Characterization of millisecond time-scale dynamics in the molten globule state of α-lactalbumin by NMR

Article No. jmbi.1999.3250 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 294, 551±560

Characterization of Millisecond Time-scale Dynamicsin the Molten Globule State of aaa-Lactalbumin by NMR

Seho Kim, Clay Bracken and Jean Baum*

Department of ChemistryRutgers UniversityPiscataway, NJ, 08854, USA

Present address: C. Bracken, DepBiochemistry, Weill Medical CollegeUniversity, New York, NY 10021, U

Abbreviations used: NMR, nuclearesonance; MG, molten globule; R2,relaxation rates; CPMG, Carr-Purcea-LA, a-lactalbumin; CD, circular dCP, cross polarization; HSQC, heterquantum coherence.

E-mail address of the [email protected]

0022-2836/99/470551±10 $30.00/0

The motional dynamics of the molten globule (MG) state of a-lactalbuminhave been characterized using 15N transverse relaxation rates (R2). Amodi®ed version of the Carr-Purcell-Meiboom-Gill (CPMG) R2 pulsesequence is proposed in order to overcome the loss of sensitivity thatarises from extreme line broadening due to complex dynamics on themillisecond time-scale. Using this pulse sequence, chemical exchangerates were extracted by examining the 15N transverse relaxation rates as afunction of CPMG delay values. The results clearly illustrate that perva-sive conformational exchange of 0.2-0.5 ms in the 15N backbone reson-ances of the molten globule state of a-lactalbumin. The temperaturedependence of the conformational exchange rates display standardArrhenius kinetic behavior between 10 and 30 �C. Estimates of the acti-vation energies range from 0.8 to 4.4 kcal/mol, indicating a low energeticbarrier to conformational ¯uctuations relative to native state proteins.The ¯uctuations and low energetic barriers may be critical for directingthe search for contacts that will result in the transition from the MG stateto the native state.

# 1999 Academic Press

Keywords: NMR; a-lactalbumin; dynamics; conformational exchange;activation barrier
*Corresponding author
Introduction

Understanding protein conformational dynamicson the slow micro- to millisecond time-scale isimportant for understanding the protein foldingprocess and may help elucidate the role of inter-mediates in folding. A general view of proteinfolding that is beginning to emerge is one in whichthe unfolded form collapses within milliseconds toa compact state (Dobson, 1994; Dill & Chan, 1997;Dobson et al., 1998). Fluctuations, on the order ofmicroseconds to milliseconds, within the inter-mediate are likely to be important in determiningthe pathway of folding to the native form or inunderstanding off-pathway processes such as

artment ofof Cornell

SA.r magnetic15N transverse

ll-Meiboom-Gill;ichroism;onuclear single

ing author:

aggregation. Conformational ¯uctuations in thecollapsed state provide a mechanism for differentresidues to come into contact thereby helping theprocess of searching for critical contacts that willresult in formation of the native state. It is import-ant to quantify the time-scale of backbone con-formational ¯uctuations and the temperaturedependence of the internal motional processes inthe collapsed state to obtain information about theenergetics of the folding process.

The initial hydrophobic collapsed state observedin native state folding reactions has been shown bynumerous laboratories to bare a striking resem-blance to the equilibrium intermediate state knownas the molten globule (MG) state (Dobson, 1994;Dill & Chan, 1997; Dobson et al., 1998). One of themost extensively studied molten globule states isthe acid denatured form of a-lactalbumin (a-LA)(Dobson, 1994; Ptitsyn, 1995; Kuwajima, 1996).a-LA is a protein composed of two domains, ana-helical domain and a b-sheet domain, with acalcium binding loop located at the juncture of thea and b-domains (Figure 1) (Pike et al., 1996). TheMG state of a-LA has been shown to contain sub-stantial secondary structure but lacks rigid tertiaryinteractions characteristic of native proteins(Kuwajima et al., 1985; Damaschun et al., 1986;

# 1999 Academic Press

mailto:[email protected]

Figure 1. X-ray crystal structure of guinea pig a-LA(Pike et al., 1996). The protein contains four a-heliceslocated in the a-domain labeled A, B, C and D, and ab-domain.

552 Millisecond Time-scale Dynamics in �-Lactalbumin

Baum et al., 1989; Kuwajima, 1989; Ptitsyn et al.,1990; Alexandrescu et al., 1993; Chyan et al., 1993;Peng & Kim, 1994; Peng et al., 1995; Schulman et al.,1995; Wu et al., 1995). Numerous studies have indi-cated that the MG state is a bipartite structure withan a-helical domain consisting of native-like sec-ondary structure and a b-domain that is largelyunstructured (Chyan et al., 1993; Peng & Kim,1994; Peng et al., 1995). Previous NMR studieshave suggested that the MG state undergoes con-formational ¯uctuations on a millisecond time-scale as evidenced by broadened lines in the NMRspectrum. However, there have been no quantitat-ive measurements to con®rm these suggestionsand there is no information about the energeticbarriers in the molten globule state (Damaschunet al., 1986; Baum et al., 1989; Chyan et al., 1993;Schulman et al., 1997).

Heteronuclear NMR spectroscopy is a powerfulapproach for studying intramolecular confor-mational dynamics (Wagner, 1993; Dayie et al.,1996; Palmer et al., 1996). In particular, slow pro-tein dynamics on the microsecond to millisecondtime-scale can be quanti®ed explicitly by 15N trans-verse relaxation rate (R2) experiments using theCarr-Purcell-Meiboom-Gill (CPMG) pulse sequence(Orekhov et al., 1994, 1995). Quantitative measure-ment of millisecond time-scale dynamics of theMG state of a-LA has been dif®cult due to overlapin the 2D heteronuclear single quantum coherence(HSQC) spectrum and a large reduction in the sen-sitivity of the signal arising from the extreme linebroadening by complex dynamical behavior. Wehave developed a variation of the CPMG R2 exper-iment that includes a heteronuclear cross polariz-ation (CP) transfer period (Krishnan & Rance,1995) to overcome these dif®culties and demon-strate that conformational exchange, on the orderof 0.2-0.5 ms, is pervasive in the 15N backbone res-onances of the molten globule state of a-lactalbu-

min. Characterization of the temperaturedependence of these internal motional processesindicates a low energetic barrier to conformational¯uctuations, in the order of 3 kcal/mol, relative tonative state proteins.

Results

Line broadening in the HSQC spectrum ofthe MG-state

The majority of the cross-peaks in the 1H-15NHSQC spectrum of the acid-denatured MG state ofguinea pig a-LA at 10 �C (Kim & Baum 1998)appear broad, suggesting substantial exchangebroadening and complex dynamical behavior(Figure 2(a) and (b)). However, a number of shar-per resonances can also be seen at chemical shiftvalues above 8 ppm indicating the presence of 15Nresonances with a variety of relaxation properties.As the temperature is raised from 25 to 40 �C, theresonances in the HSQC spectra (Figure 2) appearto sharpen progressively, suggesting that the rateof interconversion is faster at higher temperatureand that the dynamics within the protein arebecoming more homogeneous. The degree of over-lap in 2D NMR spectra makes analysis of individ-ual resonances impossible, therefore heteronuclearedited 1D NMR spectra were used to analyzeexchange effects in the MG state of the protein.This allowed signi®cant time savings and per-mitted a more thorough exploration of parameterspace.

To ensure that aggregation is not causing theNMR spectral appearance, the concentrationdependence of the R2 relaxation rates wasmeasured between 2.0 mM and 0.1 mM, and dis-played an average R2 rate which was independentof concentration. To examine lower concentrations,the CD ellipticity at 222 nm was measuredbetween 0.2 mM and 0.01 mM. Both NMR and CDspectral properties were found to be independentof concentration. This is consistent with previousequilibrium ultracentrifugation studies of humanaLA (Schulman et al., 1995), which showed that theprotein is monomeric at low pH. Since proteinaggregation is not a factor at the concentrationsused for the NMR experiments, the overlap orbroadening of resonances can be attributed toeither conformational heterogeneity or exchangedue to ¯uctuations on the micro- to millisecondtimescale (Chyan et al., 1993; Schulman et al., 1997).

Heteronuclear-cross polarization experiments

Conformational exchange processes can lead tosigni®cant reduction in spectral sensitivity and res-olution if the rate of exchange is on the same orderof magnitude as the chemical shift differencebetween distinct environments. One approach toremoving the effects of chemical exchange duringheteronuclear coherence transfer steps is by the useof cross-polarization (Krishnan & Rance, 1995). The

Figure 2. 1H-15N HSQC spectrum of a-LA in the MG state collected at (a) 25 �C, pH 2.2 and (b) 40 �C, pH 2.2.

Millisecond Time-scale Dynamics in �-Lactalbumin 553

intensity loss during periods of free precessionarises from dephasing of transverse magnetizationas the nuclei exchange between distinct chemicalenvironments. In heteronuclear NMR experiments,which rely on periods of free precession for coher-ence transfer, magnetization of non-exchangeableresonances is transferred more ef®ciently thanthose of exchanging resonances. Therefore, thedifferences in transfer ef®ciency between exchange-able and non-exchangeable resonances will biasthe intensities observed in overlapped spectra.Krishnan & Rance (1995) have shown that coher-ence transfer ef®ciency may be improved insystems undergoing chemical exchange by usingheteronuclear cross-polarization transfer of coher-ence. Cross-polarization is achieved by applicationof matched spin-lock ®elds which establishes theHartmann-Hahn condition allowing in-phasecoherence transfer between 1H and 15N nuclei.Since the nuclei are spin locked, the effective ®eldexperienced by each nucleus is strong and uniformthereby removing the effects of chemical shiftdephasing (Krishnan & Rance, 1995).

The superior performance of CP under confor-mational exchange in the MG state of a-LA canbe estimated by comparing 15N-selected pulsesequences employing an INEPT transfer periodagainst 15N-selected pulse sequences employing aheteronuclear CP transfer period. The HSQC andCP experiments are compared at low (10 �C) andhigh (40 �C) temperature in order to determine thedifference in sensitivity under exchanging (10 �C)

and non-exchanging conditions (40 �C). For theHSQC experiment, the sensitivity is lower at 10 �Cthan at 40 �C by a factor of 0.43, whereas for theCP experiment the sensitivity at 10 �C is lowerthan that at 40 �C is by only 0.8 (Table 1). This indi-cates that under exchanging conditions at 10 �C,the CP experiment is more effective than theHSQC experiment (by a factor of 1.8) in removingthe effects of chemical exchange during heteronuc-lear transfer. Therefore, the CP experiments will beincorporated into R2 experiments as they reducechemical exchange dependent losses in the coher-ence transfer ef®ciency and result in spectra thatare not selectively biased toward species withslower R2* rates. It is interesting to note that at40 �C, where chemical exchange no longer domi-nates the spectrum, the absolute intensity of theHSQC experiment is 2.2 times higher than the CPsequence (Table 1). This sensitivity difference isdue in part to the inef®cient magnetization transferin CP where completion of full cycles of the DIPSI-2 sequence resulted in an 8.1 ms transfer timerather than the optimal 5.4 ms transfer time. TheCP transfer ef®ciency may be somewhat improvedby using more recently developed shorter mixingschemes such as DIPSI-1 or Waltz-4; however, themixing scheme needs to be chosen carefully as thehomogeneity of the spin-lock ®eld is important forthe implementation of the CP sequence. Althoughthe sensitivity of the CP experiment is lower over-all than the HSQC, it has the important advantagethat the signal that is undergoing conformational

Table 1. Comparison of CP and HSQC sequences

Temperature CP HSQC CP/HSQC

10�C 1.0 1.026 0.97540�C 1.2520 2.3484 0.533Ratio (10 �C/40 �C) 0.799 0.437 1.828

Intensities are integrated areas of all amide peaks.

Figure 3. The NMR pulse sequence for 15N measure-ment of transverse relaxation utilizing 1H-15N cross-polarization transfer steps. In the pulse sequence, 90 �and 180 � pulses are represented by thin and thick bars,respectively, pulsed ®eld gradients are represented byhalf ovals, and the Hartman-Hahn transfer period isrepresented by rectangles. The cross-polarization trans-fer is accomplished by application of simultaneousDIPSI-2 periods on 1H and 15N nuclei applied to eachnuclei at 3570 Hz ®eld strength for 8.1 ms, 90 � and180 � nitrogen pulses in the CPMG sequence wereapplied at 4460 Hz ®eld strength, and 90 � and 180 � 1Hpulses were applied at 2900 Hz ®eld strength. For anal-ysis of exchange rates the CPMG sequence was repeatedn times. The effective ®eld strength of the CPMGsequence is governed by the spin-echo delay t. Thewater signal suppression is aided by application of twogradients (g1 and g2 with 21 G/cm for 2 ms) with awater-selective shaped ¯ipback pulse incorporated intoa zz-®lter. The phase cycling is f1 � x, x, ÿx, ÿx;f2 � x, ÿx, x, ÿx; and f3 � x, ÿx, ÿx, x.


exchange is not lost during the heteronuclear trans-fer period and that the CP experiment more accu-rately represents the heterogeneity of the dynamicsin the spectrum than the HSQC experiment.

Measurement of chemical exchange processesusing cross polarization relaxation experiments

A number of different 15N transverse relaxationexperiments have been proposed to measurechemical exchange processes on the micro- to milli-second time-scale (Orekhov et al., 1994, 1995; Akke& Palmer, 1996; Zinn-Justin et al., 1997; Akke et al.,1998; Loria et al., 1999). One approach is themeasurement of R2 decay rates in the presence ofspin-echo pulse trains of variable spacing t, suchas an R2

CPMG experiment. This allows the detectionof intermediate time-scale dynamics over the rangeof 102 to 104 sÿ1. The cross-polarization transferdescribed above was incorporated into the R2

CPMG

relaxation experiments, in order to improve thesensitivity of these experiments in systems thathave extreme line broadening. The standard R2

CPMG

experiment was modi®ed by substituting CP trans-fer periods for INEPT transfer periods resulting ina CP-R2

CPMG experiment (Figure 3). The CP-R2CPMG

experiment, which has improved sensitivity overthe standard R2

CPMG, is used to obtain confor-mational exchange rates on the micro- to milli-second time-scale in the MG state. Large changesin R2 rates are observed as a function of t, con®rm-

ing that chemical exchange broadening is present(Figure 4) in the MG state of a-lactalbumin.

Temperature dependence of dynamics in theMG state of aaa-lactalbumin

The temperature dependent conformationaldynamics of the MG state of a-LA are measuredusing the CP-R2

CPMG experiment and the resultsallow estimation of the activation energies for theexchange process (Palmer & Bracken, 1999). The R2

dependence on t is greater at low temperaturethan at high temperature, indicating that confor-mational exchange processes are more dominant atlow temperature than at high temperature(Figure 4). At 10 �C, the differences in R2 rates, as afunction of t, range from 20 to 3 9 sÿ1 (Figure 4(a)),whereas at 40 �C the range of R2 values is onlyfrom 10 sÿ1 to 15 sÿ1 across the 1H frequency(Figure 4(c)). There is also a dependence of the R2

rates on 1H frequency most clearly observed at10 �C, with values ranging from 20 to 27 sÿ1 up to8.1 ppm and increasing to a range of 21 to 39 sÿ1

at 7.8 ppm. The R2 dependence on the 1H frequen-cies was found to be independent of the carrierposition, pulse ®eld strengths, and the degree ofwater saturation.

Variations in the R2 values as a function ofCPMG delay t were ®t using equation (2) to obtainthe values of kex, ex, and R�2. To reduce the num-ber of parameters for the ®t to the R2 data, theassumption was made that the population of inter-converting species is equal and that pA � pB � 0.5.Because of the complex dynamical behavior andthe degree of chemical shift overlap in the MGstate, it is impossible to know exactly whichspecies are interconverting and what the relativepopulation of interconverting species is; however,it has been shown (Akke et al., 1998) that variationsin population, in the order of pA � 0.2 to 0.8, havea small effect on the exchange rates. Three par-ameter (kex, ex, and R�2) ®tting of the data at alltemperatures resulted in arbitrary changes in kex

values at higher temperatures, therefore the ex

values obtained from the three parameter ®t at10 �C were used to model the data at other tem-peratures. Since the temperature dependence of15N backbone chemical shifts in the same confor-mational state is small relative to chemical shiftchanges due to alterations in conformation(Bracken et al., 1999) this approximation shouldhave only minor effects on the estimated kex

values.

Figure 4. R2 values acquired using the CP-R2CPMG

experiment at (a) 10 �C, (b) 25 �C, and (c) 40 �C. Eachpanel shows six different R2 values as a function ofspin-echo delay t in the CPMG sequence: t � 0.154(brown), 0.25 (red), 0.35 (orange), 0.5 (green), 0.75(blue), and 1.0 (purple) ms. R2 values were measuredusing equation (1) and plotted along the 1H frequencywith 2 Hz intervals.

Figure 5. The (a) kex, (b) R�2, and (c) ex values areobtained by ®tting R2 data as a function of the delay tto equation (2). The kex, R�2, and ex values (brown) at10 �C were ®t with three independent parameters usingthe Marquart-Levenberg method, and the kex and R�2values above 10 �C were ®t with two parameters by®xing ex with the value obtained at 10 �C. The exper-imental temperatures are 10 (brown), 15 (red),20 (orange), 25 (green), 30 (blue), 35 (purple), and40 (gray) �C.


The kex, ex, and R�2 values ®t to the R2 data as afunction of delay t are shown as a function of 1Hfrequency and temperature in Figure 5. Theobserved kex values, from 10 �C to 30 �C, rangefrom 2000-5000 sÿ1 corresponding to chemical orconformational exchange processes of 0.5 ms atlow temperature and 0.2 ms at high temperature(Figure 5(a)). Above 30 �C there appears to be adiscontinuity in the kex values as a function oftemperature, with kex values ranging from 6000-8000 sÿ1. It is interesting to note that there appearsto be somewhat of a frequency dependence to thekex values with the kex values lower below 8 ppmthen above 8ppm. The differences as a function of

chemical shift are thought to be signi®cant as theerror for R�2 is estimated as 5-10 % (Figure 4) andkex is 10-15 % (Figure 5) as a function of ®eld andtemperature. The values of R�2 are relatively uni-form across the 1H frequencies, with small dipsin rates which indicate the presence of underlyingresonances with faster motional properties(Figure 5(b)). ex is approximately 500 rad/secondwhich corresponds to 1.6 ppm of 15N frequencies(Figure 5(c)). Upon increasing the temperature, R�2decreases and kex increases. The degree of exchangeline broadening in NMR is correlated to thekex/ex ratio, and severe line broadening is

Figure 6. Activation energies obtained from theArrhenius equation (equation (3)) using the values of kex

obtained from the R2 relaxation data at 10, 15, 20, 25and 30 �C. (a) The calculated Ea values versus 1H fre-quency. The error bars were estimated from MonteCarlo simulations of kex data within curve ®tting errors.(b) Two representative Arrhenius plots of kex versus 1/Tat 7.92 ppm and 8.1 ppm.


observed for values of 0.1 < kex < 10. Exchangebroadening is at a maximum when the kex/ex

ratio is equal to��2p

. Our observed kex/ex ratiosare in the range of 4.0-10.0, which indicates thatthe observed chemical exchange is in the fastexchange regime.

Activation barriers in the MG state

The temperature dependence of the exchangerates, kex, obtained from the CP-R2

CPMG experimentscan provide estimates of the activation barrier ofthe conformational ¯uctuations in the MG state.Accurate activation barrier measurements areobtained assuming that the conformation of theprotein does not change over the temperaturerange studied. CD spectra of the native and moltenglobule states were obtained as a function of tem-perature, from 5 �C to 90 �C, to determine therange of temperatures over which the confor-mation does not change. The melting pro®les wereconsistent with those described by Ptitsyn (1995),and indicate that the base-line ellipticity of the MGstate begins to change above 30 �C. The change inellipticity above 30 �C is consistent with the discon-tinuity of kex values above 30 �C (Figure 5) andindicates that the MG state begins to unfold andthat the equilibrium constant pA/pB begins tochange at this point. Based on the CD spectra ofthe MG state and the temperature dependent 2DHSQC spectra (Figure 2), it appears that the equili-brium constant remains unchanged below 30 �C,suggesting that population weighted changes ofchemical shift are not occurring but rather that therate of interconversion between states is increasing.Therefore, the temperature dependent analysis ofR2 below 30 �C may represent simply the degree ofline broadening in the spectrum. As the CD spectraare essentially invariant from 10 to 30 �C, only kex

values between 10 and 30 �C were used to obtainexchange rates as a function of temperature andestimates of activation barriers.

The kex values between 10 �C and 30 �C were ®tto the Arrhenius equation, given in equation (3), togenerate estimates of the activation energies for theexchange process. The calculated activation ener-gies are plotted in Figure 6, along with two repre-sentative curves at 7.92 and 8.1 ppm to show theerror ranges in the kex values. The Ea values rangefrom �0.8 kcal/mol to �4.4 kcal/mol and re¯ectthe change of R2 rates as a function of 1H chemicalshift seen in Figure 4. The errors below 8.0 ppmare as low as 1.0 kcal/mol, while the errors moredown®eld are quite large, due in part to lack ofprecision in estimating the faster chemicalexchange rates extracted at the higher 1H frequen-cies.

Discussion

The molten globule state of a-LA is characterizedby very complex dynamical behavior. NMRexperiments allow detection of dynamics over

different time-scales and the 15N CPMG relaxationexperiments employed here allow detection ofconformational ¯uctuations in the range ofapproximately 0.2-1.0 ms scale (Orekhov et al.,1994, 1995; Akke & Palmer, 1996; Zinn-Justin et al.,1997; Akke et al., 1998; Loria et al., 1999). The 15NCP-R2

CPMG relaxation experiments applied to a-LAindicate that this window of dynamical time-scalesexist in the MG state and that conformational¯uctuations range from 0.2 to 0.5 ms. The tempera-ture dependence of the measured conformationalexchange rates display standard Arrhenius kineticbehavior and estimated activation energies rangefrom 0.8 to 4.4 kcal/mol. The presence of severeresonance overlap in the 2D 1H-15N correlationspectra make resolution of individual peaks in the2D spectra unattainable for the MG state of a-LAat low pH, therefore faster 1D NMR methods wereemployed to allow a greater number of variablesto be changed. The results are necessarily anaverage of the relaxation of multiple amide reson-


ances and represent a distribution of relaxationand exchange rates within the protein. Since apopulation of relaxation rates are measured ratherthan speci®c rates, factors which affect the relativeintensities of the cross-peaks during the transferperiods will bias the results in favor of non-exchanging resonances. Refocused INEPT basedtransfer sequences incur signi®cant sensitivity lossdue to chemical exchange at low temperatures andresult in an arti®cial reduction in the apparentrelaxation rates. Therefore a relaxation sequenceemploying the CP transfer, which is less affectedby chemical exchange, was employed for 15Nrelaxation measurements. The intensity ofexchange broadened and non-exchange broadenedamide protons will contribute similar intensities tothe 1D envelope, making it possible to estimate theglobal R2 values for the distribution of confor-mational exchange rates contained within a-LAover a wide range of temperatures.

Despite the fact that each proton frequency mayrepresent many backbone resonances, thereappears to be an overall trend in the data of theexchange rates and activation energies, with Ea

values above 8 ppm being lower than Ea valuesbelow 8ppm. This is consistent with HSQC spectraof the MG state which display a number of sharperresonances superimposed on the broadened signalobserved above 8 ppm. The chemical shifts of thesharper resonances above 8 ppm are close to thevalues expected for unstructured peptides, andprevious studies have suggested that thebroadened peaks arise from the regions of theprotein corresponding to the partly foldedcollapsed state (Schulman et al., 1997). Therefore,we interpret the larger activation energies as corre-sponding to regions of the protein that correspondto the a-helical core of the MG state, and the lowactivation energies, above 8 ppm, correspond tothe less structured regions of the MG state or theb-domain. The low activation energies characteriz-ing the b-domain suggest that it has little or nostructural impediments on the backbone dynamics,whereas the higher activation energies correspond-ing to the core of the MG state or the a-domain,suggest that this region has certain restrictions ofthe polypeptide chain arising from the residualstructure.

Previous spectroscopic and mutagenesis exper-iments as well as molecular dynamics simulationshave indicated that the core of the MG state isstabilized primarily by non-speci®c hydrophobicinteractions (Uchiyama et al., 1995; Wu & Kim,1997; Smith et al., 1999). The magnitude of confor-mational ¯uctuations in the core of the MG statecan be related to those of native proteins by com-paring relative activation energy barriers. Typicalvalues of activation energies in native proteinshave been measured by NMR and range from 20-50 kJ/mol for backbone motion of ribonuclease H(Mandel et al., 1996), 17 kcal/mol for disul®deexchange in BPTI (Otting et al., 1993) and 57-80 kJ/mol for ring ¯ips of aromatic residues in BPTI

(Wagner, 1983). The average values of 4 kcal/molfor the backbone motions of the core of the MGstate of a-LA are smaller than those described fornative proteins. This suggests that non-speci®chydrophobic interactions may induce collectivemotions on the millisecond time-scale and that theactivation barriers describing these motions arelower than those of compact native states withspeci®c tertiary interactions.

Experimental and theoretical advances haveincreased our understanding of protein foldingand have led to concepts of energy landscapesand folding funnels. Recent developments havebrought about an increased awareness of theimportance of ensembles of conformations inunderstanding the folding process (Dill & Chan,1997; Dobson et al., 1998). We have shown that therate of interconversion between the ensemble ofconformations in the MG state is slow, in the orderof 0.5 ms, and corresponds to an activation barrierof approximately 4 kcal/mol. In studying the kin-etic folding process of a-LA, CD and NMR hasshown that there is a fast collapse within 5 ms andthen a slower folding rate to the ®nal form thattakes many seconds (Forge et al., 1999). The initialstate, obtained within 5 ms, shows that the MGproperties and the subsequent slow folding phaserepresents the cooperative two-state transitionbetween the MG and native states (Balbach et al.,1995; Arai & Kuwajima, 1996; Forge et al., 1999).The rate of interconversion of the ensemble of con-formations in the equilibrium molten globule statedescribed here is faster than the time-scale of fold-ing, indicating a consistency between the kineticfolding studies and the equilibrium state intercon-version rates. Therefore, it is possible that the com-plex dynamical behavior that is observed in theequilibrium MG state may exist in the refoldingkinetics, and that the slow conformational ¯uctu-ations on the millisecond time-scale and the lowactivation barriers may be a signature of thehydrophobic collapse. The millisecond time-scale¯uctuations and low energetic barriers may becritical for directing the search for contacts thatwill result in the transition from the MG state tothe native state.

Materials and Methods

Sample preparation

Recombinant 15N-labeled guinea pig a-LA (M90Vmutant) was prepared as described (Kim et al., 1997).The native state samples of a-LA were prepared in10 mM sodium phosphate buffer (pH 7.0), 1 mM CaCl2,and 10 % 2H2O. The MG state was obtained by adjustingthe pH to 2.2 with 0.5 M HCl. Protein concentrationswere 2.0 mM for NMR chemical exchange experimentsand 0.2 mg/ml for circular dichroism (CD) meltingexperiments. Sample concentrations were measuredusing the calculated absorbance (0.1 %) of 1.65 at280 nm.

The presence of aggregation in the MG-state wastested using both NMR and CD. The concentration


dependence of the 15N transverse relaxation rates in theMG state between 2.0 mM to 0.1 mM sample concen-tration were measured using standard 15N transverserelaxation methods (Barbato et al., 1992) at 25 �C. Lowerprotein concentrations, between 0.2 mM and 0.01 mM,were assayed by examining the CD ellipticity at 222 nmcollected on an AVIV Model 60DS CD spectrometerusing quartz cells with path lengths of 0.01 cm, and0.1 cm (Hellma GmbH & Co).

NMR spectroscopy

All NMR spectra were collected on a Varian Unity-plus 500 MHz spectrometer, equipped with a tripleresonance probe with z-axis ®eld gradient. Gradientselected 1H-15N HSQC spectra (Bax et al., 1990; Daviset al., 1992) were collected with spectral widths of 7kHz in the 1H acquisition dimension and 1.5 kHz (forthe native state) or 3 kHz (for the MG state) in theindirect 15N dimension. A heteronuclear cross-polariz-ation sequence (Ernst et al., 1991; Krishnan & Rance,1995) was employed for proton detected 1H-15N corre-lation spectra. The CP transfer was achieved using aDIPSI-2 pulse train (Shaka et al., 1988) simultaneouslyapplied to the center of amide 1H and 15N frequenciesat 3570 Hz ®eld strength. Assessment of the transferef®ciencies of the CP with the standard refocusedinept transfer period were made using, 1D HSQC and1D CP spectra acquired at 10 and 40 �C. Amide peakintensities in the HSQC and CP experiments werecompared by integration of peak intensities on exper-iments collected back-to-back.

Transverse 15N relaxation rates (R2) were acquiredusing a modi®ed version of the 2D 15N relaxationsequence by substituting the CP transfer periods in placeof the refocused INEPT transfer periods. The R2 relax-ation pulse sequence is shown in Figure 2. The 1D 15NR2 rates were collected using different spin-echo delayvalues (t) which varies the effective spin-lock ®eldstrength of the CPMG sequence. A total of 11 time pointswere collected for each R2 curve at a ®xed t delay in theCPMG period: 2.5, 9.9, 14.8, 24.6, 29.6, 39.4, 54.2, 73.9,98.6, 128.1, 197.1 ms at t � 0.154 ms; 4.0, 16.0, 24.0, 40.0,48.0, 64.0, 88.0, 120.0, 160.0, 208.0, 320.0 ms at t � 0.25,0.5, and 1.0 ms; 2.8, 11.2, 16.8, 28.0, 33.6, 44.8, 61.6, 84.0,112.0, 145.6, 224.0 ms at t � 0.35 ms; and 6.0, 24.0, 36.0,60.0, 72.0, 96.0, 132.0, 180.0, 240.0, 312.0, 480.0 ms att � 0.75 ms. Experiments were repeated at seven differ-ent temperatures: 10 �C, 15 �C, 20 �C, 25 �C, 30 �C, 35 �Cand 40 �C. Each spectrum was acquired with 8192 com-plex points, 14 kHz spectral width, 64 scans, and a ®vesecond recovery period. The large spectral width wasused for base-line correction and the long acquisitionperiod was used to eliminate possible truncationartifacts.

Data analysis

The NMR data were processed using either VNMRsoftware (Varian ) or FELIX 2.05 software (Biosym Inc.)as well as software written in-house. The 15N R2 rateswere calculated using VNMR software by exponentialcurve-®tting of relaxation data collected as a function ofrelaxation delay, shown in equation (1):

I � I1 ÿ I0eÿR2t �1�where I is the observed peak intensity, I0 is the initialspectral intensity, R2 is the observable relaxation rate,

and I1 is an offset parameter which accounts for effectsof the slowly relaxing components.

Chemical exchange rates have a relationship with R2

as a function of spin-lock ®eld strength (Bloom et al.,1965; Carver & Richards, 1972; Orekhov et al., 1994,1995; Palmer et al., 1996):

R2 � R�2 � 2pApB2exkex=�4k2

ex � o21� �2�

where R2 is the apparent transverse relaxation rate; R2 isthe intrinsic transverse relaxation rate without chemicalexchange; pA and pB are the populations occupyingchemically distinct environments A and B undergoingtwo site exchange, and these are ®xed to 0.5 by assumingequal populations; ex is the chemical shift differencebetween the exchanging sites A and B; kex is the chemicalexchange rate; and o1 is the effective spin-lock ®eldstrength when the CPMG sequence is used assumingo1 � p/2t.

Values of R2 collected as a function of the delay t at10 �C were modeled by ®tting three parameters (kex, ex,and R�2) using a Marquardt-Levenberg method (Presset al., 1992). The R2 values at other temperatures weremodeled using two parameters (kex and R2) with theindividual values of ex, obtained at 10 �C (Press et al.,1992). The conformational exchange rates (kex) weremodeled as a function of temperature using the Arrhe-nius equation shown below:

kex � AeÿEaRT �3�

where kex is the experimentally measured exchange rate;Ea is the activation energy; A is a constant, and T is theexperimental temperature given in units of Kelvin. Theerror estimates of Ea were obtained using a programwritten in-house that employed Monte Carlo simulationsusing 500 random values within the curve-®tting errorrange of kex.

Acknowledgments

We gratefully acknowledge Arthur G. Palmer III andJean Bumby for helpful discussions. S.K. and C.B. con-tributed equally to this work.

This work was supported by NIH grant GM-45302and a Camille and Henry Dreyfus Teacher-Scholaraward.

References

Akke, M. & Palmer, A. G., III (1996). Monitoring macro-molecular motions on microsecond to millisecondtime scales by R1r-R1 constant relaxation timeNMR spectroscopy. J. Am. Chem. Soc. 118, 911-912.

Akke, M., Liu, J., Cavanagh, J., Erickson, H. P. &Palmer, A. G., III (1998). Pervasive conformational¯uctuations on microsecond time scales in a ®bro-nectin type III domain. Nature Struct. Biol. 5, 55-59.

Alexandrescu, A. T., Evans, P. A., Pitkeathly, M., Baum,J. & Dobson, C. M. (1993). Structure and dynamicsof the acid-denatured molten globule state ofa-lactalbumin: a two-dimensional NMR study.Biochemistry, 32, 1707-1718.

Arai, M. & Kuwajima, K. (1996). Rapid formation of amolten globule intermediate in refolding of a-lactal-bumin. Fold. Design, 1, 275-287.


Balbach, J., Forge, V., van Nuland, N. A. J., Winder,S. L., Hore, P. J. & Dobson, C. M. (1995). Followingprotein folding in real time using NMR spec-troscopy. Nature Struct. Biol. 2, 865-870.

Barbato, G., Ikura, M., Kay, L. E., Pastor, R. W. & Bax,A. (1992). Backbone dynamics of calmodulin stu-died by 15N relaxation using inverse detected twodimensional NMR spectroscopy: the central helix is¯exible. Biochemistry, 31, 5269-5278.

Baum, J., Dobson, C. M., Evans, P. A. & Hanley, C.(1989). Characterization of a partly folded proteinby NMR methods: studies on the molten globulestate of guinea pig a-lactalbumin. Biochemistry, 28,7-13.

Bax, A., Ilura, M., Kay, L. E., Torchia, D. A. & Tschudin,R. (1990). Comparison of different mode of two-dimensional reverse-correlation NMR for the studyof proteins. J. Magn. Res. 86, 304-318.

Bloom, M., Reeves, L. W. & Wells, E. J. (1965). Spinechoes and chemical exchange. J. Chem. Phys. 42,1615-1624.

Bracken, C., Carr, P. A., Cavanagh, J. & Palmer, A. G.(1999). Temperature dependence of intramoleculardynamics of the basic and leucine zipper domainsof GCN4 - implications for the entropy of associ-ation with DNA. J. Mol. Biol. 285, 2133-2146.

Carver, J. P. & Richards, R. E. (1972). A general two-sitesolution for the chemical exchange produced depen-dence of T2 upon the Carr-Pulcell pulse separation.J. Magn. Res. 6, 89-105.

Chyan, C., Wormald, C., Dobson, C. M., Evans, P. A. &Baum, J. (1993). Structure and stability of the mol-ten globule state of guinea-pig a-lactalbumin: ahydrogen exchange study. Biochemistry, 32, 5681-5691.

Damaschun, G., Gernat, C., Damaschun, H., Bychkova,V. E. & Ptitsyn, O. B. (1986). Comparison of intra-molecular packing of a protein in native and `mol-ten globule' states. Int. J. Biol. Macromol. 8, 226-230.

Davis, A. L., Keeler, J., Laue, E. D. & Moskau, D. (1992).Experiments for recording pure-absorption hetero-nuclear correlation spectra using pulsed ®eld gradi-ents. J. Magn. Res. 98, 207-216.

Dayie, K. T., Wagner, G. & Lefevre, J. F. (1996). Theoryand practice of nuclear spin relaxation in proteins.Annu. Rev. Phys. Chem. 47, 243-282.

Dill, K. D. & Chan, H. S. (1997). From Levinthal to path-ways to funnels. Nature Struct. Biol. 4, 10-19.

Dobson, C. M. (1994). Solid evidence for moltenglobules. Curr. Biol. 4, 636-640.

Dobson, C. M., Sali, A. & Karplus, M. (1998). Proteinfolding: a perspective from theory and experiment.Angew. Chem. Int. Edit. 37, 868-893.

Ernst, M., Griesinger, C. & Ernst, R. R. (1991). Opti-mized heteronuclear cross polarization in liquids.Mol. Phys. 74, 219-252.

Forge, V., Wijesinha, R. T., Balbach, J., Brew, K.,Robinson, C. V., Red®eld, C. & Dobson, C. M.(1999). Rapid collapse and slow structural reorgani-sation during the refolding of bovine a-lactalbumin.J. Mol. Biol. 288, 673-688.

Kim, S. & Baum, J. (1998). Electrostatic interactions inthe acid denaturation of a-lactalbumin determinedby NMR. Protein Sci. 7, 1930-1938.

Kim, S., Baum, J. & Anderson, S. (1997). Productionof correctly folded recombinant [13C, 15N]-enrichedguinea pig [Val90]-a-lactalbumin. Protein Eng. 10,455-462.

Krishnan, V. V. & Rance, M. (1995). In¯uence of chemi-cal exchange among homonuclear spins in hetero-nuclear coherence-transfer experiments in liquids.J. Magn. Res. ser. A, 116, 97-106.

Kuwajima, K. (1989). The molten globule state as a cluefor understanding the fold and cooperativity ofglobular-protein structure. Proteins: Struct. Funct.Genet. 6, 87-103.

Kuwajima, K. (1996). The molten globule state of a-lac-talbumin. FASEB J. 10, 102-109.

Kuwajima, K., Hiraoka, Y., Ikeguchi, M. & Sugai, S.(1985). Comparison of the transient folding inter-mediates in lysozyme and a-lactalbumin. Biochemis-try, 24, 874-881.

Loria, J. P., Rance, M. & Palmer, A. G. P., III (1999). Arelaxation-compensated Carr-Purcell-Meiboom-Gillsequence for characterizing chemical exchange byNMR spectroscopy. J. Am. Chem. Soc. 121, 2331-2332.

Mandel, A. M., Akke, M. & Palmer, A. G., III (1996).Dynamics of ribonuclease H: temperature depen-dence of motions on multiple time scale. Biochemis-try, 35, 16009-16023.

Orekhov, V. Y., Pervushin, K. V. & Arseniev, A. S.(1994). Backbone dynamics of (1-71)bacterioopsinstudied by two-dimensional 1H-15N NMR spec-troscopy. Eur. J. Biochem. 219, 887-896.

Orekhov, V. Y., Pervushin, K. V., Korzhnev, D. M. &Arseniev, A. S. (1995). Backbone dynamics of (1-71)-and 91-36)bacterioopsin studied by two-dimensional1H-15N NMR spectroscopy. J. Biomol. NMR, 6, 113-122.

Otting, G., Liepinsh, E. & WuÈ thrich, K. (1993). Disul®debond isomerization in BPTI and BPTI(G36S): anNMR study of correlated mobility in proteins. Bio-chemistry, 32, 3571-3582.

Palmer, A. G., III & Bracken, C. (1999). Spin relaxationmethods for characterizing picosecond-nanosecondand microsecond-millisecond motions in proteins.In Applications of NMR to the Study of Structure andDynamics of Supramolecular Complexes (Pows, M.,ed.) pp. 171-191 NATO ASI Series, KluwerAcademic Publishers, Dordrecht.

Palmer, A. G., III, Williams, J. & McDermott, A. (1996).Nuclear magnetic resonance studies of biopolymerdynamics. J. Phys. Chem. 100, 13293-13310.

Peng, Z. & Kim, P. S. (1994). A protein dissection studyof a molten globule. Biochemistry, 33, 2136-2141.

Peng, Z., Wu, L. C. & Kim, P. S. (1995). Local structuralpreferences in the a-lactalbumin molten globule.Biochemistry, 34, 3248-3252.

Pike, A. C. W., Brew, K. & Acharya, K. R. (1996). Crys-tal structures of guinea-pig, goat, and bovine a-lac-talbumin highlight the enhanced conformational¯exibility of regions that are signi®cant for itsaction in lactose synthase. Structure, 4, 691-703.

Press, W. H., Teukolsky, S. A., Vettering, W. T. &Flannery, B. P. (1992). Numerical Recipes in C. 2ndedit., Cambridge University, New York.

Ptitsyn, O. B. (1995). Structure of folding intermediates.Curr. Opin. Struct. Biol. 5, 74-78.

Ptitsyn, O. B., Pain, R. H., Semisotnov, G. V., Zerovnik,E. & Razgulgaev, O. I. (1990). Evidence for a moltenglobule state as a general intermediate in proteinfolding. FEBS Letters, 262, 20-24.

Schulman, B. A., Red®eld, C., Peng, Z., Dobson, C. M.& Kim, P. S. (1995). Different subdomains are mostprotected from hydrogen exchange in the molten


globule and native states of human a-lactalbumin.J. Mol. Biol. 253, 651-657.

Schulman, B. A., Kim, P. S., Dobson, C. M. & Red®eld,C. (1997). A residue-speci®c NMR view of the non-cooperative unfolding of a molten globule. NatureStruct. Biol. 4, 630-634.

Shaka, A. J., Lee, C. J. & Pines, A. (1988). Iterativeschemes for bilinear operators: applications to spindecoupling. J. Magn. Res. 77, 274-293.

Smith, L. J., Dobson, C. M. & van Gunsteren, W. F.(1999). Side-chain conformational disorder in a mol-ten globule: molecular dynamics simulations of theA-state of human a-lactalbumin. J. Mol. Biol. 286,1567-1580.

Uchiyama, H. M., Perez-Prat, E., Watanabe, K.,Kumagai, I. & Kuwajima, K. (1995). Effects ofamino acid substitutions in the hydrophobic core of

a-lactalbumin on the molten globule state. ProteinEng. 8, 1153-1161.

Wagner, G. (1983). Characterization of the distributionof internal motions in the basic pancreatic trypsininhibitor using a large number of internal NMRprobes. Quart. Rev. Biophys. 16, 1-57.

Wagner, G. (1993). NMR relaxation and protein mobi-lity. Curr. Opin. Struct. Biol. 3, 748-754.

Wu, L. C. & Kim, P. S. (1997). Hydrophobic sequenceminimization of the a-lactalbumin molten globule.Proc. Natl Acad. Sci. USA, 94, 14314-14319.

Wu, L. C., Peng, Z. & Kim, P. S. (1995). Bipartite struc-ture of the a-lactalbumin molten globule. NatureStruct. Biol. 2, 281-286.

Zinn-Justin, S., Berthault, P., Guenneugues, M. &Desvaux, H. (1997). Off-resonance rf ®elds inheteronuclear NMR: application to the study ofslow motions. J. Biomol. NMR, 10, 363-372.

Edited by P. E. Wright

(Received 28 June 1999; received in revised form 27 September 1999; accepted 28 September 1999)

Documents

Characterization of millisecond time-scale dynamics in the molten globule state of α-lactalbumin by NMR