AngewandteChemieProtein DynamicsDOI: 10.1002/anie.201311275Probing Transient Conformational States of Proteinsby Solid-State R11 Relaxation-Dispersion NMR Spec-troscopy**Peixiang Ma, Jens D. Haller, Jrmy Zajakala, Pavel Macek, Astrid C. Sivertsen,Dieter Willbold, Jrme Boisbouvier, and Paul Schanda*AngewandteCommunications43122014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2014, 53, 4312 4317Solution-stateNMRspectroscopictechniques, andinpar-ticular so-called relaxation-dispersion (RD) NMRapproaches, have proven very successfulforstudyingmsmsmotionandcharacterizingtheexchangingshort-livedcon-formations.[1]RDNMRtechniques exploit the effect ofconformational-exchange processes on line broadening, thatis, on the relaxation rates of nuclear spin coherence (R2, R11).By quantifying spin relaxation rates in the presence ofa variable radiofrequency (rf) field, RD approaches provideinformation about relative populations and exchange rates, aswell aschemical shiftsofshort-livedconformational states,andthusaboutlocal structure.[1, 2]Inthecaseofverylargeassemblies or insoluble aggregates, where solution-stateNMRis severely challenged, magic-angle-spinning solid-state NMR (MAS ssNMR) is rapidly emerging as a tool forthe study of structure and dynamics. However, the character-izationof conformational-exchange dynamics inthe solidstate remains challenging. Herein, we show a ssNMRapproachinwhichamide-15NR11RDdata, thatis, therateof coherence decay under15N spin-lock fields of variable fieldstrengths, are quantitatively analyzed and interpreted interms of conformational dynamics, providing insight intoshort-lived states in terms of chemical shifts and bond vectororientations. Weinvestigate the robustnessof thisapproachby studying a conformational flip in crystalline ubiquitin.Conformational fluctuations between different statesexposeagivennuclearspininaproteintodifferent localenvironments, which are characterized by different bondgeometries. Asimplecase, exchangebetweentwostates, isshown in Figure 1a. As a result of conformational-exchangedynamics, agivenspinwill experienceafluctuationof itschemical shift (CS), as well as of its bond vector orientationsand, thus, dipolar coupling interactions with neighboring spinsandCSanisotropies (CSA). Insolution-state NMR,dipolarcoupling and CSAinteractions are averaged to zero byBrownianmovement (molecular tumbling). Consequently,only fluctuations of the isotropic CS are relevant whenconsidering conformational dynamics on the microsecond tomillisecondtimescale. R11RDexperimentsinsolutioncanthusonlypickupconformational dynamicsif theyinvolveachangeintheisotropic CS. Therelevant theoryis wellestablished, and the effects of exchange can be described bythe BlochMcConnell equations.[3]In MAS ssNMR, where stochastic molecular tumbling isabsent, anisotropic interactions (dipolar couplings and CSA)areperiodicallymodulatedbymagic-anglesamplespinning(MAS), and are averaged out over a sample rotation period(in thems range). This averaging leads to the line narrowingrequired for high-resolution studies. However, when consid-ering molecular dynamics and rf irradiation, as is the case inFigure 1. The effect of conformational exchange processes on15N R11relaxation rates, as revealed by numerical simulations. a) Schematicrepresentation of a two-spin NH system that is in dynamic exchangebetween a minor and a major conformation, which differ in the15Nchemical shift and the orientation of the NH bond vector. The15N CSAtensor is assumed to be collinear with the NH bond. b,c) Simulated15N R11 RD profiles obtained for this two-spin system, assumingrelative populations of pB=10%, pA=90%. Different scenarios wereassumed, regarding the15N isotropic CS (solid/dotted lines), and thechange of the bond angle orientation, q, occurring during exchange(red/black), as indicated. More simulations are shown in Figure S1.The inserts in (b,c) show enlargements of the low-rf field regime.Abstract: The function of proteins depends on their ability tosample a variety of states differing in structure and free energy.Decipheringhow thevariousthermallyaccessibleconforma-tions areconnected, andunderstandingtheir structures andrelativeenergies is crucial inrationalizingproteinfunction.Many biomolecular reactions take place within microsecondsto milliseconds, and this timescale is therefore of centralfunctional importance. Here we showthat R11relaxationdispersion experiments in magic-angle-spinning solid-stateNMR spectroscopy make it possible to investigate the thermo-dynamics andkinetics of suchexchange process, andgaininsight into structural features of short-lived states.[*] Dr. P. Ma, J. D. Haller, J. Zajakala, Dr. P. Macek, Dr. A. C. Sivertsen,Prof. Dr. D. Willbold, Dr. J. Boisbouvier, Dr. P. SchandaUniv. Grenoble Alpes, Institut de Biologie Structurale (IBS)CEA, DSV, IBS, 38027 Grenoble (France)andCNRS, IBS, 38027 Grenoble (France)E-mail: paul.schanda@ibs.frProf. Dr. D. WillboldInstitut fr Physikalische BiologieHeinrich-Heine-Universtt Dsseldorf (Germany)andICS-6: Structural BiochemistryForschungszentrum Jlich (Germany)[**] This work was financially supported by the French Research Agency(ANR-10-PDOC-011-01 ProtDynBNMR) and the European ResearchCouncil (ERC-Stg-2012-311318-ProtDyn2Function). This work usedthe platforms of the Grenoble Instruct centre (ISBG; UMS 3518CNRS-CEA-UJF-EMBL) with support fromFRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partner-ship for Structural Biology (PSB). We thank A. Krushelnitsky, T.Zinkevich, and R. Sounier for stimulating discussions and I. Ayalafor sample preparation.Supporting information for this article is available on the WWWunder http://dx.doi.org/10.1002/anie.201311275. 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co.KGaA. This is an open access article under the terms of the CreativeCommons Attribution Non-Commercial License, which permitsuse, distribution and reproduction in any medium, provided theoriginal work is properly cited and is not used for commercialpurposes.AngewandteChemie4313 Angew. Chem. Int. Ed. 2014, 53, 4312 43172014 The Authors. Published by Wiley-VCHVerlag GmbH & Co. KGaA, Weinheim www.angewandte.orgR11RDexperiments, thevarioustime-dependentprocesses(sample rotation, rf irradiation, and conformationalexchange) may interfere, leading to different decay processes.Consequently, the situation is more complex than in solution-stateNMRexperiments.[4]BeforeconsideringexperimentalimplementationsofMASssNMRR11RDexperiments, wethus investigate the properties of15NR11decay in anexchangingsystemundergoingMASbynumerical simula-tions.Figure 1b,c shows simulated15N R11 RD profiles for a1H15Nspinpairthat undergoesexchangebetweentwostates,populated to 90% and 10%, respectively, and which is subjectto MAS and a15N spin-lock rf field of variable amplitude. Weassumed different exchange scenarios that involve either onlya fluctuation of the15N spins isotropic CS, or a change of theNH bond orientation within the molecular frame of reference,or both. The results of these simulations can be summarizedas follows: 1) If exchange takes place between two states thatdiffer only in their isotropic CS, but not in bond orientations,R11RDprofilescanbedescribedbytheBlochMcConnellformalism(blackcurvesinFigure 1b,c), asinsolution-stateNMR experiments.[1a]2) If bond angle fluctuations areinvolved upon conformational exchange (thus reorientingthe dipolar coupling and CSA interactions), the situation getsmorecomplex. R11rateconstants inthis caseareoverallincreased, because the fluctuating anisotropic interactionsinduce relaxation.[5]Of particular note for the presentdiscussion is the fact that the increase of R11rates isparticularlypronouncedwhenthespin-lockfieldstrength(n1) approaches the sample rotation frequency (nMAS), that is,close to the so-called rotary resonance conditions.[6]Importantly, as shown in Figure 1 and Figure S1 andreportedpreviously,[4, 5]conformational exchange broadenstheserotaryresonancerecouplingconditions, andtheR11relaxation rate in the vicinity of the rotary resonanceconditions depends on the kinetics of the exchange, therelative populations of the involved states, and the jump anglebetweenthedifferentconformations. Takentogether, thesesimulationsdemonstratethatMASssNMRR11RDexperi-ments mayproviderichinformationabout conformationalexchange, and that they may report not only on chemical shiftfluctuations between the states (as is the case in solution-stateNMR measurements), but also on the bond angles by whichashort-livedexcitedstatediffers fromthepredominantground-state conformation.Exploiting this potential of MAS ssNMR R11 RD experi-ments toquantitatively analyze conformational exchange inproteins has so far been hampered by the fact that R11 decayrates in ssNMR not only contain a dynamics-related compo-nent, butalsohavesubstantialcontributionsfromcoherentdecaymechanisms(i.e. dipolardephasing).Dipolardephas-ing contributes to R11 decay particularly in multispin systems,such as proteins with numerous proton spins. Extracting thedynamics-related part of R11decay rates thus requiressuppressingthisdipolar-dephasingpart. Asshownrecently,athighMASfrequenciesofroughly40 kHzorhigher, andspin-lock fields above 15 kHz this dipolar dephasing issignificantly reduced and appears negligible even in theproton-rich environment of a protein;[7]however, the require-ment for highspin-lockfields eliminates thepossibilityofobservingR11RDinthewindowbelow510 kHzspin-lockfield, where RD profiles are particularly sensitive to isotropicCS fluctuations.We circumvent these limitations here by employing highdegreesof sampledeuteration, whichstronglyreducesthedipolar-dephasingcontributionto15NR11rates.[8]Inhighlydeuterated samples, that is, samples that are fully deuteratedat non-exchangeablesites, andinwhichtheexchangeable(amide)sitesare(partly)reprotonated, theprotondipolarcoupling network is strongly diluted, allowing for high-resolutionhighlysensitiveproton-detectedssNMRspectra,and long15N coherence life times.[8, 9]Figure 2a showsrepresentative examples of residue-wise R11RDprofilesobtained on a sample of deuterated microcrystalline ubiquitinthat has been reprotonated at exchangeable sites to 50%.15N R11 data have been measured at a MAS frequency of39.5 kHz, and spin-lock rf field strengths of 215 kHz, that is,farfromthen =1rotaryresonancecondition(whichisat39.5 kHz). The reported rate constants are on-resonance R11,thatis, theR1contributioninthetiltedrotatingframehasbeen eliminated (using standard formulae,[1a]see the Support-ing Information).Forthevastmajorityofresidues, forexample, residuesIle3, Leu15, Lys 33, and Gly47 in Figure 2a, we find that theRD profiles are flat over the entire range of rf field strengths.This finding confirms that coherent dephasing is indeedefficiently suppressed by deuteration and fast MAS; it furthersuggests that nonflat R11 RD profiles can safely be ascribed tomotional processes. Indeed, forafewresiduesweobservenonflat R11RDcurves, that is, increased relaxation rateconstants at low spin-lock rf fields, as expected for the BlochMcConnell regime of exchange. The residues with suchnonflat15NR11RDprofiles arelocatedinawell-definedregionofubiquitin, intheN-terminalpartofthehelixanda loop that is hydrogen-bonded to this helix (residues Ile23,Lys 27, Glu51, Asp52, Arg54, Thr 55; see Figure 3 andFigure S5). Conformational exchangeinthis regionof theprotein has been reported in numerous studies in solution,[10]and we have recently also reported conformational exchangein this part of the protein in microcrystals from CarrPurcellMeiboomGill (CPMG)RDandmultiple-quantumrelaxa-tion experiments.[11]In order to obtain quantitative insight into the exchangeprocess occurring in this part of the protein, we have fitted anexchange model to the R11 RD profiles of these residues usingthe BlochMcConnell formalism. The application of thisformalism is strictly speaking not correct in MAS ssNMR ifbondreorientationis involved(cf. Figure 1). However, itappears justifiedherebythefact that theRDcurves ofFigure 2 were collected in a range of rf fields well outside therotary-resonanceconditions; bondanglefluctuations haveonly very small impact on R11 RD in this regime, especiallywhenchangesinthebondvectororientationsaresmall(cf.Figure 1). That bondanglefluctuations areindeedrathersmall is justified a posteriori with independent measurements(see below).We fitted a two-state exchange model to the R11-derivedRDprofiles of residues23, 27, 51, 52, 54, and55(Figure 2 and Figure S8). Because of their close spatialAngewandteCommunications4314 www.angewandte.org2014 The Authors. Published by Wiley-VCHVerlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2014, 53, 4312 4317proximity, we assumed that these residues are involved in thesame exchange process. Consequently, the exchange rateconstant, kex=kAB+kBA and minor-state population, pB, wereassumedtobeidenticalforalltheseresidues, andonlythechemical-shift differences between the major and minorstates, Dd, which are sensitive to changes in the localenvironment, were assumed to be specific to individualresidues. Solid lines in Figure 2a show best-fit curves of thisfit. Thefit yields anexchangerateconstant kexof 8600 1700 s1and a minor-state population pBof 3.1 1.2%.Residue-wisechemical-shiftdifferencesDdareinthe rangeof 25 ppm (all values are reported in Table S1). In order toinvestigate the reliability of these results, which wereobtainedfromasingleR11RDmeasurement, weexploredtheinclusionof additional, independent datasets: a firstpossibility, oftenusedinsolution-stateNMR, wouldbethemeasurementofR11RDatadditional staticmagneticfieldstrengths (requiring, however, access to another spectrometerequipped with a fast-MAS probe). As an alternative, we usehereacombinedfit withCPMGRDdata, obtainedpre-viouslyundersimilarconditionsoffast MASanddeutera-tion.[11]CPMG RD is sensitive to exchange processes onmsms timescales, making a combined fit with R11 possible. Suchan analysis of the present R11 RD data with CPMG data, alsoobtained at a magnetic field strength of 14.1 T, yields values ofDd that are very similar to those obtained from the above fitof only R11 RD data (see Table S1). The obtained exchangerate kex is 2900 140 s1, and the population pB is 9.3 0.6%.Although these values slightly differ from the values obtainedfrom fitting only a single R11 RD data set (where kex=8600 1700 s1, pB=3.1 1.2%) it is noteworthy that the fit curvesof thecombinedR11/CPMGfit, shownas orangelines inFigure 2a, arealmostindistinguishablefromthefitsofR11data only, which shows that the present data are in excellentagreement with independent CPMG data. The differences ofthefittedparameterspointtothewell-knownfactthatitisFigure 3. Residues involved in the conformational exchange process inubiquitin. a) Residue-wise chemical-shift differences Dd (obtainedfrom data in Figure 2a) and b) jump angles q (obtained from Fig-ure 2b) are plotted onto the structure of ubiquitin crystals used in thisstudy (PDB 3ons). Amides 24 and 25 (black spheres) are invisible inNH correlation spectra, presumably due to exchange broadening.[11]c) Residue-wise differences of the NH orientations in the crystalstructure used here (type-II b-turn) and in a structure featuring a type-Ib-turn (PDB 1ubi). These angles were obtained by aligning the twostructures to all secondary structure elements and extracting thedirection of the respective NH bonds.Figure 2. Experimental15N RD data obtained on perdeuterated ubiquitin at 300 K, obtained from proton-detected experiments. Plotted is the on-resonance R11, that is, corrected for chemical-shift offset (see the Supporting Information). a) RD profiles obtained at 39.5 kHz MAS and low spin-lock rf field strengths of 215 kHz. Solid black lines show the result of a BlochMcConnell fit of a two-state exchange model using only R11-derived data, while orange lines are derived from a fit that includes CPMG data for residues 23, 27, and 55 at 600 MHz, as reported earlier (seeFigure S8). Straight dashed lines (constant R11 rate) in panels (a) and (b) show the relaxation rate constant obtained at 39.5 kHz MAS and 15 kHzspin-lock field strength, which is considered as free from exchange effects. b) RD profiles obtained at 20 kHz MAS on a fully deuterated, 20%reprotonated sample of ubiquitin. Solid lines show simulated R11 RD profiles assuming an exchange rate kex=2900 s1and population pB=9.3%.All available RD profiles, as well as experimental details are shown in the Supporting Information.AngewandteChemie4315 Angew. Chem. Int. Ed. 2014, 53, 4312 43172014 The Authors. Published by Wiley-VCHVerlag GmbH & Co. KGaA, Weinheim www.angewandte.orgdifficult to disentangle all fit parameters froma singlemeasurement (here, a single B0field strength).TheR11RDexperiment has advantages over thepre-viously proposed CPMG RD experiment:[11]In solids, even atfast MAS and high degrees of deuteration, measured R2 ratescontain a substantial amount of dipolar dephasing, in contrastto R11 (see Figure S10).[7]This has two important consequen-ces: 1) Due to the more rapid coherence decay, sensitivity inthe CPMG experiment[11]is significantly lower than in the R11experiment. 2) The remaining coherent contributions to theeffective R2 rates in a CPMG experiment are almost, but notentirely independent of the CPMG frequency.[11]Thus,observed variations of the effective R2 in the CPMG experi-ment maytosomeextent beartifactual. Wenotethat thetimescales probed by R11and CPMGRDexperimentsoverlap but are not exactly identical, such that the approachesshould be considered as complementary.Insolution, thesamepart of ubiquitinundergoescon-formational exchange, but theprocessismuchfasterthanobserved here. Even at a temperature 20 K lower than whatwas used here, the exchange occurs much faster than here (kex1250025000 s1).[10]At 298 K, asimilar temperaturetothat used here, the R11 RD profile in solution for example, forIle23 is flat (to within 0.5 s1),[10a]which is in stark contrast tothe pronounced dispersion of R11 seen in crystals in this study.This finding unequivocally establishes that the motionalprocess in microcrystals is slower than in solution. Intermo-lecular contacts are at the origin of this slowdown, acting asadditional energy barriers for motion in the crystal.[11]Having thus established that R11 RD experiments at highMAS frequencies can provide information about the thermo-dynamics andkinetics of exchange, as well as site-specificchemical-shift values of the minor states, akin to the situationin solution-state NMR experiments, we investigated whetheradditional structural information about the minor state maybeobtained. AsFigure 1shows,15NR11RDprofilesinthevicinity of the rotary resonance condition(n1nMAS) aresensitive to the angle by which the NH bond (i.e. the dipolarcouplingand15NCSA)isalteredupontheconformationaltransition. For technical reasons we refrained from perform-ing such measurements at high MAS frequencies, because therequired high spin-lock field strengths (n1nMAS) wouldchallenge the integrity of hardware and sample. Instead, weperformedexperimentsat lowerMASfrequency(20 kHz),where the rotary-resonance condition is met at lower rf field.Toensurethatcoherentcontributionstothe15NR11decayremain negligible even at slower MAS, we employed a higherdegreeof deuteration(20%reprotonationof amidesitesinstead of 50%); the larger volume of the sample rotor thatcanbeusedatlowerMASfrequencycompensatesfortheresulting sensitivity loss. Figure 2b shows representativeexamplesof15NR11RDprofiles obtainedatspin-lock fieldstrengthsof1219 kHz. Manyoftheresidues, suchasIle3,Leu15, Lys 33, and Gly47 in Figure 2b, show flat RD profilesover the entire range of sampled spin-lock field strengths, oronlyshowanincreasewhenthespin-lockfieldstrengthiswithin 23 kHz of the n =1 rotary resonance condition.Interestingly, theobtainedplateauvaluesaresimilartothevalues obtained at fast MAS, implying that coherent contri-butions to R11 are indeed efficiently suppressed, even at thelower MAS frequency. An increase of R11 when approachingthe rotary resonance condition (n1=nMAS) can be understoodfrompartial recouplingofdipolarcouplingandCSAinter-actions.[6]It could thus arise even in the absence of msconformational exchange. Strikingly, however, those residuesthathavethestrongestdependencyofR11onthespin-lockfield strength are the ones for which we have detectedconformational exchange also through the isotropic CSmechanism, namely residues 23, 27, 51, 52, 54, and 55(Figure 2). Based on the simulations in Figure 1, whichreveal that the angle of reorientation upon exchange impactsthe RDprofiles, we estimated the jump angles that canexplaintheRDprofiles of Figure 2b. Graycurves inFig-ure 2b show simulations that assume different angles.Extractedjumpanglesvarybetweentheresidues, andthelargest angular fluctuations are observed for residues Asp52and Arg54 (20308), while residues Ile23, Lys 27, Glu51, andThr 55 show smaller reorientational motion along theexchange process, with jump angles of 108 or below. ResidueGly53 has spectral overlap and no data are available.It is interestingtocomparethesedatawithstructuralmodelsof theexchangeprocess. AsdeducedfromvariousNMRdata,[10]mutations,[12]and inspections of differentcrystal structuresof ubiquitin, theproposedmechanismofthe exchange process involves an approximately 1408flip ofpeptide plane D52/G53, and a rearrangement of the hydrogenbonding between this loop and the helix comprising residues2333. Figure 3showsthat inthemicrocrystal usedinthisstudy the NH bond of residue 53 points towards the helix (aso-called type-II b-turn); in most other structures deposited inthe Protein Data Bank it points outward (type-Ib-turn, seeFigure S9). Wespeculatethattheconformational exchangeobserved in our sample may be a transition between these twotypes of b-turns. Totest this hypothesis, weextractedtheangles by which the NH orientations differ in representativeX-raystructures featuringtype-I andtype-II b-turns. Thelargest differences in the NHorientations are found forAsp52 and Arg54, which are neighboring the peptide plane ofGly53 that is flipped. Residues 23, 27, 51, and 55 show smallerdifferences intheir respectiveNHbondorientations (Fig-ure 3c). This is in good qualitative agreement with our data(Figure 2b), which also reveal the largest reorientationalmotion for Asp52 and Arg54, and significantly smaller jumpangles for the other residues involved in the exchange process.These data indicate that R11 RD data can provide structuralinsight into excited states, in addition to chemical-shift data.This feature, together with the increased capabilities ofrelatingchemical shiftstostructure, maybeofgreatvalueindeterminingthestructuresof short-livedconformations,including systems not suitable for atomic resolution studies bysolution-state NMRspectroscopy, such as very large orinsoluble proteins.Received: December 30, 2013Published online: March 18, 2014AngewandteCommunications4316 www.angewandte.org2014 The Authors. Published by Wiley-VCHVerlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2014, 53, 4312 4317.Keywords: protein dynamics relaxation dispersion solid-state NMR spectroscopy transient conformations ubiquitin[1] a) A. G. 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