Protein folding and unfolding studied at atomicresolution by fast two-dimensional NMR spectroscopyPaul Schanda*, Vincent Forge†, and Bernhard Brutscher*‡

*Institut de Biologie Structurale Jean-Pierre Ebel, 41 Rue Jules Horowitz, Commissariat a l’Energie Atomique (CEA), Centre National de la RechercheScientifique, Universite Joseph-Fourier, 38027 Grenoble Cedex 9, France; and †CEA, Direction des Sciences du Vivant-Institut de Recherches enTechnologies et Sciences pour le vivant, Laboratoire de Chimie et Biologie des Metaux (Unite Mixte de Recherche 5249), 17 Rue des Martyrs,38054 Grenoble Cedex 9, France

Edited by S. Walter Englander, University of Pennsylvania School of Medicine, Philadelphia, PA, and approved May 23, 2007 (received for reviewMarch 6, 2007)

Atom-resolved real-time studies of kinetic processes in proteinshave been hampered in the past by the lack of experimentaltechniques that yield sufficient temporal and atomic resolution.Here we present band-selective optimized flip-angle short tran-sient (SOFAST) real-time 2D NMR spectroscopy, a method thatallows simultaneous observation of reaction kinetics for a largenumber of nuclear sites along the polypeptide chain of a proteinwith an unprecedented time resolution of a few seconds. SOFASTreal-time 2D NMR spectroscopy combines fast NMR data acquisi-tion techniques with rapid sample mixing inside the NMR magnetto initiate the kinetic event. We demonstrate the use of SOFASTreal-time 2D NMR to monitor the conformational transition of�-lactalbumin from a molten globular to the native state for a largenumber of amide sites along the polypeptide chain. The kineticbehavior observed for the disappearance of the molten globuleand the appearance of the native state is monoexponential anduniform along the polypeptide chain. This observation confirmsprevious findings that a single transition state ensemble controlsfolding of �-lactalbumin from the molten globule to the nativestate. In a second application, the spontaneous unfolding of nativeubiquitin under nondenaturing conditions is characterized byamide hydrogen exchange rate constants measured at high pH byusing SOFAST real-time 2D NMR. Our data reveal that ubiquitinunfolds in a gradual manner with distinct unfolding regimes.

hydrogen/deuterium exchange � molecular kinetics ��-lactalbumin � ubiquitin

A detailed description of the structural changes occurringduring the folding and unfolding of proteins still remains a

challenging objective in biophysics. The understanding of thefundamental mechanisms of the folding process will also shedlight on the factors leading to protein misfolding responsible forneurodegenerative diseases such as Alzheimer’s and Parkinson’sdiseases (1). The ideal method to study protein folding/unfoldingprovides structural information at atomic resolution and issensitive to changes occurring on time scales ranging frommicroseconds to minutes, a task that no single technique is ableto fulfill. NMR spectroscopy is especially well adapted to obtaindetailed atomistic information about the mechanisms, kinetics,and energetics of the folding/unfolding process for virtuallyevery nuclear site in the protein. NMR methods are sensitive tomolecular dynamics occurring over a wide range of time scales(Fig. 1a). Although steady-state NMR methods are well suitedto characterize equilibrium molecular dynamics occurring on asubsecond time scale (2, 3), unidirectional processes are beststudied by real-time NMR (4, 5), where a series of NMR spectrais recorded during the reaction (Fig. 1b). Two difficulties have sofar hampered the widespread application of real-time NMR tothe study of protein folding. The first problem is the low intrinsicsensitivity of NMR at ambient temperature, a consequence ofthe small transition energies involved, compared with otherspectroscopic techniques. Recent progress in NMR technology(magnetic field strength, cryogenic probes, and electronics, for

example), however, has greatly enhanced the sensitivity of NMRexperiments, allowing acquisition of 1D spectra of proteins atsubmillimolar concentration at �1–10 s�1 rates. The secondlimitation concerns the time requirements to record higher-dimensional (�2D) NMR spectra indispensable to resolve thelarge number of nuclear resonances in a protein. Whereas a 1DNMR spectrum can be obtained in �1 s within a single scan byphysical detection of the NMR time domain signal induced in areceiver coil, the recording of a 2D NMR data set requiresnumerous repetitions (typically �100) of the basic pulse se-quence to sample the evolution of the nuclear spins in theadditional time dimension (6). In the past, this requirement haslimited the application of real-time 2D NMR methods to rela-tively slow kinetic processes with characteristic time constants ofminutes to hours. A major challenge for real-time NMR istherefore to achieve reduced acquisition times that will allowextension of real-time NMR methods to the missing NMR timewindow of seconds to minutes (Fig. 1a). Doing so will make thetechnique applicable to the study of a larger variety of molecularkinetics. Here we introduce band-selective optimized flip-angleshort transient (SOFAST) real-time 2D NMR, a method thatallows site-resolved studies of kinetic processes in proteins witha time resolution down to a few seconds. We demonstrate thepotential of this method for the study of protein folding andunfolding reactions.

Results and DiscussionSOFAST Real-Time 2D NMR Spectroscopy. To overcome the inherenttime limitation of 2D NMR spectroscopy, two conceptually differ-ent approaches can be envisaged. First, the number of scansrequired to sample the multidimensional time space may be re-duced by using spectral aliasing, nonlinear data sampling tech-niques, spatial frequency encoding, or Hadamard-typefrequency-space spectroscopy (7, 8). Second, accelerating the re-covery of spin polarization between scans by means of optimizedpulse sequences (9–11) or the addition of relaxation agents to thesolution (12, 13) allows higher repetition rates of the pulse se-quence. Here, we propose a fluid turbulence-adapted (FTA) ver-sion of 1H-15N 2D SOFAST-heteronuclear multiple quantum co-herence (HMQC) (10, 14) to follow a kinetic process for individualamide sites in a protein [see supporting information (SI) Fig. 4].

Author contributions: V.F. and B.B. designed research; P.S. performed research; V.F. con-tributed new reagents/analytic tools; P.S. analyzed data; and P.S. and B.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Abbreviations: FTA, fluid turbulence-adapted; HMQC, heteronuclear multiple quantum co-herence; MG, molten globule; SOFAST, band-selective optimized flip-angle short transient.

‡To whom correspondence should be addressed at: Laboratoire de RMN, Institut deBiologie Structurale Jean-Pierre Ebel, 41 Rue Jules Horowitz, 38027 Grenoble Cedex 1,France. E-mail: bernhard.brutscher@ibs.fr.

This article contains supporting information online at www.pnas.org/cgi/content/full/0702069104/DC1.

© 2007 by The National Academy of Sciences of the USA

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Longitudinal relaxation optimization in FTA-SOFAST-HMQCallows the use of scan times of �100 ms without a significantreduction in sensitivity (signal to noise per unit time), resulting inexperimental times on the order of 5 s per 2D 1H-15N correlationspectrum, which shows one correlation peak for each nonprolineresidue of the protein. The advantage of the SOFAST approachwith respect to other fast 2D NMR techniques, such as Hadamardor spatial frequency encoding, is the higher sensitivity provided bythis technique for the short acquisition times of a few seconds.High-quality 2D spectra are obtained by the FTA-SOFAST-HMQC experiment on a modern high-field NMR spectrometer forprotein samples in the submillimolar concentration range, as illus-trated in Fig. 1b for a 0.2 mM sample of ubiquitin recorded in a 4-sacquisition time. If faster sampling of the kinetic time domain isdesired, this minimal experimental time required for FTA-SOFAST-HMQC can be reduced further to �1 s by using com-puter-optimized spectral aliasing in the 15N dimension (15).

To benefit fully from the increased time resolution providedby the FTA-SOFAST-HMQC experiment for real-time kineticstudies, a device is required that allows initiation of the kineticevent inside the magnet. The triggering of kinetic reactions canbe achieved either by a sudden change of the protein state itself,

e.g., by a photo-induced excitation or cleavage of chemical bonds(16, 17), or by a change of environment, such as the solventcomposition, temperature, or pH (4, 18). Here, we opted for aninitiation of the reaction by an abrupt change in the solventconditions achieved by rapid mixing of two solutions inside theNMR magnet (5, 19). The fast mixing device used for this work(SI Fig. 5a) allows complete mixing in �300 ms as demonstratedby dye injection experiments into water solutions (SI Fig. 5b). Adrawback of the fast injection is the presence of turbulences ofthe liquid over times significantly longer than the mixing time.Many modern NMR experiments make use of pairs of pulsed-field gradients for spectral artifact suppression. In the presenceof bulk liquid motion, incomplete refocusing of spin coherencecaused by translational diffusion during the time delay betweenthe two pulsed-field gradients yields uniformly reduced NMRsignal intensities. The FTA-SOFAST-HMQC experiment hasbeen optimized for minimal signal loss in the presence of bulkliquid motion, yielding dead times after the injection of �2 s (SIFig. 6). We have chosen two conceptually different applicationsto demonstrate the potential of SOFAST real-time 2D NMR toprovide accurate measures of kinetic rate constants for a largenumber of amide sites. The first example concerns the folding of�-lactalbumin from a molten globular (MG) to the native state.In the second application, the unfolding kinetics experienced byindividual residues of ubiquitin under native equilibrium condi-tions are investigated.

Conformational Transition Kinetics of �-Lactalbumin from an MG tothe Native State Studied in Real Time. The protein �-lactalbuminserves as a model system to investigate the structural transitionfrom a partially folded to the native state by using SOFASTreal-time 2D NMR. This protein has been used extensively as amodel system for protein-folding studies. The structure of native�-lactalbumin (14 kDa) comprises two domains, one containingfour �-helices and a short 310 helix, and the other containing athree-stranded �-sheet and another 310 helix. Here, we focusedon the Ca-free (apo) form of �-lactalbumin. Under destabilizingconditions such as low pH, addition of cosolvents, high temper-ature, or combinations thereof, apo-�-lactalbumin exists in anMG state. The MG state is characterized by the absence oflong-lived tertiary structure, but it still contains a high degree ofsecondary structure (20, 21), with a radius of gyration only�10% larger than the native state (22, 23). The MG state of�-lactalbumin has recently attracted considerable interest whenit was realized that it may act as an important component causingapoptosis of tumor cells (24).

Pioneering work on the use of time-resolved NMR methodsfor monitoring protein refolding was performed on �-lactalbu-min (5, 23, 25–27). One-dimensional 1H NMR spectra recordedduring the refolding reaction of apo-�-lactalbumin providedevidence that the MG state, accumulated during the early stagesof the folding reaction, shows similar spectral characteristics(poorly dispersed and broad resonances) and therefore similardynamic properties of the conformational ensemble to thoseobserved for the MG state stabilized at acidic pH (25). Addi-tional diffusion-edited and NOE transfer NMR measurementsallowed monitoring the compactness of the protein and theestablishing of native tertiary contacts, respectively (23, 27). Thefolding kinetics of apo-�-lactalbumin have also been studiedpreviously at a residue level by line shape analysis of individualcross-peaks detected in a single 2D NMR spectrum recordedduring the refolding event (26). This technique, however, islimited to relatively slow kinetics (several minutes) and is proneto large experimental uncertainties. In the study of Balbach et al.(26), folding rates for a total of 25 backbone amides could bequantified with an estimated experimental error of �25%.Because of the small number and the high uncertainties of themeasured rate constants, these data did not allow definite

Fig. 1. SOFAST real-time 2D NMR closes the seconds time gap for NMRstudies of protein dynamics. (a) Time scales of biophysical processes such asprotein folding or amide hydrogen exchange, and NMR methods available tostudy the kinetics of these processes at atomic resolution. (b) Principle ofSOFAST real-time 2D NMR. A series of 2D FTA-SOFAST-HMQC spectra isrecorded after initiating a kinetic change in the protein state. Each cross-peakreports on the local structure and dynamics at the site of a single amide group.The bottom spectrum has been recorded in an experimental time of 4 s on a0.2 mM 15N-labeled sample of ubiquitin at a magnetic field strength of 18.8 Tby using a cryogenically cooled probe.

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exclusion of the presence of differential folding kinetics alongthe polypeptide chain. To illustrate the improvements providedby SOFAST real-time 2D NMR for the measurement of residue-specific folding rates, we monitored the same refolding reactionas Balbach et al. from the MG state, formed at pH 2, to the nativestate. Refolding was initiated by a sudden pH jump (from 2 to8), and the reaction was monitored by a series of FTA-SOFAST-HMQC spectra recorded at a 0.1 s�1 rate. Spectra acquiredbefore injection, immediately after, and 2 min after injection areshown in Fig. 2a. The center spectrum in Fig. 2a provides asnapshot of the transient MG state under native conditions. Thebroad and weak signals in this spectrum are indicative oflarge-scale dynamics occurring on a micro- to millisecond timescale. These data are in agreement with this transient MG state

being a compact, highly dynamic ensemble of conformationalstates. All NMR signals observed during the folding process canbe assigned either to the MG or the native state. No additionalpeaks indicative of a significantly populated folding intermediatewere detected, in agreement with previous findings (23, 26, 27).The refolding kinetics could be quantified for 92 of 121 backboneamide sites in the protein from intensity measurements of wellresolved cross-peaks in the 1H-15N correlation spectra (Fig. 2b).In addition, and in contrast to the studies by Balbach et al. (23,26, 27), the intensity decay of five cross-peaks characteristic forthe MG state could be quantified. Under the experimentalconditions chosen (15°C, pH 8, Ca2�-free), the NMR intensitydecay and buildup curves can be fitted to monoexponentialfunctions. Fitting the data to more complex, e.g., biexponentialor stretched exponential functions, does not yield statisticallysignificant improvements. The folding time constants measuredfor individual amide sites of the native protein and the MG stateare identical (�f � 109 � 5 s) within the experimental uncer-tainty. The obtained folding kinetics are consistent with resultsfrom fluorescence measurements (SI Fig. 7), yielding a charac-teristic time constant of 110 � 10 s for the establishment ofnative tertiary structure. The good agreement with the fluores-cence measurements demonstrates the accuracy obtained bySOFAST real-time 2D NMR experiments.

The MG state of apo-�-lactalbumin explores a large confor-mational space, possibly including the presence of native andnonnative secondary structures (20). Therefore, the transitionfrom this MG state to the native state requires a large decreasein conformational entropy, known as entropy bottleneck effect(28, 29), which is the main reason for the significantly slowerfolding kinetics observed for the apo form compared with theholo form of �-lactalbumin (30). The high precision of rateconstants (error of �5–10%) obtained in our work for a largenumber of individual amide sites allows us to draw someconclusions on the energy landscape and folding pathways. Thefinding of equal kinetic rates for the buildup of the native stateand the disappearance of the MG state indicates a transitionbetween only two states, the MG ensemble representing a largenumber of conformational substates interconverting on themicro- to millisecond time scale, and the native state. In otherwords, the establishment of the native tertiary structure is notaccompanied by a change in the structural and dynamic prop-erties of the MG ensemble as folding proceeds. These observa-tions are in agreement with the assumption of a smooth energylandscape where the folding rate is controlled by a singletransition state ensemble originating from the conformationalentropy bottleneck effect. The existence of a unique transitionstate is in agreement with conclusions drawn from other bio-physical folding experiments (30).

Ubiquitin Unfolding Kinetics Under Equilibrium Conditions Revealedby EX1 Amide Hydrogen Exchange Measurements. To illustrate fur-ther the potential of SOFAST real-time 2D NMR to detectdifferential kinetic behavior along the polypeptide chain, we haveinvestigated the unfolding kinetics of human ubiquitin under non-denaturing equilibrium conditions by amide hydrogen/deuterium(H/D) exchange methods. H/D exchange is a powerful tool for thestudy of protein structure, folding, unfolding, and binding (31, 32).The exchange between amide and solvent hydrogens requires thatthe amide group is in an exchange-competent, solvent-accessibleconformation, which can be achieved by local, subglobal, or globalprotein unfolding fluctuations (‘‘opening reactions’’) that tran-siently break the exchange-protecting structures, e.g., hydrogenbonds. The measurement of amide H/D exchange rates thusprovides residue-specific information on the otherwise invisiblemanifold of partially or globally unfolded excited conformationalstates populated at a very low level. Despite their low populationunder native conditions, such high-energy states may be crucial for

Fig. 2. Folding of �-lactalbumin studied by SOFAST real-time 2D NMR. (a)FTA-SOFAST-HMQC spectra of bovine �-lactalbumin at pH 2.0 (Left), immedi-ately after a sudden pH jump to pH 8.0 that triggers folding (Center), and 120 safter injection (Right). Each spectrum shows the sum of two acquisitions of10.9-s duration. Peaks corresponding to the MG state that disappear duringfolding are annotated. (b) Refolding kinetics of bovine �-lactalbumin from theMG state to the native state. The measured peak intensities are plotted as afunction of the folding time. Shown are three residues situated in loop (K17),�-sheet (Q44), and �-helical regions (V91) that are indicated on the structure(Protein Data Bank ID code 1F6R). In addition, the signal decay observed for apeak assigned to the MG state is shown. Solid lines represent best fits to athree-parameter exponential function. A histogram shows the measuredfolding time constants for 92 residues in the native state as well as the 5 ratesmeasured for the disappearance of the MG state.

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protein function (33, 34), or they may represent intermediate stateson the protein folding pathway (35). Especially interesting are H/Dexchange measurements in the so-called EX1 regime, where themeasured exchange rate constants directly reflect the kinetics of theunfolding reaction at individual amide sites (32). EX1 conditionsare generally reached at high pH. However, for most proteins, H/Dexchange at high pH is too fast to measure by conventional 2Dreal-time NMR. Therefore, previous EX1 studies had to focus ona small number of well protected amide sites. It has been shown thatthe EX1 exchange rates measured for a few slowly exchangingamide sites in ubiquitin correspond to the rate of global unfoldingobserved with other biophysical methods (36). The SOFASTreal-time 2D NMR method, presented here, allows extension of theH/D exchange measurements under EX1 conditions to a signifi-cantly larger number of amide sites, yielding a more comprehensivepicture of the potentially heterogeneous unfolding processes alongthe polypeptide chain. Representative exchange curves measuredfor the 76-residue protein ubiquitin at pH 11.95 and 25°C are shownin Fig. 3. For all amides, even in the most protected parts of theprotein, exchange time constants �ex �200 s were observed. Thehigh quality of the data allowed accurate quantification of exchangekinetics down to �ex �5 s, with a detection limit of �ex �1 s. This timeresolution proved to be sufficient to measure exchange rates foramides in secondary structural elements, whereas H/D exchange inloop regions was generally completed during the dead time of theexperiment. In the top drawing of Fig. 3, the measured exchangerates are color-coded on the ribbon structure of ubiquitin. Theslowest H/D exchange is observed for residues in strands I and II ofthe �-sheet (residues 3–5 and 15) and the central part of the �-helix(residue 27), whereas amide hydrogens located in other parts of themolecule, comprising strands III–V, exchange much faster. Inter-estingly, a gradual increase in the kinetics of opening fluctuationsacross the �-sheet is observed, indicating noncooperative unfoldingevents in this �-sheet, reminiscent of a hydrophobic zipper foldingmechanism (37).

Over the past 20 years, ubiquitin has been used as a modelsystem for the study of protein stability and folding with a varietyof biophysical techniques, denaturation methods, protein engi-neering studies, and computational approaches (38). Therefore,it is interesting to compare the results presented here withprevious findings on the thermodynamic stability, folding, andunfolding kinetics of ubiquitin under different experimentalconditions. H/D exchange rates measured under EX2 conditions(39, 40) provide a measure of the thermodynamic equilibriumconstant K � kopen/kclose, rather than a kinetic rate constant asobtained under EX1 conditions. Interestingly, the residue-specific equilibrium constants also show differential stabilityamong the secondary structural elements of ubiquitin, withnotably the first two �-strands and the �-helix being the moststable parts. The kinetic information obtained from H/D ex-change measurements under EX1 conditions complements theseformer results, indicating that the differential thermodynamicstability observed for ubiquitin is mainly determined by theunfolding kinetics (opening rates). Differential noncooperativeunfolding has also been observed by NMR studies of ubiquitinunder denaturing conditions. The populations of nonnativeconformational states can be enhanced by changing the temper-ature, pressure, or pH, or by adding alcohol cosolvents. Acold-denaturation study by Wand et al. (41, 42) shows for reversemicelle-encapsulated ubiquitin that the mixed �-sheet is pro-gressively destabilized from the C-terminal side in the temper-ature range from �20° to �30°C. In an NMR study by Cordierand Grzesiek (43), the change in hydrogen bond strength withincreasing temperature was measured. The N terminus of�-strand V was found to be the least thermally stable, whereasstrands I and II remain stable even at elevated temperatures.Spectral changes observed upon high-pressure denaturation atambient temperature indicate the presence of a partially un-

folded intermediate in which the C-terminal part (residues70–76) as well as residues 33–42 and residue 8 are denatured(44). The higher stability of the secondary structural elements inthe N-terminal part of ubiquitin (�I-�II strand and �-helix) is alsoevidenced by the observation of a partially structured state (Astate) at low pH in a 60%/40% methanol/water mixture (45). Inthe A state, the N-terminal part of ubiquitin retains a native-likesecondary structure, whereas the C-terminal part undergoes aconformational transition form the native state to an ensembleof conformational states with high helical propensity (46).Finally, a recent time-resolved infrared (IR) study (47) revealeda complex thermal unfolding behavior of ubiquitin spanning awide range of time scales, indicating a gradual, rather than acooperative, unfolding process. All experimental data from both

Fig. 3. H/D exchange data obtained for human ubiquitin at pH 11.95 by usingSOFAST real-time 2D NMR. (Upper) The measured exchange rates are color-coded on the ubiquitin structure. (Lower) Examples of exchange curves cor-responding to different exchange regimes together with the fitted exchangetime constants. For residues color-coded in white, no signal decay was ob-served because the cross-peak intensity has decreased to its plateau valueduring the dead time of the experiment.

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equilibrium and transient unfolding studies support the pictureof noncooperative unfolding events taking place in the nativeenvironment. The C-terminal part, comprising �-strands III–V,is significantly less stable, with unfolding rates that are up to 2orders of magnitude higher than those observed for �-strands Iand II, and the �-helix in the N-terminal part. The observedconformational dynamics under native conditions and the pres-ence of partially unfolded conformational states may be ofimportance for the biological function of ubiquitin because it hasbeen shown that incorporation of a disulfide bridge betweenresidues 4 and 66, stabilizing the connection between strands Iand V of the �-sheet, leads to a 70–80% decrease of activity insignaling proteolysis (48).

SOFAST real-time 2D NMR closes the gap (seconds tominutes) on the kinetic time scale (see Fig. 1), which makespossible H/D exchange measurements for a large number ofamide sites at any desired pH by 2D NMR methods. Thecombined interpretation of H/D exchange rates measured underEX1 as well as the more common EX2 conditions presents anattractive tool to access residue-specific kinetic folding andunfolding rates of proteins under native (physiological) condi-tions. In concert with other NMR data, providing the sameatomic resolution, e.g., spin relaxation measurements or NMRspectra recorded under denaturing conditions, H/D exchangemeasurements will help us to gain a deeper insight into thenature of partially unfolded conformational states present undernative conditions and the rates of interconversion between thoseexcited states and the native state.

In summary, we have demonstrated that SOFAST real-time2D NMR allows the accurate measurement of kinetic rateconstants up to �1 s�1 simultaneously for a large number ofamide sites in a protein. The time frame ranging from a fewseconds to roughly a minute used to be inaccessible to conven-tional 2D NMR methods. We have illustrated the potential ofSOFAST real-time 2D NMR to provide accurate residue-specific kinetic information on protein folding and unfoldingevents either by direct real-time studies of folding/unfolding orindirectly by real-time measurements of H/D exchange kinetics.The kinetic rates measured for the refolding of �-lactalbumin areuniform throughout the protein, indicating a smooth energylandscape for the transition from the MG to the native state. Onthe contrary, the kinetic data collected for the protein ubiquitinindicate a highly noncooperative unfolding behavior undernative conditions, in agreement with previous results from NMRand IR spectroscopy. The methodology presented here can beeasily applied to protein systems with molecular masses rangingbetween �5 and �20 kDa where deuteration is not a prereq-uisite for obtaining well resolved 1H-15N correlation spectra.Here, we have focused on the application of SOFAST real-time2D NMR to the study of protein folding and unfolding, but themethod also offers opportunities for residue-specific kineticmeasurements of other unidirectional events occurring on a timescale of seconds, such as binding, enzymatic reactions, andchemical exchange. Combined with recent advances towardNMR studies of proteins inside intact living cells (49, 50),SOFAST real-time 2D NMR may also become a powerfulexperimental tool for in situ time- and site-resolved observationof kinetic events, such as protein folding and posttranslationalmodifications in a cellular environment.

Materials and MethodsSOFAST Real-Time 2D NMR. All NMR experiments were performedon an INOVA spectrometer (Varian, Palo Alto, CA) operating at800-MHz 1H frequency and equipped with a cryogenic probe. Tokeep the dead time as short as possible, the acquisition of the firstspectrum was started after the dead time of the mixing required tostabilize the fluid turbulence, which was �2 s (see SI Fig. 6). Toachieve high time resolution while retaining good spectral quality

without the need of advanced spectral processing, we used alternatephase cycling in subsequent spectra as follows. Each experiment wasperformed with only one scan per increment in t1, ensuring highestrepetition rates of experiments, and the sign of the 15N excitationpulse phase and receiver phase in FTA-SOFAST-HMQC wasalternated between subsequent experiments. Adding two succeed-ing spectra (1 and 2, 2 and 3, 3 and 4, . . . ) along the kinetic timedimension removes spectral artifacts such as axial peaks or t1 noiseat the water frequency and improves the base line, allowing a moreaccurate and precise measurement of spectral parameters, such aspeak positions and intensities. This procedure does not change thekinetic information contained in the data and the time resolutionas illustrated in SI Fig. 8. The only price to pay is an effective deadtime that is longer by half the duration of one basic 2D experiment.All data were extracted as peak intensities and fit to a three-parameter exponential function. Uncertainties in the obtained fitparameters were estimated from Monte Carlo simulations by using100 data sets generated by varying the intensity value of each datapoint within three times the spectral noise.

Real-Time Folding of Bovine �-Lactalbumin. Nitrogen-15 labeledbovine �-lactalbumin (M90V) was expressed and purified as de-scribed previously (51). The MG state was prepared by dissolving8.8 mg of protein in 350 �l of H2O/D2O (10:1) and adjusting the pHto 2.0 with 1 M HCl solution. Fifty microliters of a refolding buffersolution [H2O/D2O (10:1)] containing 800 mM Tris and 80 mMEDTA (pH 9.1) was loaded in the injection device, and refoldingwas initiated inside the spectrometer by rapid injection into theprotein solution. The folding reaction was followed by a series ofFTA-SOFAST-HMQC experiments of 10.9-s duration with a scantime of 130 ms and 40 complex data points in t1 (80 scans per 2Dspectrum). The final pH measured after the folding reaction was8.0, and the final protein concentration was 1.55 mM. NMRassignments were taken from ref. 27. The reproducibility of thereal-time folding data has been evaluated by repeating the exper-iment twice. The difference in the rate constants obtained from thetwo measurement series is smaller than the experimental errorestimated from Monte Carlo simulations of a single data set.

Amide Hydrogen Exchange Measurements in Ubiquitin. For theSOFAST real-time 2D NMR amide hydrogen exchange mea-surements, 2.5 mg of human ubiquitin was dissolved in 50 �l ofH2O buffer containing 50 mM glycine and loaded into theinjection device. H/D exchange was initiated by injection of theprotein solution into 350 �l of D2O buffer, resulting in a finalprotein concentration of 0.7 mM. The resulting pH, corrected forthe electrode isotope effect, was 11.95. The decay of peakintensities caused by exchange was followed by recording a seriesof FTA-SOFAST-HMQC spectra of 6.7-s duration with a scantime of 112 ms, and recording 30 complex data points (60 scans).NMR assignments at pH 11.95 were obtained from publisheddata at pH 6.6 (52), and a series of 1H-15N SOFAST-HMQCspectra was recorded in the pH range 7–12.

SOFAST NMR makes use of longitudinal amide 1H relaxationenhancement to increase the experimental sensitivity for highrepetition rates of the experiment (10, 14). Shorter spin-latticerelaxation times are achieved by using amide-proton selectiveradiofrequency pulses that leave the polarization of other 1Hspins, e.g., aliphatic and water 1H, unperturbed. The energy putinto the system is then efficiently dissipated within the dipolar-coupled 1H spin network by spin diffusion (NOE) effects. In thecourse of H/D exchange monitored by real-time 2D NMR, amidehydrogens are progressively exchanged for deuterons. This ex-change alters the 1H spin coupling network in the protein fromone 2D experiment to the other, which influences the relaxationproperties of the amide 1H spins and raises the question aboutthe accuracy of the measured exchange rates by using SOFASTreal-time 2D NMR. We have addressed this issue by both

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experiment and computer simulations. The results, shown in SIFig. 9, indicate that the systematic error introduced by thechanging 1H spin network remains small compared with thestatistical uncertainty and can be safely neglected.

We thank Dr. Keith Brew (University of Miami) and Dr. StephanGrzesiek (Biozentrum Basel) for providing the expression vectors of

bovine �-lactalbumin and ubiquitin, and Isabel Ayala and Cecile Giustini(Institut de Biologie Structurale Grenoble) for help with the preparationof the isotope-labeled protein samples. This work was supported bythe Commissariat a l’Energie Atomique, the Centre National de laRecherche Scientifique, the French Research Agency Agence Nationalde la Recherche (ANR) Grant ANR-05-JCJC-0077, and EuropeanCommission (EU-NMR) Grant RII3-026145. P.S. acknowledges supportfrom the French ministry of Education, Research, and Technology.

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11262 � www.pnas.org�cgi�doi�10.1073�pnas.0702069104 Schanda et al.

Protein folding and unfolding studied at atomicresolution by fast two-dimensional NMRspectroscopy

Schanda and Forge. 10.1073/pnas.0702069104.

Supporting InformationFiles in this Data Supplement:SI Figure 4SI Figure 5SI Figure 6SI Figure 7SI Figure 8SI Figure 9

SI Figure 4

Fig. 4. Fluid turbulence-adapted band-selective optimized flip-angle short transient heteronuclear multiple quantumcoherence (FTA-SOFAST-HMQC) experiment used for SOFAST real-time 2D NMR measurements. The small angle(a< 90°) 1H pulse (filled symbol) is applied with a PC9 shape (1), whereas for the 1H 180° pulses (open symbol) aniSNOB-5 shape (2) is used. The a angle is typically set between 40° and 60° depending on the scan time chosen. Thetransfer delays D are set to 1/(2JHN), and the small delay d takes into account spin evolution during the PC9 pulse andis adjusted to half of the PC9 pulse length. For 15N chemical shift editing in t1, the basic sequence is repeated n timesusing short interscan delays (between 1 and 50 ms) resulting in scan times of ~100 ms. Heteronuclear decoupling isperformed by using WURST-40 (3). The pulsed-field gradients (PFGZ) are applied for 100 ms with a strength oftypically 30 G/cm (1 G = 0.1 mT). In the original SOFAST-HMQC pulse sequence (4, 5), the delay between thegradients encloses the indirect evolution time t1, with typical maximum values of 20-30 ms. Therefore, turbulence ofthe solution, which stems from rapid injection, leads to incomplete refocusing and thus to loss of magnetization, whichmeans that the signal in a series of experiments builds up only slowly after injection (see SI Fig. 6), a fact that clearlycompromises spectral quality and interpretation of kinetic data. In contrast, in the FTA-SOFAST-HMQC sequenceshown here, the pulsed-field gradients are moved to the end of the pulse sequence, and an additional 1H refocusingpulse is added. While retaining excellent water suppression and high sensitivity, this modification reduces the timedelay between gradients to 1-2 ms. The pulse sequence code in Varian language is available from the authors onrequest.

SI Figure 5

Fig. 5. Practical implementation of rapid mixing inside the NMR spectrometer. (a) Sketch of the fast injection deviceused for SOFAST real-time 2D NMR applications. (b) Photographs of the NMR sample tube taken after fast injectionof 50 ml of blue dye into 350 ml of water with a rapid injection device. Homogeneous mixing is completed within »100ms.

SI Figure 6

Fig. 6. Buildup of signal intensity after rapid sample injection measured by a gradient-echo NMR pulse sequence withdifferent dephasing times. Fifty microliters of a 2 M glycine solution (in D2O) was injected in 350 ml of D2O, and thesignal intensity of the glycine Ha peak was followed as a function of time by using a 1D gradient spin-echo pulsesequence (90° pulse-PFGz-t/2-180° pulse-t/2-PFGz-acquisition). This sequence is sensitive to translational diffusioneffects and thus to the fluid turbulences created by the fast injection. It mimics the situation found in more complexNMR experiments such as SOFAST-HMQC. Results are shown for dephasing times of t = 20 ms (filled squares),corresponding to the situation in standard SOFAST-HMQC, and t = 1 ms (open circles), corresponding to the situationin FTA-SOFAST-HMQC. Stable conditions are reached in less than ~2 s with the short dephasing time, whereas ~15 selapse until equilibrium is reached with the longer delay, emphasizing the interest of FTA-SOFAST-HMQC when usinga rapid injection device.

SI Figure 7

Fig. 7. Comparison of folding kinetics of a-lactalbumin measured by fluorescence (black squares) and SOFASTreal-time 2D NMR spectroscopy (red circles). The protocol used for the fluorescence measurements (proteinconcentration, pH jump, solvent) is identical to that used for the time-resolved NMR experiments, except for theinitiation of the folding reaction. For the fluorescence measurements, manual mixing yielded a dead time of ≈1 min.The excitation wavelength is 295 nm (1-nm bandwidth) and the fluorescence change is monitored at 340 nm (3-nmbandwidth). Nonoptimal sample volume and the resulting small detection window, as well as autoquenching effectscaused by the high sample concentration, explain the relatively low signal-to-noise ratio obtained by the fluorescenceexperiment. A high protein concentration was chosen to mimic the NMR conditions, resulting in a small volume for theavailable amount of 2 mg of 15N-labeled protein. The same recombinant 15N-labeled a-lactalbumin has been used forNMR and fluorescence experiments because the recombinant protein (expressed in Escherichia coli) is less stable thanthe natural protein, and this has an effect on the refolding kinetics (6). The experimental data were fitted tomonoexponential decay shown as red and black lines for NMR and fluorescence data, respectively.

SI Figure 8

Fig. 8. Additional spectral processing used in SOFAST real-time 2D NMR. (a) Coaddition of subsequent 2D spectrausing alternate phase cycling. (Left and Center) First two FTA-SOFAST-HMQC spectra recorded at time 0 and Dt afterinjection of ubiquitin into a H2O solution. The phases of the 15N excitation pulse and the receiver have been alternatedfor subsequent experiments. As a result of this phase cycling, artifacts have opposite sign in the two spectra, whereasthe signals of interest are of the same sign. Addition of the two spectra thus removes the artifacts (Right). If the seriesof recorded spectra is replaced by the sum of successive spectra, e.g. (1 + 2), (2 + 3), (3 + 4), ..., this results in cleanerspectra without affecting the time resolution of the kinetic data. (b) Effect of this coaddition approach on the extractedrate constants for monoexponential intensity changes. The black solid line represents the exponential change in peakintensity that is sampled at a rate determined by the acquisition time of one spectrum, as indicated by black circles.Coadding two successive spectra corresponds to replacing two data points by their linear average and assigning this

summed value to the central time point in between the experimentally measured data points. These data points (whichfor reasons of clarity were scaled in the figure) are indicated as green circles, and the linear approximation connectingthe experimental data points is indicated by black straight lines. The monoexponential rate determined by thesecoadded data points corresponds exactly to the actual rate, which is explained by the fact that these data points areshifted by a constant offset independent of time.

SI Figure 9

Fig. 9. Evaluation of the accuracy of measured H/D exchange time constants tex using SOFAST real-time 2D NMR. (a)tex values measured by using the new FTA-SOFAST-HMQC sequence of SI Fig. 4 are compared with results obtainedby using standard HSQC-based 2D NMR methods. H/D exchange was monitored for 24 h for ubiquitin at 25°C at twodifferent pH values: 7.3 (black circles) and 9.8 (red circles). HSQC (experiment time, 3 min 15 s; recycle delay, 0.9 s)and FTA-SOFAST-HMQC (experiment time, 25 s; recycle delay, 50 ms) spectra were recorded in an interleavedmanner resulting in a 0.14 min-1 sampling rate of the exchange process. H/D exchange was initiated as explained in"Materials and Methods." Under these experimental conditions, the data recorded at pH 9.8 (red circles) represent the15 most slowly exchanging amides, whereas the data measured at pH 7.3 allowed quantification of the exchange ratesfor less protected amide sites. The good correlation of these data (correlation coefficient of R = 0.997) illustrates thehigh accuracy obtained by the SOFAST approach despite the changing 1H spin-coupling network arising from thepartial deuteration of amide sites during the exchange process. (b) The experimental results shown in (a) may berationalized by computer simulations. The buildup of amide 1H polarization has been calculated for ubiquitin bynumerical integration of the Solomon equations describing the spin dynamics in the dipolar-coupled 1H network (7)assuming a magnetic field strength of 18.8 T and different rotational correlation times tc of the protein. Two extremecases have been considered: (i) all backbone amide sites are protonated (solid curves), and (ii) only the observed amidesite is protonated while all others are deuterated (dashed curves). The result of these simulations is that polarizationrecovery is slightly faster for the second case. This can be understood by the fact that fewer amide protons are excitedin the molecule, and therefore less energy has to be evacuated from the spin system to restore the equilibriumpolarization. Nevertheless, for the fast-pulsing conditions used here (indicated by an arrow) and the tumblingcorrelation time of ubiquitin at 25°C ( tc »4 ns), the difference in the restored polarization is negligible (<1%). Forlarger proteins, the difference increases (see curves obtained for tc = 15 ns) because of the more efficient spin diffusion.However, if higher repetition rates (shorter recovery times) are used for such large proteins, the difference becomesagain negligible.

Kupce E, Freeman R (1994) J Magn Reson A 108:268-273.1.Kupce E, Boyd J, Campbell ID (1995) J Magn Reson B 106:300-303.2.Kupce E, Freeman R (1995) J Magn Reson A 115:273-276.3.Schanda P, Brutscher B (2005) J Am Chem Soc 127:8014-8015.4.Schanda P, Kupce E, Brutscher B (2005) J Biomol NMR 33:199-211.5.Ishikawa N, Chiba T, Chen LT, Shimizu A, Ikeguchi M, Sugai S (1998) Protein Eng 11:333-335.6.Ishima R, Shibata S, Akasaka K (1991) J Magn Reson 91:455-465.7.

Copyright ©2008 by the National Academy of Sciences

S2

Fig. 4. FTA-SOFAST-HMQC experiment used for SOFAST real-time 2D NMR

measurements. The small angle (α < 90°) 1H pulse (filled symbol) is applied with a PC9 shape

(1), while for the 1H 180° pulses (open symbol) an iSNOB-5 shape (2) is used. The α angle is

typically set between 40° and 60° depending on the scan time chosen. The transfer delays Δ are

set to 1/(2JHN), and the small delay δ takes into account spin evolution during the PC9 pulse, and

is adjusted to half of the PC9 pulse length. For 15N chemical shift editing in t1, the basic

sequence is repeated n-times using short inter-scan delays (between 1 and 50 ms) resulting in

scan times of ~100ms. Heteronuclear decoupling is performed using WURST-40 (3). The

pulsed field gradients (PFGZ) are applied for 100 µs with a strength of typically 30 G/cm. In the

original SOFAST-HMQC pulse sequence (4, 5), the delay between the gradients encloses the

indirect evolution time t1, with typical maximum values of 20-30ms. Therefore, turbulence of the

solution, that stems from rapid injection leads to incomplete refocusing and thus to loss of

magnetization. This means that the signal in a series of experiments builds up only slowly after

injection (see SI Fig 6), a fact that clearly compromises spectral quality and interpretation of

kinetic data. In contrast, in the FTA-SOFAST-HMQC sequence shown here, the pulsed field

gradients are moved to the end of the pulse sequence and an additional 1H refocusing pulse is

added. While retaining excellent water suppression and high sensitivity this modification

reduces the time delay between gradients to 1-2 ms. The pulse sequence code in Varian

language is available from the authors upon request.

S3

Fig. 5. (a) Sketch of the fast injection device used for SOFAST real-time 2D NMR applications.

(b) Photographs of the NMR sample tube taken after fast injection of 50 µL blue dye into 350

µL of water using a rapid injection device. Homogeneous mixing is completed within

approximately 100 milliseconds.

S4

Fig. 6. Buildup of signal intensity after rapid sample injection measured by a gradient-echo

NMR pulse sequence using different dephasing times. 50 µL of a 2M glycine solution (in D2O)

were injected in 350 µL D2O and the signal intensity of glycine's Hα-peak was followed as a

function of time using a 1D gradient spin-echo pulse sequence (90° pulse – PFGz – τ/2 - 180°

pulse – τ/2 – PFGz – acquisition). This sequence is sensitive to translational diffusion effects,

and thus to the fluid turbulences created by the fast injection. It mimics the situation found in

more complex NMR experiments such as SOFAST-HMQC. Results are shown for dephasing

times of τ = 20 ms (filled squares), corresponding to the situation in standard SOFAST-

HMQC, and τ = 1 ms (open circles), corresponding to the situation in fluid-turbulence-adapted

FTA-SOFAST-HMQC. Stable conditions are reached in less than ~ 2 s using the short

dephasing time, while ~ 15 s elapse until equilibrium is reached when using the longer delay,

emphasizing the interest of FTA-SOFAST-HMQC when using a rapid injection device.

S7

Fig. 8. Comparison of folding kinetics of α-lactalbumine measured by fluorescence (black

squares) and SOFAST real-time 2D NMR spectroscopy (red circles). The protocol used for the

fluorescence measurements (protein concentration, pH-jump, solvent...) is identical to that used

for the time-resolved NMR experiments, except for the initiation of the folding reaction. For the

fluorescence measurements manual mixing yielded a dead time of about 1 minute. The

excitation wavelength is 295 nm (bandwidth: 1 nm) and the fluorescence change is monitored at

340 nm (bandwidth: 3 nm). Non-optimal sample volume, and the resulting small detection

window, as well as auto-quenching effects because of the high sample concentration explain the

relatively low signal-to-noise ratio obtained by the fluorescence experiment. A high protein

concentration was chosen to mimic the NMR conditions, resulting in a small volume for the

available amount of 2 mg 1 5N-labeled protein. The same recombinant 1 5N-labeled α-lactalbumin

has been used for NMR and fluorescence experiments because the recombinant protein

(expressed in E. Coli) is less stable than the natural protein, and this has an effect on the

refolding kinetics (6). The experimental data were fitted to mono-exponential decay shown as

red and black lines for NMR and fluorescence data, respectively.

S5

Fig. 7. Additional spectral processing used in SOFAST real-time 2D NMR. (a) Co-addition of

subsequent 2D spectra using alternate phase cycling. Left and central panel: first two FTA-

SOFAST-HMQC spectra recorded at time 0 and Δt after injection of ubiquitin into a H2O

solution. The phases of the 1 5N excitation pulse and the receiver have been alternated for

subsequent experiments. As a result of this phase cycling, artifacts have opposite sign in the two

spectra, whereas the signals of interest are of the same sign. Addition of the 2 spectra thus

removes the artifacts (right panel). If the series of recorded spectra is replaced by the sum of

successive spectra, e.g. (1+2) , (2+3), (3+4), … , this results in cleaner spectra without affecting

the time resolution of the kinetic data. (b) Effect of this co-addition-approach on the extracted

rate constants for mono-exponential intensity changes. The black solid line represents the

exponential change in peak intensity that is sampled at a rate determined by the acquisition time

of one spectrum, as indicated by black circles. Co-adding two successive spectra corresponds to

replacing two data points by their linear average and assigning this summed value to the central

time point in between the experimentally measured data points. These data points (which for

S8

Fig. 9. Evaluation of the accuracy of measured H/D exchange time constants τex using SOFAST

real-time 2D NMR. (a) τex values measured using the new FTA-SOFAST-HMQC sequence of

Fig. 4 are compared to results obtained using standard HSQC-based 2D NMR methods. H/D

exchange was monitored during 24 hours for ubiquitin at 25°C at two different pH values : 7.3

(black circles), and 9.8 (red circles). HSQC (expt. time: 3 min 15 s, recycle delay: 0.9 s) and

FTA-SOFAST-HMQC (expt. time: 25 s, recycle delay: 50 ms) spectra were recorded in an

interleaved manner resulting in a 0.14 min-1 sampling rate of the exchange process. H/D

exchange was initiated as explained in the Methods section. Under these experimental

conditions, the data recorded at pH 9.8 (red circles) represent the 15 most slowly exchanging

amides, whereas the data measured at pH 7.3 allowed quantifying the exchange rates for less

protected amide sites. The good correlation of these data (correlation coefficient of R=0.997)

illustrate the high accuracy obtained by the SOFAST approach despite the changing 1H spin

coupling network arising from the partial deuteration of amide sites during the exchange

process. (b) The experimental results shown in (a) may be rationalized by computer simulations.

The buildup of amide 1H polarization has been calculated for ubiquitin by numerical integration

of the Solomon equations describing the spin dynamics in the dipolar-coupled 1H network (7)

assuming a magnetic field strength of 18.8 T, and different rotational correlation times τc of the