1412). Note that in this study the exchange of W36 Hε1 proton with solvent, as quantified by e-PHOGSY, was mistakenly described as water NOE.

  8. In applications involving unfolded proteins, the 1HN,15N spectral map generally offers better resolution than other 2D spectra. However, even in the case of small proteins, such as drkN SH3, there is a significant number of peak overlaps. In principle, this problem can be overcome with the help of reduced dimensionality schemes utilizing 13C′ and 13Cα chemical shifts (Brutscher et al. (1994) J. Magn. Reson. Ser. B 105:77–82; Tugarinov et al. (2004) J. Biomol. NMR 30:347–352).

  9. Note that, in principle, heating effects can be compensated for (Wang and Bax 1993 J. Biomol. NMR 3:715–720).

  10. Note that this constant refers to the deuterium-to-proton exchange catalyzed by \( {\text{OH}}^{-} \). In the context of our measurement, one is interested in deuterium-to-proton exchange catalyzed by both \( {\text{OH}}^{-} \) and \( {\text{OD}}^{-} \). The difference, however, appears to be subtle.

References

  • Bai YW, Milne JS, Mayne L, Englander SW (1993) Primary structure effects on peptide group hydrogen exchange. Proteins Struct Funct Genet 17:75–86

    Google Scholar 

  • Baxter NJ, Williamson MP (1997) Temperature dependence of 1H chemical shifts in proteins. J Biomol NMR 9:359–369

    Google Scholar 

  • Best RB, Vendruscolo M (2006) Structural interpretation of hydrogen exchange protection factors in proteins: characterization of the native state fluctuations of CI2. Structure 14:97–106

    Google Scholar 

  • Bezsonova I, Singer A, Choy WY, Tollinger M, Forman-Kay JD (2005) Structural comparison of the unstable drkN SH3 domain and a stable mutant. Biochemistry 44:15550–15560

    Google Scholar 

  • Böhlen JM, Rey M, Bodenhausen G (1989) Refocusing with chirped pulses for broadband excitation without phase dispersion. J Magn Reson 84:191–197

    Google Scholar 

  • Brutscher B, Simorre JP, Caffrey MS, Marion D (1994) Design of a complete set of two-dimensional triple-resonance experiments for assigning labeled proteins. J Magn Reson Ser B 105:77–82

    Google Scholar 

  • Buck M, Radford SE, Dobson CM (1994) Amide hydrogen exchange in a highly denatured state. Hen egg-white lysozyme in urea. J Mol Biol 237:247–254

    Google Scholar 

  • Cavanagh J, Rance M (1992) Suppression of cross-relaxation effects in TOCSY spectra via a modified DIPSI-2 mixing sequence. J Magn Reson 96:670–678

    Google Scholar 

  • Chevelkov V, Faelber K, Diehl A, Heinemann U, Oschkinat H, Reif B (2005) Detection of dynamic water molecules in a microcrystalline sample of the SH3 domain of α-spectrin by MAS solid-state NMR. J Biomol NMR 31:295–310

    Google Scholar 

  • Chevelkov V, Zhuravleva AV, Xue Y, Reif B, Skrynnikov NR (2007) Combined analysis of 15N relaxation data from solid- and solution-state NMR spectroscopy. J Am Chem Soc 129:12594–12595

    Google Scholar 

  • Choy WY, Forman-Kay JD (2001) Calculation of ensembles of structures representing the unfolded state of an SH3 domain. J Mol Biol 308:1011–1032

    Google Scholar 

  • Choy WY, Mulder FAA, Crowhurst KA, Muhandiram DR, Millett IS, Doniach S, Forman-Kay JD, Kay LE (2002) Distribution of molecular size within an unfolded state ensemble using small-angle X-ray scattering and pulse field gradient NMR techniques. J Mol Biol 316:101–112

    Google Scholar 

  • Connelly GP, Bai YW, Jeng MF, Englander SW (1993) Isotope effects in peptide group hydrogen exchange. Proteins Struct Funct Genet 17:87–92

    Google Scholar 

  • Covington AK, Robinson RA, Bates RG (1966) Ionization constant of deuterium oxide from 5 to 50 degrees. J Phys Chem 70:3820–3824

    Google Scholar 

  • Covington AK, Paabo M, Robinson RA, Bates RG (1968) Use of glass electrode in deuterium oxide and relation between standardized pD (pad) scale and operational pH in heavy water. Anal Chem 40:700–706

    Google Scholar 

  • Crowhurst KA, Forman-Kay JD (2003) Aromatic and methyl NOEs highlight hydrophobic clustering in the unfolded state of an SH3 domain. Biochemistry 42:8687–8695

    Google Scholar 

  • Crowhurst KA, Tollinger M, Forman-Kay JD (2002) Cooperative interactions and a non-native buried Trp in the unfolded state of an SH3 domain. J Mol Biol 322:163–178

    Google Scholar 

  • Dalvit C (1996) Homonuclear 1D and 2D NMR experiments for the observation of solvent-solute interactions. J Magn Reson B 112:282–288

    Google Scholar 

  • Dalvit C, Hommel U (1995) Sensitivity-improved detection of protein hydration and its extension to the assignment of fast-exchanging resonances. J Magn Reson B 109:334–338

    Google Scholar 

  • Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on unix pipes. J Biomol NMR 6:277–293

    Google Scholar 

  • Delepierre M, Dobson CM, Karplus M, Poulsen FM, States DJ, Wedin RE (1987) Electrostatic effects and hydrogen exchange behavior in proteins. The pH dependence of exchange rates in lysozyme. J Mol Biol 197:111–122

    Google Scholar 

  • Desvaux H, Birlirakis N, Wary C, Berthault P (1995) Study of slow molecular motions in solution using off-resonance irradiation in homonuclear NMR. 2. Fast chemical exchange processes. Mol Phys 86:1059–1073

    ADS  Google Scholar 

  • Dobson CM, Lian L-Y, Redfield C, Topping KD (1986) Measurement of hydrogen exchange rates using 2D NMR spectroscopy. J Magn Reson 69:201–209

    Google Scholar 

  • Dötsch V, Wider G, Siegal G, Wüthrich K (1995) Interaction of urea with an unfolded protein—the DNA-binding domain of the 434-repressor. FEBS Lett 366:6–10

    Google Scholar 

  • Eichmüller C, Skrynnikov NR (2005) A new amide proton R 1ρ experiment permits accurate characterization of microsecond time-scale conformational exchange. J Biomol NMR 32:281–293

    Google Scholar 

  • Emsley L, Bodenhausen G (1990) Gaussian pulse cascades—new analytical functions for rectangular selective inversion and in-phase excitation in NMR. Chem Phys Lett 165:469–476

    ADS  Google Scholar 

  • Emsley L, Bodenhausen G (1992) Optimization of shaped selective pulses for NMR using a quaternion description of their overall propagators. J Magn Reson 97:135–148

    Google Scholar 

  • Englander SW, Poulsen A (1969) Hydrogen-tritium exchange of random chain polypeptide. Biopolymers 7:379–393

    Google Scholar 

  • Ernst RR, Bodenhausen G, Wokaun A (1987) Principles of nuclear magnetic resonance in one and two dimensions. Oxford University Press, Oxford

    Google Scholar 

  • Evanics F, Bezsonova I, Marsh J, Kitevski JL, Forman-Kay JD, Prosser RS (2006) Tryptophan solvent exposure in folded and unfolded states of an SH3 domain by 19F and 1H NMR. Biochemistry 45:14120–14128

    Google Scholar 

  • Farrow NA, Zhang OW, Forman-Kay JD, Kay LE (1994) A heteronuclear correlation experiment for simultaneous determination of 15N longitudinal decay and chemical exchange rates of systems in slow equilibrium. J Biomol NMR 4:727–734

    Google Scholar 

  • Farrow NA, Zhang OW, Forman-Kay JD, Kay LE (1995) Comparison of the backbone dynamics of a folded and an unfolded SH3 domain existing in equilibrium in aqueous buffer. Biochemistry 34:868–878

    Google Scholar 

  • Farrow NA, Zhang OW, Forman-Kay JD, Kay LE (1997) Characterization of the backbone dynamics of folded and denatured states of an SH3 domain. Biochemistry 36:2390–2402

    Google Scholar 

  • Forge V, Wijesinha RT, Balbach J, Brew K, Robinson CV, Redfield C, Dobson CM (1999) Rapid collapse and slow structural reorganisation during the refolding of bovine α-lactalbumin. J Mol Biol 288:673–688

    Google Scholar 

  • Gal M, Schanda P, Brutscher B, Frydman L (2007) UltraSOFAST HMQC NMR and the repetitive acquisition of 2D protein spectra at Hz rates. J Am Chem Soc 129:1372–1377

    Google Scholar 

  • Gemmecker G, Jahnke W, Kessler H (1993) Measurement of fast proton exchange rates in isotopically labeled compounds. J Am Chem Soc 115:11620–11621

    Google Scholar 

  • Gold V, Lowe BM (1967) Measurement of solvent isotope effects with glass electrode. I. The ionic product of D2O and D2O-H2O mixtures. J Chem Soc A:936–943

    Google Scholar 

  • Griesinger C, Ernst RR (1987) Frequency offset effects and their elimination in NMR rotating-frame cross-relaxation spectroscopy. J Magn Reson 75:261–271

    Google Scholar 

  • Grzesiek S, Bax A (1992) An efficient experiment for sequential backbone assignment of medium-sized isotopically enriched proteins. J Magn Reson 99:201–207

    Google Scholar 

  • Grzesiek S, Bax A (1993a) Amino-acid type determination in the sequential assignment procedure of uniformly 13C/15N-enriched proteins. J Biomol NMR 3:185–204

    Google Scholar 

  • Grzesiek S, Bax A (1993b) The importance of not saturating H2O in protein NMR: application to sensitivity enhancement and NOE measurements. J Am Chem Soc 115:12593–12594

    Google Scholar 

  • Hansen PE (2000) Isotope effects on chemical shifts of proteins and peptides. Magn Reson Chem 38:1–10

    Google Scholar 

  • Harbison GS, Roberts JE, Herzfeld J, Griffin RG (1988) Solid-state NMR detection of proton exchange between the bacteriorhodopsin Schiff-base and bulk water. J Am Chem Soc 110:7221–7223

    Google Scholar 

  • Hawkes GE, Randall EW, Hull WE, Gattegno D, Conti F (1978) Qualitative aspects of hydrogen-deuterium exchange in 1H, 13C, and 15N nuclear magnetic resonance spectra of viomycin in aqueous solution. Biochemistry 17:3986–3992

    Google Scholar 

  • Henry GD, Sykes BD (1993) Saturation transfer of exchangeable protons in 1H-decoupled 15N INEPT spectra in water. Application to the measurement of hydrogen exchange rates in amides and proteins. J Magn Reson B 102:193–200

    Google Scholar 

  • Henry GD, Weiner JH, Sykes BD (1987) Backbone dynamics of a model membrane protein: measurement of individual amide hydrogen exchange rates in detergent-solubilized M13 coat protein using 13C NMR hydrogen-deuterium isotope shifts. Biochemistry 26:3626–3634

    Google Scholar 

  • Hitchens TK, McCallum SA, Rule GS (1999) A JCH-modulated 2D (HACACO)NH pulse scheme for quantitative measurement of 13Cα-1Hα couplings in 15N, 13C-labeled proteins. J Magn Reson 140:281–284

    ADS  Google Scholar 

  • Hvidt A, Nielsen SO (1966) Hydrogen exchange in proteins. Adv Protein Chem 21:287–386

    Google Scholar 

  • Hwang TL, Mori S, Shaka AJ, van Zijl PCM (1997) Application of phase-modulated CLEAN chemical EXchange spectroscopy (CLEANEX-PM) to detect water-protein proton exchange and intermolecular NOEs. J Am Chem Soc 119:6203–6204

    Google Scholar 

  • Hwang TL, van Zijl PCM, 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

    Google Scholar 

  • Jeener J, Meier BH, Bachmann P, Ernst RR (1979) Investigation of exchange processes by 2-dimensional NMR spectroscopy. J Chem Phys 71:4546–4553

    ADS  Google Scholar 

  • Jensen MR, Kristensen SM, Led JJ (2007) Elimination of spin diffusion effects in saturation transfer experiments: application to hydrogen exchange in proteins. Magn Reson Chem 45:257–261

    Google Scholar 

  • Kanelis V, Donaldson L, Muhandiram DR, Rotin D, Forman-Kay JD, Kay LE (2000) Sequential assignment of proline-rich regions in proteins: application to modular binding domain complexes. J Biomol NMR 16:253–259

    Google Scholar 

  • Kateb F, Pelupessy P, Bodenhausen G (2007) Measuring fast hydrogen exchange rates by NMR spectroscopy. J Magn Reson 184:108–113

    ADS  Google Scholar 

  • Kohn JE, Millett IS, Jacob J, Zagrovic B, Dillon TM, Cingel N, Dothager RS, Seifert S, Thiyagarajan P, Sosnick TR, Hasan MZ, Pande VS, Ruczinski I, Doniach S, Plaxco KW (2004) Random-coil behavior and the dimensions of chemically unfolded proteins. Proc Natl Acad Sci USA 101:12491–12496

    ADS  Google Scholar 

  • Koide S, Jahnke W, Wright PE (1995) Measurement of intrinsic exchange rates of amide protons in a 15N-labeled peptide. J Biomol NMR 6:306–312

    Google Scholar 

  • Kriwacki RW, Hill RB, Flanagan JM, Caradonna JP, Prestegard JH (1993) New NMR methods for the characterization of bound waters in macromolecules. J Am Chem Soc 115:8907–8911

    Google Scholar 

  • Lesage A, Böckmann A (2003) Water-protein interactions in microcrystalline Crh measured by 1H–13C solid-state NMR spectroscopy. J Am Chem Soc 125:13336–13337

    Google Scholar 

  • Lesage A, Gardiennet C, Loquet A, Verel R, Pintacuda G, Emsley L, Meier BH, Böckmann A (2008) Polarization transfer over the water-protein interface in solids. Angew Chem Int Ed 47:5851–5854

    Google Scholar 

  • Linderstrøm-Lang K (1955) Deuterium exchange between peptides and water. Chem Soc (London) Spec Publ 2:1–20

    Google Scholar 

  • LiWang AC, Bax A (1996) Equilibrium protium/deuterium fractionation of backbone amides in U-13C/15N labeled human ubiquitin by triple resonance NMR. J Am Chem Soc 118:12864–12865

    Google Scholar 

  • Loftus D, Gbenle GO, Kim PS, Baldwin RL (1986) Effects of denaturants on amide proton exchange rates: a test for structure in protein fragments and folding intermediates. Biochemistry 25:1428–1436

    Google Scholar 

  • Marion D, Wüthrich K (1983) Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H–1H spin-spin coupling constants in proteins. Biochem Biophys Res Commun 113:967–974

    Google Scholar 

  • Marsh JA, Forman-Kay JD (2009) Structure and disorder in an unfolded state under nondenaturing conditions from ensemble models consistent with a large number of experimental restraints. J Mol Biol 391:359–374

    Google Scholar 

  • McConnell HM (1958) Reaction rates by nuclear magnetic resonance. J Chem Phys 28:430–431

    ADS  Google Scholar 

  • Melacini G, Kaptein R, Boelens R (1999) Editing of chemical exchange-relayed NOEs in NMR experiments for the observation of protein-water interactions. J Magn Reson 136:214–218

    ADS  Google Scholar 

  • Modig K, Liepinsh E, Otting G, Halle B (2004) Dynamics of protein and peptide hydration. J Am Chem Soc 126:102–114

    Google Scholar 

  • Mok YK, Elisseeva EL, Davidson AR, Forman-Kay JD (2001) Dramatic stabilization of an SH3 domain by a single substitution: roles of the folded and unfolded states. J Mol Biol 307:913–928

    Google Scholar 

  • Mori S, Abeygunawardana C, van Zijl PCM, Berg JM (1996) Water exchange filter with improved sensitivity (WEX II) to study solvent-exchangeable protons. Application to the consensus zinc finger peptide CP-1. J Magn Reson B 110:96–101

    Google Scholar 

  • Mori S, van Zijl PCM, Shortle D (1997) Measurement of water-amide proton exchange rates in the denatured state of staphylococcal nuclease by a magnetization transfer technique. Proteins Struct Funct Genet 28:325–332

    Google Scholar 

  • Nishimura C, Dyson HJ, Wright PE (2008) The kinetic and equilibrium molten globule intermediates of apoleghemoglobin differ in structure. J Mol Biol 378:715–725

    Google Scholar 

  • Otting G, Liepinsh E (1995) Protein hydration viewed by high-resolution NMR spectroscopy: implications for magnetic resonance image contrast. Accounts Chem Res 28:171–177

    Google Scholar 

  • Otting G, Liepinsh E, Wüthrich K (1991) Protein hydration in aqueous solution. Science 254:974–980

    ADS  Google Scholar 

  • Reimer U, Scherer G, Drewello M, Kruber S, Schutkowski M, Fischer G (1998) Side-chain effects on peptidyl-prolyl cis/trans isomerisation. J Mol Biol 279:449–460

    Google Scholar 

  • Roder H, Elöve GA, Englander SW (1988) Structural characterization of folding intermediates in cytochrome c by H-exchange labelling and proton NMR. Nature 335:700–704

    ADS  Google Scholar 

  • Schleucher J, Wijmenga SS (2002) How to detect internal motion by homonuclear NMR. J Am Chem Soc 124:5881–5889

    Google Scholar 

  • Schulman BA, Redfield C, Peng ZY, Dobson CM, Kim PS (1995) Different subdomains are most protected from hydrogen exchange in the molten globule and native states of human α-lactalbumin. J Mol Biol 253:651–657

    Google Scholar 

  • Shaka AJ, Keeler J, Frenkiel T, Freeman R (1983) An improved sequence for broad-band decoupling: WALTZ-16. J Magn Reson 52:335–338

    Google Scholar 

  • Shaka AJ, Barker PB, Freeman R (1985) Computer-optimized decoupling scheme for wideband applications and low-level operation. J Magn Reson 64:547–552

    Google Scholar 

  • Shaka AJ, Lee CJ, Pines A (1988) Iterative schemes for bilinear operators—application to spin decoupling. J Magn Reson 77:274–293

    Google Scholar 

  • Shang ZG, Swapna GVT, Rios CB, Montelione GT (1997) Sensitivity enhancement of triple-resonance protein NMR spectra by proton evolution of multiple-quantum coherences using a simultaneous 1H and 13C constant-time evolution period. J Am Chem Soc 119:9274–9278

    Google Scholar 

  • Sklenár V, Piotto M, Leppik R, Saudek V (1993) Gradient-tailored water suppression for 1H–15 N HSQC experiments optimized to retain full sensitivity. J Magn Reson A 102:241–245

    Google Scholar 

  • Skrynnikov NR, Ernst RR (1999) Detection of intermolecular chemical exchange through decorrelation of two-spin order. J Magn Reson 137:276–280

    ADS  Google Scholar 

  • Spera S, Ikura M, Bax A (1991) Measurement of the exchange rates of rapidly exchanging amide protons. Application to the study of calmodulin and its complex with a myosin light chain kinase fragment. J Biomol NMR 1:155–166

    Google Scholar 

  • Tollinger M, Skrynnikov NR, Mulder FAA, Forman-Kay JD, Kay LE (2001) Slow dynamics in folded and unfolded states of an SH3 domain. J Am Chem Soc 123:11341–11352

    Google Scholar 

  • Tugarinov V, Choy WY, Kupce E, Kay LE (2004) Addressing the overlap problem in the quantitative analysis of two dimensional NMR spectra: Application to 15N relaxation measurements. J Biomol NMR 30:347–352

    Google Scholar 

  • van de Ven FJM, Janssen HGJM, Graslund A, Hilbers CW (1988) Chemically relayed nuclear Overhauser effects: connectivities between resonances of nonexchangeable protons and water. J Magn Reson 79:221–235

    Google Scholar 

  • Vendruscolo M, Paci E, Dobson CM, Karplus M (2003) Rare fluctuations of native proteins sampled by equilibrium hydrogen exchange. J Am Chem Soc 125:15686–15687

    Google Scholar 

  • Vuister GW, Bax A (1992) Resolution enhancement and spectral editing of uniformly 13C-enriched proteins by homonuclear broadband 13C decoupling. J Magn Reson 98:428–435

    Google Scholar 

  • Wagner G, Wüthrich K (1982) Amide proton exchange and surface conformation of the basic pancreatic trypsin inhibitor in solution: studies with two-dimensional nuclear magnetic resonance. J Mol Biol 160:343–361

    Google Scholar 

  • Wang AC, Bax A (1993) Minimizing the effects of radiofrequency heating in multidimensional NMR experiments. J Biomol NMR 3:715–720

    Google Scholar 

  • Wang AC, Grzesiek S, Tschudin R, Lodi PJ, Bax A (1995) Sequential backbone assignment of isotopically enriched proteins in D2O by deuterium-decoupled HA(CA)N and HA(CACO)N. J Biomol NMR 5:376–382

    Google Scholar 

  • Wirmer J, Peti W, Schwalbe H (2006) Motional properties of unfolded ubiquitin: a model for a random coil protein. J Biomol NMR 35:175–186

    Google Scholar 

  • Xu J, Millet O, Kay LE, Skrynnikov NR (2005) A new spin probe of protein dynamics: nitrogen relaxation in 15N–2H amide groups. J Am Chem Soc 127:3220–3229

    Google Scholar 

  • Xue Y, Podkorytov IS, Rao DK, Benjamin N, Sun HL, Skrynnikov NR (2009) Paramagnetic relaxation enhancements in unfolded proteins: theory and application to drkN SH3 domain. Protein Sci 18:1401–1424

    Google Scholar 

  • Yao J, Chung J, Eliezer D, Wright PE, Dyson HJ (2001) NMR structural and dynamic characterization of the acid-unfolded state of apomyoglobin provides insights into the early events in protein folding. Biochemistry 40:3561–3571

    Google Scholar 

  • Zangger K, Armitage IM (1998) Sensitivity-enhanced detection of fast exchanging protons by an exchange-edited gradient HEHAHA-HSQC experiment. J Magn Reson 135:70–75

    ADS  Google Scholar 

  • Zhang OW, Forman-Kay JD (1997) NMR studies of unfolded states of an SH3 domain in aqueous solution and denaturing conditions. Biochemistry 36:3959–3970

    Google Scholar 

  • Zhang OW, Kay LE, Olivier JP, Forman-Kay JD (1994) Backbone 1H and 15N resonance assignments of the N-terminal SH3 domain of drk in folded and unfolded states using enhanced-sensitivity pulsed-field gradient NMR techniques. J Biomol NMR 4:845–858

    Google Scholar 

  • Zheng ZW, Gryk MR, Finucane MD, Jardetzky O (1995) Investigation of protein amide-proton exchange by 1H longitudinal spin relaxation. J Magn Reson B 108:220–234

    Google Scholar 

  • Zhou JY, van Zijl PCM (2006) Chemical exchange saturation transfer imaging and spectroscopy. Prog NMR Spectrosc 48:109–136

    Google Scholar 

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Acknowledgments

We are thankful to Joseph Marsh for helpful discussions. The research has been funded through the NSF grants MCB-044563 and CHE-0723718 to N.R.S.

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  1. Veniamin Chevelkov

    Present address: Forschunginstitut für Molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, 13125, Berlin, Germany

  2. D. Krishna Rao

    Present address: Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX, 77555-0647, USA

Authors and Affiliations

  1. Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN, 47907-2084, USA

    Veniamin Chevelkov, Yi Xue, D. Krishna Rao & Nikolai R. Skrynnikov

  2. Program in Molecular Structure and Function, Hospital for Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8, Canada

    Julie D. Forman-Kay

  3. Department of Biochemistry, University of Toronto, 1 King’s College Circle, Toronto, ON, M5S 1A8, Canada

    Julie D. Forman-Kay

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Chevelkov, V., Xue, Y., Krishna Rao, D. et al. 15NH/D-SOLEXSY experiment for accurate measurement of amide solvent exchange rates: application to denatured drkN SH3. J Biomol NMR 46, 227–244 (2010). https://doi.org/10.1007/s10858-010-9398-8

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