Native-unlike Long-lived Intermediates along the Folding Pathway of the Amyloidogenic Protein  2Microglobulin Revealed by Real-time Two-dimensional NMR

Native-unlike Long-lived Intermediates along the FoldingPathway of the Amyloidogenic Protein �2-MicroglobulinRevealed by Real-time Two-dimensional NMR*□S

Received for publication, September 17, 2009, and in revised form, December 4, 2009 Published, JBC Papers in Press, December 22, 2009, DOI 10.1074/jbc.M109.061168

Alessandra Corazza‡1, Enrico Rennella‡, Paul Schanda§2, Maria Chiara Mimmi‡, Thomas Cutuil§, Sara Raimondi¶�,Sofia Giorgetti¶�, Federico Fogolari‡, Paolo Viglino‡, Lucio Frydman**, Maayan Gal**, Vittorio Bellotti¶�,Bernhard Brutscher§, and Gennaro Esposito‡3

From the ‡Department of Biomedical Science and Technology, University of Udine, Piazzale Kolbe 4, 33100 Udine, Italy, the§Institut de Biologie Structurale Jean-Pierre Ebel, 41 Rue Jules Horowitz, UMR5075 CNRS-Universite Joseph Fourier-Centre d’EtudeAtomique, 41, rue Jules Horowitz, 38027 Grenoble Cedex 9, France, the ¶Department of Biochemistry, University of Pavia, ViaTaramelli 3b, 27100 Pavia, Italy, the �Biotechnology Laboratory, Fondazione IRCCS, PoliclinicoS, Matteo, 27100 Pavia, Italy, and the**Department of Chemical Physics, Weizmann Institute of Science, Rehovot 76100, Israel

�2-microglobulin (�2m), the light chain of class I major his-tocompatibility complex, is responsible for the dialysis-relatedamyloidosis and, in patients undergoing long term dialysis, thefull-length and chemically unmodified �2m converts into amy-loid fibrils. The protein, belonging to the immunoglobulinsuperfamily, in common to other members of this family, expe-riences during its folding a long-lived intermediate associated tothe trans-to-cis isomerization of Pro-32 that has been addressedas the precursor of the amyloid fibril formation. In this respect,previous studies on the W60G �2m mutant, showing that thelackofTrp-60prevents fibril formation inmild aggregating con-dition, prompted us to reinvestigate the refolding kinetics ofwild type and W60G �2m at atomic resolution by real-timeNMR. The analysis, conducted at ambient temperature by theband selective flip angle short transient real-time two-dimen-sional NMR techniques and probing the �2m states every 15 s,revealed a more complex folding energy landscape than previ-ously reported for wild type �2m, involving more than a singleintermediate species, and shedding new light into the fibrillo-genic pathway. Moreover, a significant difference in the kineticscheme previously characterized by optical spectroscopicmeth-ods was discovered for the W60G �2mmutant.

Among the amyloidogenic proteins that have been most fre-quently studied over the last years, there is �2-microglobulin(�2m)4 that is responsible for dialysis-related amyloidosis.

�2m, the non-polymorphic light chain of class I major histo-compatibility complex, is a small protein that converts intofibrils without the necessity of any chemicalmodification. Fibrilformation occurs in vitro, and most probably also in vivo,through the intact protein. This allows extending experimentalconclusions from in vitro studies to the natural process. Moreimportantly, �2m recapitulates exquisitely the paradigm of thepartially unfolded intermediate, bridging the native fold andthe fibrillar conformation, as traditionally invoked to explainthe conformational transition from the native fold to the amy-loid. Therefore, a detailed characterization of all intermediatestates occurring along the folding pathway is of great impor-tance to understand the process of fibril formation.A kinetic scheme of �2m refolding entailing two fast steps,

burst phase and fast phase, prior to reaching a slow conversionphase from the intermediate to the native state, was firstreported by Chiti et al. (1). This long-lived �2m refolding inter-mediate, termed I2 or IT by different authors, has long beenrecognized as an effective fibril-competent species, formerlyregarded as an ensemble of species (1) and later as a singlespecies (2–4). It has been shown that IT contains a non-nativetrans peptide bond betweenHis-31 and Pro-32 that slowly con-verts into cis conformation during the final refolding step (3).The isomerization occurs with minor rearrangements of theprotein toward the native structure from an already native-likestate (3–5). The native-like intermediate has been proposed asthe effective fibril-competent species (4, 6). However, the rela-tionship between folding and fibrillogenesis is not so simplebecause controversial data have been reported about the fibril-logenesis properties of somemutant proteins, containing trans� angles at position 31. In fact, the experimental data of Jahn etal. (4) show that the mutant P32G does not promote fibrillo-genesis in its native form, based on the positive correlationbetween the intermediate concentration and the extent offormed fibrils (4). It follows that the presence of a trans peptidebond between residues 31 and 32 within the native structure of�2m is not a sufficient condition to induce fibrillar aggregation;rather, some partial unfolding, perhaps associated with oligo-merization, is needed to trigger amyloid formation. This infer-ence was further reinforced by Sakata et al. (7), who showed

* This work was supported by grants from Ministero dell’Istruzione,dell’Universita e della Ricerca (FIRB Grant RBNE03PX83 and RBRN07BMCTand PRIN 2007_XY59ZJ_002), the European Union (EU) (EURAMY ProjectLSHM-CT-2005-037525; EU-NMR Contract 026145), and the French AgenceNationale pour la Recherche (Grant ANR-08-BLAN-0210).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Tables 1 and 2 and Figs. 1 and 2.

1 To whom correspondence may be addressed. Tel.: 39-0432-494322; Fax:39-0432-494301; E-mail: [email protected].

2 Present address: Laboratorium fuer Physikalische Chemie ETH Hoengger-berg, 8093 Zurich, CH, Switzerland.

3 To whom correspondence may be addressed. Tel.: 39-0432-494322; Fax:39-0432-494301; E-mail: [email protected].

4 The abbreviations used are: �2m, �2-microglobulin; SOFAST, selective flipangle short transient; HMQC, heteronuclear multiple quantum coherence;FTA, fluid turbulence-adapted; I, intermediate; N, native; U, unfolded state.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 8, pp. 5827–5835, February 19, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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that, in contrast to P32G, the mutant P32V does not undergoany amyloid transition. They also pointed out the necessity ofintroducing a coupling between the trans-cis isomerization andthe adjacent kinetic step and suggested a minimal foldingmodel (7). The apparently monoexponential unfolding patternof the spectroscopic traces was thus rationalized as the conse-quence of the occurrence of two processes with similar timing:unfolding and trans-cis peptidyl-prolyl isomerization betweenpositions 31 and 32. Recently, further evidence of a substan-tially native-like structure of the refolding intermediate for theconstant domain of an antibody light chain (CL) was providedby NMR experiments, performed at very low temperature, andmolecular dynamics simulations (8). It is worth noting that�2m and CL belong to the same immunoglobulin superfamily,but despite the presence of a slow refolding intermediate, CLdoes not convert into amyloid fibrils.Traditionally, protein folding studies have been performed

by following the reaction by optical spectroscopies such as fluo-rescence or circular dichroism coupled with efficient mixingdevices to obtain a good temporal resolution. Although thesemethods are characterized by high sensitivity and good timeresolution, they do not provide detailed residue-resolved infor-mation on the folding process because of their reliance on onlyvery few detectable probes. Multidimensional NMR spectros-copy overcomes some of these limitations as kinetic and struc-tural information can be obtained in real-time during therefolding process for a large number of sites within the protein.A major breakthrough for the application of real-time NMR toatom-resolved studies of protein folding has been the in-troduction of fast and ultrafast multidimensional NMR tech-niques (9, 10). In particular, the SOFAST-HMQC (11–13) andultraSOFAST-HMQC (14, 15) experiments allow the recordingof two-dimensional 1H-15N correlation spectra of proteins at�0.1–1 s�1 repetition rates, thus providing the required timeresolution for the study of kinetic molecular processes occur-ring on the seconds-to-minutes time scale.A number of mutations of �2m have been exploited to reveal

possible determinants in the fibrillogenesis phenomenon; inparticular, Trp-60 was identified by molecular dynamics simu-lations as one of the essential residues for the aggregation pro-cess (16). In this work, SOFAST real-time NMR techniques areemployed to gain more insight into the folding process of wildtype and W60G �2m at ambient temperature. The results,obtained by probing the folding every 15 s at atomic level, showthat the process is more complex than previously reported andseems to involve more than a single long-lived intermediate.Moreover, our NMR data reveal a significant difference in thefolding pathway of W60G �2m when compared with wild type�2m, which was not detectable by optical spectroscopic meth-ods (17).

EXPERIMENTAL PROCEDURES

Proteins—Expression and purification of wild type andW60G�2mwas carried out as reported previously (17, 18) withadditional 15N uniform labeling. A methionine residue wasalways present at the N-terminal position of all recombinantproducts. All the recombinant proteins gave a single specieswhen assayed by electrospray-ionization mass spectrometry.

Refolding Protocol—The pH-jump protocol here adopted hasalready been reported by Kameda et al. (3). The protein wasunfolded in the denaturing solution (23mMHCl, 1.5 M urea, pH2.2), and the pH was determined to range between 2.4 and 3.0.The refolding was started by injecting 100�l of refolding buffer(300 mM phosphate, 1.5 M urea, pH 7.4) into 360 �l of unfolded�2m solution using a fast injection device for rapidlymixing thetwo solutions inside the NMR magnet (13). At the end of thefolding process and of the measurements, the final pH wasdetermined to range between 6.6 and 6.8. The temperature ofall refolding measurements was 23.9 °C.NMR Data Acquisition and Processing—All NMR experi-

ments were performed with a Varian INOVA spectrometeroperating at 800 MHz (1H frequency), equipped with a cryo-genic probe. The refolding process was started inside the spec-trometer, using an injection device, as already described else-where (13). The reactions were then followed through a seriesof FTA-SOFAST-HMQC spectra (13) of about 15-s duration.Short interscan delays could be used because of the efficient T1relaxation, resulting in overall single-scan times of �100 ms(11). Nitrogen decoupling was achieved by the use of aWURST-40 train (19), whereas the States et al. (20) schemewasemployed for sign discrimination with respect to the nitrogencarrier frequency. Each experiment was performed with onlyone scan for T1 increment, and phase cycling was performed insubsequent spectra to eliminate artifacts, as described before(13). The experiments were processed with NMRPipe (21) Inthe indirect dimension F1, the original data set of 60 real pointswas extended by linear prediction (30 points) and zero-filled to128 points, and a squared sine-bell functionwas employed,witha shift of �/4. In the direct dimension F2, a sine-bell functionwith a shift of 2�/5 was employed. Following two-dimensionalFourier transform, a fourth order polynomial baseline correc-tion was applied.NMR Data Analysis—Spectra inspection, peak assignment,

and volume calculation were performed using NMRView (22).Different classes of peaks, detailed under “Results,” were ana-lyzed. Here, it is worth noting that for native (N) and interme-diate (I) species peaks, the boxes, used in volumemeasurementsto confine the peak areas, comprise only one peak, whereas forI�N peaks, the boxes are bigger and contain two peaks belong-ing to I and N species. As a consequence, the error on the mea-sured volumes is likely to be higher for the peaks of the latterclass than for those of the first two classes. NMR assignmentswere taken from Biological Magnetic Resonance Bank (BMRB)entries 3078 (23) and 15480 (18), with slight adjustments aris-ing from thermal variation of the chemical shifts. Fits of thetime course of the peak volumes were done usingMathematica6 (Wolfram Research). Statistical analysis of the fit goodnesswas performed using Student’s t test for the fitting parameters,using F-tests on the sum of the squares of the residuals, andcomparing the adjusted R-square values.

RESULTS

A Single Slow Phase Does Not Account for �2m Folding—In arecent study (17), and in agreement with published data (1, 2),we confirmed the change in tryptophan fluorescence emissionduring the folding process of wild type�2m, consistentwith the

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presence of a slowphase ascribed to Pro-32 isomerization (3, 4).Analogous experiments were performed on theW60Gmutant,forwhich a reduced association and aggregation propensity hadbeen revealed (17). Unlike wild type�2m, no folding slow phasecould be detected through fluorescence spectra for thismutant,suggesting thatW60G �2m reaches a native-like conformationin a couple of minutes, at least as sensed by the fluorescenceprobes. It is worth noting that the fluorescence changes uponfolding of �2m are essentially due to Trp-95 that becomes bur-ied in the native conformation, whereas Trp-60 contributesonly marginally because it remains solvent-exposed also in thefolded state, as shown by Kihara et al. (24).To gain further insight into the folding process of �2m spe-

cies, we used real-time two-dimensional SOFAST NMR spec-troscopy. The refolding of wild type andW60G �2m was stud-ied at 23.9 °C starting from protein samples in 1.5 M urea atpH � 2.2. To increase the timing efficiency, the refolding reac-tionwas initiated inside themagnet (13) by rapidly injecting therefolding phosphate buffer at pH� 7.4. The pH that was finallyachieved ranged from 6.6 to 6.8 for the different samples. Therefolding process was followed by acquisition of a series ofSOFAST-HMQC spectra recorded at a 0.07 s�1 rate using asequence optimized for the use of a fast mixing device (FTA-SOFAST-HMQC) (13). The measurements were repeatedtwice for each protein (wild type andW60Gmutant) to ensurereproducibility of the results and to evaluate their accuracy. Fig.1 shows NMR spectra recorded before injection, immediatelyafter injection, and once the refolding process was completed.Surprisingly, the NMR data obtained for the wild type and

W60G proteins showed similar features, especially the pres-ence of two protein states characterized by distinct sets of peaksthat can be ascribed to the native state (N) and to an interme-diate state (I). Thus, in contrast to fluorescence data, our NMRdata reveal the presence of a long-lived folding intermediate forboth wild type and W60G �2m. The absence of unfolded state(U) peaks in the very first NMR spectra acquired after initiatingthe refolding process indicates that the conformational transi-tion fromU to I was completed within the dead time (�10 s) ofthe kinetic experiment.For amore detailed quantitative analysis of theNMRdata, we

classified the observed NMR correlation peaks in three differ-ent classes. 1) The first class was pure native state peaks (N) forwhich the chemical shift values match those of native �2munder equilibrium conditions. In this class, we included onlythewell isolated peakswith a volume ratio between the first andthe last spectrum below 15%. These peaks report on the growthof the native state population over time. 2) The second classwaspure intermediate state peaks (I) for which the chemical shiftvalues differ from both the unfolded and the folded states.These peaks correspond to amide sites with a local chemicalenvironment in the intermediate state that is different from theone in the native state. The decay of these peaks is related to thedecrease of the intermediate species (I) over time. 3) The thirdclass was peaks arising from overlap of intermediate and nativespecies resonances (I�N) due to similar chemical environ-ments of the corresponding amide groups in the I-state and theN-state. Intensity changes over time observed for this class ofpeaks provide useful information on the presence of additional

folding intermediates and differences in local dynamicsbetween the two states.This classification enabled the identification of 27/27 (class

N), 9/18 (class I), and 18/18 (class I�N) peaks in the spectra ofwild type/W60G �2m, respectively. Examples of residue-spe-cific kinetic folding curves obtained from the volume changesof class N, I, and I�N peaks over time are shown insupplemental Fig. 1. As expected, within each peak family, thesame kinetic behavior is observed within the experimental error.Therefore, to increase the precision of the quantitative kineticanalysis, the volumes for all peaks of a given class, N, I, and (I�N),wereadded.Theresultingkinetic tracesare showninFig.2 forwildtype andW60G �2m. In the case of a simple two-state transitionfrom I to N, one would expect a monoexponential behavior forboth, the decrease of I-state, and the increase of N-state, and as aconsequence, no change in the (N�I) peak volumes over time.Fitting reveals that amonoexponential curve does not account fortheobserved foldingkinetics of theN-state, particularly in the caseof the wild type protein. This observation is supported by the sta-tistical Student’s t test and by the analysis of the residuals (supple-mental Tables 1 and 2), according towhich a biexponentialmodelleads to a significant improvement in the fit quality and reliability.The statistical tests reject with very high probability the redun-dancy of the biexponential with respect to the monoexponentialmodel.According to all the examined data sets for both wild type

and W60G �2m, two slow folding phases are required toaccount for the formation of the native state (Fig. 2, a and b, andTable 1) with a first rate constant on the order of 10�3 s�1, k2,and a second, slower one on the order of 10�4 s�1, k3. Animportant difference between the wild type and mutant pro-teins is observed with respect to the amplitudes of the twokinetic phases. For the W60G mutant, the slower phase onlyaccounts for about 5%, whereas it is more pronounced in thewild type protein with values ranging from 16 to 25%, depend-ing on the experimental data set used (Table 1). Instead, thedecay of the observed intermediate species follows a monoex-ponential kinetics for both �2m variants (Fig. 2, c and d, andTable 1). This decay is characterized, in the case of wild type�2m, by a slightly slower rate constant (k1 � (1.56� 0.14) 10�3

s�1) than for the mutant (k1 � (2.35 � 0.07) 10�3 s�1). Inter-estingly, the decay rate of the intermediate species for wild type�2m does not match any of the rates obtained from the biexpo-nential fitting of the native species buildup, whereas for W60G�2m, the rates k1 and k2 are equal, within the statisticaluncertainty.The presence of two different kinetic constants suggests the

existence of an additional intermediate species that does notgive rise to detectable NMR signals. This conclusion is furthersupported by the kinetic traces obtained from the third class ofpeaks (I�N), shown in Fig. 2, e and f, for wild type and W60G�2m, respectively. For both proteins, the signal intensity ofclass I�Npeaks, and consistently the sumof I-state andN-statepopulations, are not constant throughout the experimentalobservation interval. Again, this observation points toward thepresence of some “invisible” species that converts into thenative state together with the distinctly observed slow refoldingintermediate I.

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The data from class I�N peaks were fitted for the wild typeprotein to a biexponential equation, yielding rate constants of(4.6 � 0.1) � 10�3 s�1 and (6.25 � 0.95) � 10�4 s�1. These valuesare of the same order of magnitude as those extracted from the

native state growth curve (classNpeaks). For theW60Gmutantprotein, amonoexponential function satisfactorily accounts forthe class I�N peak data, with a kinetic rate constant of (1.6 �0.2) � 10�3 s�1. The contribution of the additional NMR-invis-

FIGURE 1. Refolding of wild type and W60G �2m studied by two-dimensional FTA-SOFAST-HMQC experiments. a–f, NMR spectra acquired before thestarting of the refolding reaction (a and d), after 25 s (b and e) and after 2 h (c and f) for wild type and W60G �2m, respectively. Negative peaks (in red) are dueto spectral folding. In the zoomed regions N, I, and I�N peaks are boxed in blue, green, and red, respectively.

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ible species to the folding process can be estimated to (35� 1)%and (14 � 1)% for wild type and W60G �2m, respectively. Aswith the other peak classes, the robustness of the fitting is con-firmed by analysis of the residuals.The increase in peak volume observed for class I�N peaks

during the refolding process may also be due to pronounceddifferences in the overall rotational tumbling, e.g. due to partial

aggregation or internal mobility ofthe protein in the I-states and theN-states, resulting in different spinrelaxation properties and thus inunequal signal intensities in theNMR spectra even for equal popula-tions of the two states. To investi-gate whether differences in amide1H and 15N relaxation between theN-state and the I-state are at the ori-gin of the missing peak intensities,we evaluated line widths of typicalcross peaks assigned to the N-stateand the I-state. 1H and 15N linewidths are identical within experi-mental error, thus weakening thedifferential relaxation property ar-gument as a possible explanation forthe signal gain observed for classI�N peaks as folding proceeds. Inaddition, refolding measurementsperformed with longer recyclingdelay (d1) give the same results asobtained with shorter d1 (see sup-plemental Fig. 2), which furtherrules out the differential relaxationrate argument.The real-time two-dimensional

NMR data provide clear evidencethat the slow folding phase observedfor �2m is not a simple two-stateprocess from an intermediate stateaccumulated during the first sec-onds of the folding process to thenative state but that additionallong-lived intermediate states arerequired to explain the observedfolding kinetics. The burst phaseamplitude, corresponding to theamount of protein that folds directlyto the native state via a pathway thatdoes not require any slow phase orlong-lived intermediate, has beencalculated by extrapolating to zerothe fitting curve of the N-state vol-umes. This burst phase amplitude isabout (5.6 � 1.3)% for wild type�2m and slightly higher (10.6 �3.4)% for the W60G mutant. Thesevalues are significantly lower thanthe 20% figure previously obtained

by Kameda et al. (3) for wild type �2m also from real-timeNMR measurements, although under different experimentalconditions.Possible Kinetic Schemes for �2m Folding—Different models

have been proposed for �2m refolding, ranging from simplelinear two-step schemes (1, 3) to parallel models with two orthree intermediate species (4, 7). As already pointed out above,

FIGURE 2. Kinetics fittings for the global data set of wild type (WT) and W60G �2m. a–f, kinetics of nativestate formation (a and b), intermediate species decay (c and d), and native and intermediate summation (e andf) for wild type (a, c, and e) and W60G �2m (b, d, and f). For each class, the sum of the measured peak volumesis plotted as a function of time. Black circles, experimental data; orange line, monoexponential fits; green lines,biexponential fits; orange circles, monoexponential residual; green circles, biexponential residuals.

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a scheme involving a single intermediate species is not suffi-cient to explain the biexponential growth of the native species.Furthermore, a parallel model (Fig. 3b), i.e. a scheme with twodifferent intermediates that convert into the native state alongparallel pathways, resembling the one that entails two slowsteps proposed by Goto and co-workers (7), also has to be dis-carded in the case of wild type �2m. The main reason is theimpossibility of enforcing, in amechanismwith two intermedi-ates, the three significantly different kinetic constants resultingfrom the fitting of the N- and I-state peaks. Other differentschemes involving two intermediates were evaluated unsuc-cessfully, leading to similar functional forms that essentiallyconverge into equations with only two apparent rates. In fact,the solution of the set of differential equations describing anykinetic scheme implies the correspondence between n eigen-values (apparent kinetic constants) and the presence of n�1species. Only a more complex parallel model involving at leastfive states, e.g. of the type illustrated in Fig. 3a (Model A), couldsatisfy our kinetic data. Additional experimental information,not available from our NMR data, would be required, however,to select a specific mechanism among the many possible five-state kinetic schemes and to calculate themicroscopic rate con-stants. A five-statemodel was previously proposed by Radford’sgroup (4), but a direct numerical comparison with our datawere not viable, due to different experimental temperatures. Inessence, our analysis, exclusively based on the decrease of I andthe growth of the native species, cannot exclude schemes otherthan model A (Fig. 3a) but must include more than two inter-mediate species, all NMR-silent but one.A simpler parallel model with two intermediate species (Fig.

3,Model B) accounts for the observedW60G folding kinetics. Irepresents the most populated intermediate species (on aver-age (75 � 5)%) that converts to N with a microscopic kinetic

FIGURE 3. Plausible folding mechanisms of wild type and W60G �2m. a, apossible five-state scheme consistent with wild type �2m refolding evidence.U, N, and I represent the unfolded, native, and NMR-visible, intermediate spe-cies, respectively. The two NMR-silent intermediates I* and I** may be eithersingle-molecule species or oligomeric forms. The sketched mechanism is aminimal scheme of five species that accounts for the four significantly differ-ent apparent kinetic constants (eigenvalues) that are necessary to fit the dataset. Any system of kinetic differential equations with a solution of n eigenval-ues implies the presence of (n � 1) species. The presented scheme is consis-tent with our data also in the limiting reduction to the kinetic model B, underthe assumption that I**3 I is very fast. As the available data do not enable usto discriminate among the possible permutations of a five-species scheme,no further attempt was made to extract numerical kinetic constants. b, a par-allel model that satisfactorily accounts for W60G mutant refolding data. Thethree satisfyingly different apparent kinetic constants fitting the data set leadto a model with four species involved, three observable, U, I, and N, andanother unobservable named I*. N, I, and I�N kinetic traces are best-fitted byapparent kinetic constants �1 � (2.35 � 0.05) � 10�3 s�1 and �2 � (1.60 �0.05) � 10�4 s�1 under the assumption ��, �� �� �1, �2. The population distri-bution of the different pathways are given as percentages.

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�3

(5�

2)�

10�4

89.6

�0.9

3.2

�0.9

7.2

�2.3

I�N

(1.80

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)�10

�3

85.3

�2.4

W60

G—

seco

ndset

I(2.28

�0.02

)�10

�3

Nmon

o(2.06

�0.01

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�3

15.7

�0.1

Nbiexp

(2.22

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�3

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81.9

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�4.2

Two-dimensional NMR Reveals the Folding Pathway of �2m

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rate constant of 2.35 � 10�3 s�1, whereas I* represents the leastpopulated ((14 � 5)%) and NMR-silent intermediate and con-verts to N at a rate of 1.6 � 10�3 s�1. A further (11 � 3)% ofunfolded W60G �2m transforms directly into N without pass-ing through any of these intermediate species, conceivablybecause of an already attained cis configuration at the His-31–Pro-32 � angle. Note that the five-state model A reduces to thesimplermodel B assumed for themutantW60G if the step fromI** to I is very fast.Structural Properties of the Intermediate Species—The real-

time NMR study reported here allowed us to identify at leasttwo distinct long-lived intermediates of �2m folding, both forthe wild type species and for the W60G mutant. These twointermediate states have completely different spectral signa-tures. Although the I-state is directly observed in the NMRspectra as a set of 1H-15N correlation peaks characteristic of awell defined globular conformation, the I* together with I**states do not give rise to any detectable NMR signal.Even without the sequential resonance assignment of the

I-state, some structural information is obtained from the anal-ysis of its spectral pattern, observed at the beginning of the slowfolding phase. In both �2m variants, some peaks of this inter-mediate species were found to overlap those of the native state(Fig. 4a), indicating that for those amides, the local chemicalenvironment in the I-state is close to that in the N-state. Thedistribution of amide groups with similar chemical shifts in theI- andN-states clusters in precise locations of themolecule (Fig.4b), at the opposite side of Pro-32, which has a cis peptide con-figuration in the native state but may have not yet adopted thisconfiguration in the intermediate species. Indeed, the magni-tude of the decay rate of the native-like intermediate is typical ofproline peptide bond isomerization (25). The native-like inter-mediate is thus likely to possess a trans configuration at Pro-32,a structure similar to that of mutant P32A (a crystallographicdimer, PDB entry 2F8O (6)). Further work is in progress todetermine the solution structure of the intermediate throughfast multidimensional NMR techniques. All these observationsare consistent with the previously reported results (3, 4) thatstressed the key role of trans-cis isomerization as the rate-lim-iting step for the folding of�2m. It is interesting to note that thepeak integrals of the native-like intermediate are lower in wildtype than in W60G �2m (Fig. 4c). This observation correlateswith the population levels of additional species present at thebeginning of the slow folding phase, especially for wild type�2m, as inferred from the multiexponential kinetics of nativestate formation.The structural nature of the additional, presumably native-

unlike, intermediates is unknown. By NMR, we were only ableto observe the native-like intermediate but not the other spe-cies. Twomechanisms can reasonably account for the substan-tial line broadening determining the NMR invisibility of theadditional intermediate species: either a conformationalequilibrium at unfavorable exchange rates (�s to ms range),plausibly within an ensemble of unfolded forms, or an exten-sive association/aggregation process of the same forms.Finally, because all the slow folding rates found in this workare compatible, as mentioned, with a proline peptide bondisomerization, it is reasonable to assume that a trans config-

uration for the � angle preceding Pro-32 is also present inthe additional intermediate(s).

DISCUSSION

The present work takes advantage of the recent advances infast multidimensional NMR techniques to gain a more detailedview into the folding process of�2m at ambient temperature. Asimilar approach was recently applied to bovine �-lactalbumin(13), where a good agreement in the measured folding kineticswas found between NMR and fluorescence methods.Instead, in this work, a surprising mismatch between NMR

and fluorescencewas found; in contrast to CD and fluorescencedata (17), our NMR results reveal the presence of a slow phasefor W60G �2m. Moreover, the presence of at least two foldingslow phases for wild type �2m had neither been recognizedbefore nor considered in any of the previously proposed foldingkinetic schemes (1, 3, 4).Kameda et al. (3) performed a similar analysis on wild type

�2m using conventional HSQC NMR experiments at 2.8 °C,but they were unable to detect the multiexponential kineticsof native state formation we observed at ambient tempera-ture. Apart from differences in the experimental tempera-ture that could profoundly affect the folding mechanism, thelower time resolution of conventional NMR experiments incomparison with the SOFAST counterparts could also jus-tify the different ability to go deeper into the fine details ofthe kinetic processes. A further difference between the pres-ent analysis and the results reported by Kameda et al. (3)regards the values of the burst phase amplitude. We foundburst phase amplitudes around 5%, i.e. significantly belowthe previously reported value of 20% (3). Again, the differ-ences in data collection and modeling could well be respon-sible for this discrepancy.Close inspection of theNMRpattern observed for the I inter-

mediate enabled recognizing a native-like structure in theregion opposite to Pro-32, in agreement with previous findings(3, 4). On the contrary, the residues close to Pro-32, the facingN-terminal end, and theDE loop of the observable intermediatespecies could not be identified,mainly because this is the regionwhere most structural differences are expected in comparisonwith the native state. An additional reason could be the exten-sive broadening of these resonances because of the unfavorablerates of conformational exchange, whose remnants are alsopresent in the fully folded wild type protein, as revealed byR2/R1 relaxation data (17).As detailed above, the biexponential growth of the native

species and the apparent monoexponential decay of the inter-mediate species, characterized by three different apparentrates, rule out a model involving only two intermediate speciesand prompt us to propose a more complex mechanism. Theproposed five-state kinetic scheme can account for the discrep-ancy between optical and NMR results observed upon compar-ingwild type andW60G�2m. In thismodel, three intermediatespecies are included, one, I, observable byNMR and two others,I* and I**, corresponding to NMR-silent species. No data areavailable for I* and I**, but the failure to observe them in theNMR spectra could be due to unfavorable conformationaldynamics and/or oligomerization.

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As experienced by W60G mutant, the parallel foldingscheme (model B) predicts a preferential pathway, with I accu-mulating more than 5-fold with respect to I*. This is consistentwith a virtually undetectable folding slow phase forW60G�2min fluorescence spectra and demonstrates that the I intermedi-ate, highly native-like, is fluorometrically indistinguishablefrom the native state. In other words, fluorescence does notdiscriminate between natively folded species and native-likeintermediates of �2m. Hence, the slow phase observed by fluo-rescence must be a native-unlike, NMR-silent species. In wildtype �2m, a slow folding phase is detected by fluorescencebecause the additional long-lived I* and I** intermediates thataccumulate to a higher extent must be structurally differentfrom the N-state in the vicinity of residue Trp-95.Therefore, although the NMR undetectability of I* and I**

gives some indications about their oligomeric and dynamicproperties, the fluorescence response suggests that I* and I**

are intermediate states that have not yet reached a native-likepacking of the hydrophobic core. The higher population of thenative and native-like conformations, N and I, for W60G, incomparison with wild type �2m, correlates with the higherthermodynamic stability of the mutant protein (17).The presence of folding intermediates and the fibrillogenesis

propensity of a protein is regarded as an important correlationwhen studying amyloidosis (26, 27). Along this view, Chiti et al.(2) proposed that I2, a folding intermediate associated with theslow process, is involved in the amyloid deposition, on the basisof the enhanced seed elongation during the folding; later, thisidea was recovered by other groups (3, 4, 24). Controversialcases are represented, however, by the different amyloidogenicpropensity of P32G and P32V �2m (4, 7) or by the inability ofthe CL antibody to form fibrils (8).

In this work, we show that the correlation between foldingand fibrillogenesis for �2m is more complex than previously

FIGURE 4. Structural characterization of the native-like intermediate. a, representative peaks of the intermediate species that were found to overlap thoseof the native species; black and red contours, respectively, depict the initial and final spectrum i.e. acquired after the start and at the end of the refolding process.b, distribution of amide groups exhibiting native-like chemical shifts drawn as spheres over a schematic representation of �2m structure (PDB 1JNJ). c, volume(first spectrum)/volume (last spectrum) of native-like intermediate peaks for wild type (orange-filled bars) and W60G (empty bars) �2m. Average values anderrors were calculated from two sets of experiments for each variant. The bar in position 101 refers to the indolic group of Trp-95.

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believed. In fact, we demonstrate that more than a single inter-mediate species is transiently populated during the folding slowprocess of �2m and that not all of them correlate with fibrillo-genic capability. In particular, the inability of W60G �2m toelongate fibrils in 20% trifluoroethanol (17) appears coupledwith the occurrence of a predominant native-like intermediatespecies and with a very reduced occurrence of native-unlikeintermediate(s). The latter species transiently populates duringthe folding slow phase of wild type �2m that, in fact, is capableof elongating fibrils in vitro. Because both native-like andnative-unlike species seem related to trans-cis isomerization ofthe peptide bond preceding Pro-32, our findings, although con-firming the crucial role of that peptide bond conversion in the�2m folding, cast some doubts on previous claims for amyloidformation to proceed via a native-like intermediate (4).On the basis of the data in our hands, however, it is not

possible to unequivocally identify theNMR-silent species as theconformer(s) responsible in vivo for the onset of the pathology.Future work will focus on this problem but also on a moredetailed characterization of all the species involved in the fold-ing slow phase, also from a structural point of view.

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C. M., Bellotti, V., and Taddei, N. (2001) J. Mol. Biol. 307, 379–3912. Chiti, F., De Lorenzi, E., Grossi, S.,Mangione, P., Giorgetti, S., Caccialanza,

G., Dobson, C. M., Merlini, G., Ramponi, G., and Bellotti, V. (2001) J. Biol.Chem. 276, 46714–46721

3. Kameda, A., Hoshino, M., Higurashi, T., Takahashi, S., Naiki, H., andGoto, Y. (2005) J. Mol. Biol. 348, 383–397

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5. Corazza, A., Pettirossi, F., Viglino, P., Verdone, G., Garcia, J., Dumy, P.,Giorgetti, S., Mangione, P., Raimondi, S., Stoppini, M., Bellotti, V., andEsposito, G. (2004) J. Biol. Chem. 279, 9176–9189

6. Eakin, C. M., Berman, A. J., and Miranker, A. D. (2006) Nat. Struct. Mol.Biol. 13, 202–208

7. Sakata, M., Chatani, E., Kameda, A., Sakurai, K., Naiki, H., and Goto, Y.(2008) J. Mol. Biol. 382, 1242–1255

8. Feige,M. J., Groscurth, S.,Marcinowski,M., Yew, Z. T., Truffault, V., Paci,E., Kessler, H., and Buchner, J. (2008) Proc. Natl. Acad. Sci. U.S.A. 105,13373–13378

9. Freeman, R., and Kupce, E. (2003) J. Biomol. NMR 27, 101–11310. Malmodin, D., and Billeter, M. (2005) Prog. Nucl. Magn. Reson. Spectrosc.

46, 109–12911. Schanda, P., and Brutscher, B. (2005) J. Am. Chem. Soc. 127, 8014–801512. Schanda, P., Van Melckebeke, H., and Brutscher, B. (2006) J. Am. Chem.

Soc. 128, 9042–904313. Schanda, P., Forge, V., and Brutscher, B. (2007) Proc. Natl. Acad. Sci.

U.S.A. 104, 11257–1126214. Gal, M., Schanda, P., Brutscher, B., and Frydman, L. (2007) J. Am. Chem.

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Bellotti, V., and Esposito, G. (2007) Biophys. J. 92, 1673–168117. Esposito, G., Ricagno, S., Corazza, A., Rennella, E., Gumral, D., Mimmi,

M. C., Betto, E., Pucillo, C. E., Fogolari, F., Viglino, P., Raimondi, S., Gior-getti, S., Bolognesi, B., Merlini, G., Stoppini, M., Bolognesi, M., and Bel-lotti, V. (2008) J. Mol. Biol. 378, 887–897

18. Esposito, G., Michelutti, R., Verdone, G., Viglino, P., Hernandez, H., Rob-inson, C. V., Amoresano, A., Dal Piaz, F., Monti, M., Pucci, P., Mangione,P., Stoppini, M., Merlini, G., Ferri, G., and Bellotti, V. (2000) Protein Sci. 9,831–845

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(1995) J. Biomol. NMR 6, 277–29322. Blevins, R. A., and Johnson, B. A. (1994) J. Biomol. NMR 4, 603–61423. Okon, M., Bray, P., and Vucelic, D. (1992) Biochemistry 31, 8906–891524. Kihara,M., Chatani, E., Iwata, K,, Yamamoto, K.,Matsuura, T., Nakagawa,

A., Naiki, H., and Goto, Y. (2006) J. Biol. Chem. 281, 31061–3106925. Cheng, H. N., and Bovey, F. A. (1977) Biopolymers 16, 1465–147226. Chiti, F., and Dobson, C. M. (2006) Annu. Rev. Biochem. 75, 333–36627. Chiti, F., and Dobson, C. M. (2009) Nat. Chem. Biol. 5, 15–22

Two-dimensional NMR Reveals the Folding Pathway of �2m

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Supporting Information

The monoexponential and the biexponential models for the native state formation were compared by statistical analysis of the fitting parameters and

residuals distributions. Two set of data for each protein, from independent series of measurements, were processed and analyzed.

set df tA2 tA3 tk2 tk3 tNeq pA2 pA3 pk2 pk3 pNeq

wt 1st 318 -62.0851 -24.483 44.1875 4.29785 88.2726 8.12E-180 3.80E-75 8.80E-138 2.30E-05 1.02E-225

wt 2nd 437 -42.2262 -17.5629 34.0894 16.8946 483.12 2.48E-156 1.24E-52 3.54E-125 1.20E-49 0.00E+00

W60G 1st 488 -46.7747 -1.24337 42.2439 1.52216 641.996 1.88E-182 2.14E-01 3.94E-165 1.29E-01 0.00E+00

W60G 2nd 489 -80.4548 -6.22323 53.1178 2.65231 226.577 3.64E-284 1.05E-09 3.26E-205 8.25E-03 0.00E+00

Table 1. Two-tailed t-Student tests for the 5 fitting parameters of the biexponential model (32

2 3

−−− = − −

k tk t

eqN( t ) N A e A e ). The table reports

t-values for the null hypothesis (i.e. each parameter value is zero) and the corresponding probabilities. All parameters are significantly different from

zero, except those relative to the slower phase for the 1st set of W60G β2m (A3 and k3) that present, respectively, a probability of 21 and 13% to be

zero.The degrees of freedom (df) are equal to the size of each data set minus 5.

set SSRmono SSRbi F P TSS R2mono R2

bi R2adj,mono R2

adj,bi

wt 1st 2.25E+12 7.72E+11 304.515271 1.31E-74 2.31E+14 0.99026 0.99666 0.99020 0.99662

wt 2nd 8.51E+11 2.22E+11 619.597825 2.69E-128 6.05E+13 0.98593 0.99633 0.98586 0.99630

W60G 1st 1.74E+11 1.68E+11 8.48003518 2.40E-04 3.21E+13 0.99458 0.99476 0.99456 0.99472

W60G 2nd 2.65E+11 2.14E+11 58.4170842 1.78E-23 3.58E+13 0.99261 0.99403 0.99258 0.99398

Table 2. One-tailed F-tests comparing the sum of the squares of the residuals (SSR) for the mono exponential ( 2

2

−− = −

k t

mono eqN ( t ) N A e ) and

5-parameters bi exponential (32

2 3

−−− = − −

k tk t

bi exp eqN ( t ) N A e A e ) models. The F-value is given by (SSRmono – SSRbi) ⋅ (set size – 5) / (2 ⋅

SSRbi) and has a F-distribution with [2, (set size – 5)] degrees of freedom. In all cases, the low P values bring to the rejection of the null hypothesis,

i.e. that the slower phase is absent. Total sum of squares (TSS), R-square and adjusted R-square values also are reported. Adjusted R-square values

are equal to 1 – [SSR ⋅ (set size – 1)]/[TSS ⋅ (set size – number of fitting parameters)] and indicate the goodness of a fitting model.

Figure Legends

Figure 1. Examples of refolding kinetics of wt β2m for selected residues. Panels A, B, C show the time evolution of amide cross peaks belonging to

class N, class I and class I+N, respectively. The rate constants k1, k2, k3, k4, k5 and amplitudes A2, A3, A4, A5 are reported for the different residues.

The kinetic traces are fitted according the following equations: A) N(t) – Neq = -[A2exp(-k2t) + A3exp(-k3t)] , B) I(t) = A1 exp (-k1t) and C) [I+N](t) –

[I+N]eq = -[A4exp(-k4t) + A5 exp(-k5t)], where Neq and [I+N]eq are the values for the N and [I+N] species at the end of the refolding reaction. The burst

phase, bp, is obtained by extrapolating the curves to time 0.

Figure 2. Comparison of wt β2m folding kinetics data obtained using different relaxation delays (d1) in the acquisition of FTA-SOFAST-HMQC

spectra. Except for the different d1 values the experimental protocol is as the one described in the Methods. Kinetics of native state formation a),

intermediate species decay b), native and intermediate summation c). In black and are red indicated the data points acquired, respectively, with short

(d1 = 10 ms) and long (d1 = 1s) relaxation delay. For each class, the sum of the measured peak volumes is plotted as a function of time.

Figure 1

Figure 2

Frydman, Maayan Gal, Vittorio Bellotti, Bernhard Brutscher and Gennaro EspositoCutuil, Sara Raimondi, Sofia Giorgetti, Federico Fogolari, Paolo Viglino, Lucio

Alessandra Corazza, Enrico Rennella, Paul Schanda, Maria Chiara Mimmi, ThomasNMR

-Microglobulin Revealed by Real-time Two-dimensional2βAmyloidogenic Protein Native-unlike Long-lived Intermediates along the Folding Pathway of the

doi: 10.1074/jbc.M109.061168 originally published online December 22, 20092010, 285:5827-5835.J. Biol. Chem. 

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