Structural and Dynamics Characteristics of Acylphosphatase from Sulfolobus solfataricus in the...

Structural and Dynamics Characteristics of Acylphosphatasefrom Sulfolobus solfataricus in the Monomeric State and inthe Initial Native-like Aggregates*□S

Received for publication, November 4, 2009, and in revised form, March 8, 2010 Published, JBC Papers in Press, March 11, 2010, DOI 10.1074/jbc.M109.082156

Katiuscia Pagano‡, Francesco Bemporad§1, Federico Fogolari‡¶, Gennaro Esposito‡¶, Paolo Viglino‡¶,Fabrizio Chiti§¶, and Alessandra Corazza‡¶2

From the ‡Department of Biomedical Sciences and Technologies, University of Udine, Piazzale Kolbe 4, 33100 Udine, Italy, the§Department of Biochemical Sciences, University of Firenze, Viale Morgagni 50, 50134, Firenze, Italy, and the ¶ConsorzioInteruniversitario Istituto Nazionale di Biostrutture e Biosistemi, Viale Medaglie d’Oro 305, 00136 Rome, Italy

It has previously been shown that the acylphosphatase fromSulfolobus solfataricus is capable of forming amyloid-like aggre-gates under conditions in which the native structure is main-tained and via the transient formation of native-like aggregates.Basedon thepreviously determinedNMRstructure of thenativeprotein, showing a ferredoxin-like fold and the peculiar pres-ence of an unstructured N-terminal segment, we show here, at amolecular level using NMR spectroscopy, that indeed S. solfa-taricus acylphosphatase remains in a native-like conformationwhen placed in aggregating conditions and that such a native-like structure persists when the protein forms the initial aggre-gates, at least within the lowmolecularweight species. The anal-ysis carried out under different solution conditions, based onthe measurement of the combined 1H and 15N chemical shiftsand hydrogen/deuterium exchange rates, enabled the most sig-nificant conformational changes to be monitored upon transferof the monomeric state into aggregating conditions and uponformation of the initial native-like aggregates. Importantincreases of the hydrogen/deuterium exchange rates through-out the native protein, accompanied by small and localizedstructural changes, in the monomeric protein were observed.The results also allow the identification of the intermolecularinteraction regions within the native-like aggregates, thatinvolve, in particular, theN-terminal unstructured segment, theapical region including strands S4 and S5 with the connectingloop, and the opposite active site.

Proteins and peptides have a generic propensity to form wellorganized aggregates characterized by a fibrillar morphologyand an extended cross-� structure, generally referred to asamyloid-like fibrils (1, 2). This process is important for a num-

ber of reasons. From a physicochemical perspective, it repre-sents an essential feature of the behavior of polypeptide chainsthat need to be fully understood for a thorough characterizationof the nature of proteins (3). From a more biological perspec-tive, formation of amyloid fibrils, or intracellular inclusionswith structurally related characteristics, is associated with over40 pathological conditions in humans (4, 5). It is also a majorproblem in biotechnology because the large scale expression ofproteins potentially interesting to the market often results intheir self-assembly in inclusion bodies with amyloid-like struc-tural features (4).A large body of experimental data supports the hypothesis

that globular proteins, normally adopting a well defined three-dimensional fold, need to unfold, at least partially, to becomeaggregation-competent (2, 5). However, it is also becomingincreasingly evident that some normally folded proteins canaggregate from native-like states directly accessible throughthermal fluctuations of the native state and with no need oftransitions across the energy barrier for unfolding (reviewed inRef. 6).One of the proteins shown tohave an ability to aggregatedirectly from the native-like state is the acylphosphatase fromSulfolobus solfataricus (Sso AcP).3 The structure of native SsoAcP has been solved with both NMR and x-ray crystallography(7). Similarly to other acylphosphatases so far characterized,Sso AcP has a ferredoxin-like fold and thus consists of twoparallel �-helices packed against a five-stranded �-sheet. How-ever, by contrast with other acylphosphatases of known struc-ture, Sso AcP contains an 11-residue, unstructured segment atthe N terminus (7). In the presence of 15–25% (v/v) 2,2,2-tri-fluoroethanol (TFE), 50 mM acetate buffer, pH 5.5, 25 °C, SsoAcP has been shown to aggregate within 30min into structuredspherical and chain-like protofibrils (8). Protofibril formation isalso observed in the absence of TFE under the same conditions ofpH and temperature, although the process requires weeks in thiscase (8).Theadditionof inorganicphosphate, a competitive inhib-itor binding to the active site of the enzyme, stabilizes the nativefold of the protein and inhibits protofibril formation (9).Under conditions in which protofibril formation occurs

more rapidly (50 mM acetate buffer, 20% (v/v) TFE, pH 5.5,

* This work was supported by MIUR Grants PRIN 2007 XY59ZJ, PRIN M3E2T2,FIRB RBNE03PX83, and FIRB RBRN07BMCT and European Union EURAMYProject LSHM-CT-2005-037525.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table S1 and Figs. S1–S4.

1 Supported in part by the Accademia dei Lincei and by a long term fellowshipfrom the Federation of the European Biochemical Societies. Presentaddress: Dept. of Chemistry, University of Cambridge, Lensfield Road, Cam-bridge CB2 1EW, United Kingdom.

2 To whom correspondence should be addressed: Dept. of Biomedical Sci-ences and Technologies, University of Udine, P. le Kolbe 4, 33100 Udine,Italy. Tel.: 390432494322; Fax: �390432494301; E-mail: [email protected].

3 The abbreviations used are: Sso AcP, S. solfataricus acylphosphatase; TFE,2,2,2-trifluoroethanol; CR, congo red; ThT, thioflavin T; PF, protection fac-tor; DOSY, diffusion ordered NMR spectroscopy; H/D, hydrogen/deuteri-um; HSQC, heteronuclear single quantum coherence.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 19, pp. 14689 –14700, May 7, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

MAY 7, 2010 • VOLUME 285 • NUMBER 19 JOURNAL OF BIOLOGICAL CHEMISTRY 14689

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25 °C), it has been shown that Sso AcP adopts initially, beforeaggregation starts, an essentially native fold (8, 10). Twosequential phases have been detected in the aggregation pro-cess (11). In a first rapid phase, the native protein aggregateswithin 2 min into oligomers in which the individual moleculesstill possess considerable enzymatic activity. These oligomericspecies do not bindCongo red, thioflavin T (ThT), or 1-anilino-8-naphtalenesulfonic acid and are not yet characterized by thehigh content of �-sheet structure typical of amyloid aggregates(11). These initial native-like aggregates convert directly,within 30 min, into spherical and chain-like protofibrils. Theselatter species have a diameter of 3–5 nm; have no enzymaticactivity; bind Congo red, ThT, and 1-anilino-8-naphtalenesul-fonic acid; and have a large �-sheet structure content (11).Importantly, both phases of aggregation are more rapid thanthe unfolding reaction determined for the monomeric, nativestate under the same conditions, indicating that unfolding isnot a necessary step in the process.A combination of limited proteolysis experiments (10), pro-

tein engineering (10, 12, 13), and specific kinetic tests with pep-tides resembling theN-terminal segment (12) have put forwarda model in which the rate-determining step for the first aggre-gation phase consists in the collision of theN-terminal segmentwith the globular unit of SsoAcP, probablywith�-strand 4 (12).By contrast, the further conversion of the native-like assembliesinto protofibrils appears to involve a cooperative and position-independent conversion of the proteinmolecules present in theinitial aggregates (10). Importantly, however, the rates of bothaggregation phases correlate with the free energy change ofunfolding in the groups ofmutants investigated, indicating thatcooperative fluctuations are required for the formation of boththe initial aggregates and the subsequent protofibrils (10).Because aggregation of Sso AcP into amyloid-like protofi-

brils involves the formation, as a first detectable step, of assem-blies in which the individual protein molecules retain a native-like conformation, it is important to explore the structure anddynamics of the native protein under conditions in which pro-tofibril formation occurs, with an aim of comparing these char-acteristics with those determined under conditions in whichaggregation does not occur. In this work we have used NMRspectroscopy to detect possible structural changes in six differ-ent conditions, chosen on the basis of their ability to stabilizethe native state, populate the monomeric, native-like state thatis prone to aggregation, or facilitate the detection of the native-like structure within the early aggregates.

MATERIALS AND METHODS

Expression and Purification of Sso AcP—Sso AcP was ex-pressed and purified as previously described (14). The uni-formly 15N-labeled proteinwas produced and purified byASLA(Riga, Latvia) according to the protocol previously optimized(14). The resulting sequence was GSMKKWSDTEVFEMLKR-MYARVYGLVQGVGFRKFVQIHAIRLGIKGYAKNLPDGSV-EVVAEGYEEALSKLLERIKQGPPAAEVEKVDYSFSEYKGEF-EDFETY. The initial GS dipeptide (labeled Gly�1 and Ser0)does not belong to the protein and is due to the gene cloninginto the pGEX-2T plasmid. UV absorption was employed to

determine Sso AcP concentration using an extinction coeffi-cient at 280 nm (�280) of 1.246 ml mg�1 cm�1.Aggregation Kinetics of Sso AcP—Aggregation kinetics of Sso

AcP were studied by ThT fluorescence under different experi-mental conditions. In brief, the reaction was started by dilutinga solution containing 10 mM Tris, pH 8.0, and 10 mg ml�1

native Sso AcP to reach a final protein concentration equal to0.4 mg ml�1. Four different final conditions were analyzed: (A)50 mMNaCl, 50 mM phosphate buffer, pH 5.7; (B) 50 mMNaCl,50 mM phosphate buffer, 20% (v/v) TFE, pH 5.7; (C) 50 mM

NaCl, 50mMacetate buffer, pH5.7; and (D) 50mMNaCl, 50mM

acetate buffer, 20% (v/v) TFE, pH 5.7. Conditions from A to Dwill be named in the text as phosphate, phosphate-TFE, acetate,and acetate-TFE, respectively. All of the samples were incu-bated at 25 °C. At several times, 60 �l of the sample were addedto 440 ml of 25 �M ThT, 25 mM phosphate buffer, pH 6.0, at25 °C. The resulting fluorescence was measured with aPerkinElmer LS-55 fluorimeter (Wellesley, MA) thermostattedwith a Thermo-HAAKE F8 bath (Karlsruhe, Germany). Excita-tion and emission wavelengths were 440 and 485 nm, respec-tively. The resulting plot of ThT fluorescence (expressed asratio between the observed signal and ThT fluorescence in theabsence of protein) versus timewas fitted to a single exponentialequation of the following form,

Fl�t� � A � B exp��kt� (Eq. 1)

where Fl(t) is the fluorescence observed at time t, A is the fluo-rescence at the apparent equilibrium, B is the change of fluo-rescence during the exponential phase, k is the apparent rateconstant, and t is the time. Before starting the experiment, thesample was centrifuged at 16,000� g for 5min, and the proteinconcentration was measured.NMR Experimental Conditions and Chemical Shift Analysis—

In addition to the four experimental conditions aforemen-tioned (conditions A–D), NMR experiments were also per-formed in the following: (E) 50 mM acetate buffer, 3 mM phos-phate, and (F) 50mM acetate buffer, 3 mM phosphate, 20% (v/v)TFE. 50 mM NaCl was present in all samples; the pH wasadjusted to 5.7. These conditions will be referred to as“pseudoacetate” and “pseudoacetate-TFE,” respectively.In every experimental condition listed, we acquired

[1H,15N]HSQC spectra, which can be considered as the finger-print of every protein. NMR experiments were acquired on aBruker Avance 500 spectrometer on 0.4 mM Sso AcP samples.For the assignment conventional proton 15N decoupled homo-nuclear two-dimensional total correlation spectroscopy andnuclear Overhauser effect spectroscopy, two-dimensional[1H,15N]HSQC, HSQC-total correlation spectroscopy, andthree-dimensional [1H,15N]HSQC-nuclear Overhauser effectspectroscopy at 37 and 25 °C were recorded. Typically a sweepwidth of 12 and 35 ppm, 1024–2048 � 256–512 data pointswere used at proton and nitrogen frequency, respectively. Dataacquired and processed using Topspin (Bruker Biospin) wereapodized with a squared sinebell shifted by 90° and polynomialbase-line correction. The assignment was performed into theFelix (Accelrys, San Diego, CA) and Sparky (T. D. Goddard andD. G. Kneller, SPARKY3, University of California, San Fran-

Conformational Changes in Native-like Aggregates of Sso AcP

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cisco) framework. The combined 1H,15N chemical shiftchanges of backbone amides and Asn, Gln side chains amines,andArg side chains amides in the different buffering conditionsversus phosphate were analyzed.NMRExchangeMeasurements—3mg of protonated Sso AcP

were dissolved in 500 �l of deuterated buffer at pH* 5.7. (pH*refers to the pH measured in a D2O solution, uncorrected forthe deuterium isotope effect.) A series of [1H,15N]HSQC exper-iments with 1k � 60 data points, 8 or 16 scans (480 or 960s/spectrum) were recorded at 25 °C to follow amide decayingsignals over time in the six previously mentioned conditions. Thetotal duration of the experiments ranges from 3 to 4 days. To rel-atively quantify protein concentration in solution one-dimen-sional spectra using the ERETICTM (Bruker Biospin) were inter-leaved between HSQC experiments. The ERETICTM technique(electronic reference to access in vivo concentration) is a quantifi-cation experiment that uses an electronic signal as a reference.The data were processed with NMRPipe (15) using a 90°

shifted sinebell apodization function and standard linear pre-diction and analyzed with NMRViewJ 7.0.8 (16). The lag timebetween Sso AcP dissolution and the starting of the first mea-surement was �13 min in phosphate and 10 min in bothpseudoacetate and acetate buffer, respectively; therefore fastexchanging amides were not detected, and a lower limit fortheir kinetic constant kex was estimated to be �10�3 s�1. Wewill refer to these amides as fast. In the opposite time limit veryslow exchanging amide protons were still detectable after sev-eral days, and an upper limit for the kinetic constant was eval-uated to be �10�9 s�1. We will refer to these amides as slow.For the remaining HN, referred in the text as intermediates, anexponential fitting permitted reliable evaluation of the kex, andthe values are reported as a logarithm of protection factor(Log(PF)). To obtain the experimental exchange rates, kex, wemeasured, inside the NMRViewJ framework, the volumes ofpeaks at different deuteration times and fitted the data to anexponential function through a nonlinear fitting procedure.Protection factors PF were calculated as PF � kint/kex, wherekint is the intrinsic rate, observed in unstructured peptides (17).Diffusion Ordered NMR Spectroscopy (DOSY) Experiments—

To measure the diffusion coefficient of Sso AcP in solution at37 °C, the convection compensated two-dimensional doublestimulated echo sequence (18) was used, and a matrix of 2k inthe F2 dimension by 80 points in F1where the gradient strengthvaries linearly from 2 to 95% of its maximum value (61.1 gauss/cm). The length of the diffusion gradient and the stimulatedgradient were optimized for each sample to give a total decay ofthe signal of �95%. The water suppression was achieved withthe addition of a sculptingmodule (19). The self-diffusion coef-ficient D can be evaluated from the diffusion decay of the pro-tein signal according to the Stejskal and Tanner equation (20)I � I0e�D(�g�)2(�-�/3-/2), where � is the gyromagnetic ratio, gis the gradient strength, � is the applied gradient duration, isthe correction rephasing time, and � is the echo time. The datawere processed using a 90° sinesquare function in F2, and theanalysis along F1 was realized using the Bruker Dosy softwareand fitting the signal decay with a single exponential.In the presence of TFE, massive precipitation prevents the

measurement of the diffusion coefficient. In fact during the

experiment the signal intensity decays not only because ofthe strength of the gradient but also because of sample precip-itation. To overcome the problem the DOSY was acquired as aseries of 80 one-dimensional double stimulated echo experimentsat different gradient strengths interleaved with one-dimensionalspectrawith ERETICTM.Aprecipitation index, obtained from theratiobetween theprotein and theERETICsignal,wasused to scalethe DOSY spectra that were subsequently transformed in a two-dimensional matrix and processed as described.The experiments were carried out in the above reported dif-

ferent buffering and cosolvent conditions, except for the ace-tate case, where only 15% (v/v) TFE was added to prevent a toomassive signal loss forbidding the direct result comparison.Moreover, a normalization was performed measuring the tri-methylsilylpropionic acid self-diffusion coefficient in each ofthe different experimental conditions that exhibit different vis-cosity (data reported in supplemental Table S1). Experimentaluncertainties were estimated by comparing the variation ofmeas-ured decay constants between individual recorded frequencies(21).Molecular Dynamics Simulations—A molecular dynamics

simulation of Sso AcP has been performed starting from theNMR structure (firstmodel in the entry with ProteinData Bankcode 1Y9O). The structure was protonated when needed andneutralized as described elsewhere (22). Sodium phosphatemolecules were added by generating first the phosphate ion andthen adding the sodium as described for the protein. The singlemolecule was then randomly rotated and placed at 18 Å spacedpoints in a cubic grid of 90 � 90 � 90 Å3 surrounding theprotein. The system was then relaxed by 200 conjugate gradi-ents minimization steps. The dielectric was set to 10.0 to min-imize the effect of missing solvent, and the cut-off was 12.0 Å.Theminimized systemwas solvated using themodule solvate inthe VMD software package (23) in a box with margins at 4.0 Ådistance from any solute atom. The systems contained �65000atoms. The solvent molecules were relaxed, keeping the solute(including the ion) fixed, by molecular dynamics simulation(22). The system without restraints was energy minimized by300 conjugate gradients minimization steps. The system wasthen heated to 27 °C in 2 ps, and a further 118 ps of simulationwas run to let the system equilibrate, and finally production runcould start. The system was then simulated for 5 ns.In all molecular dynamics simulation, the temperature was

kept constant through a simple velocity rescaling procedure,whereas the pressure was controlled through a Berendsen bath(24) using a relaxation time of 100 fs. The resulting concentra-tions of phosphate and protein were 309 and 2.6 mM, respec-tively. All of the structural analyses, in particular root meansquare deviations, secondary structure, and angular orderparameter analyses, have been performed using the programMOLMOL (25). The analysis of solute phosphate contacts wasperformedwith home-written C programs. The definition for acontact was a distance between heavy atoms less than the sumof van der Waal’s radii plus 1 Å.

RESULTS

Sso AcP Does Not Aggregate in Phosphate Buffer—In a firstpreliminary experiment, the time course of Sso AcP aggrega-




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tion was studied by means of ThT fluorescence in phosphateand acetate buffer, both in the absence and in the presence of20% (v/v) TFE. Sso AcP induces an 11-fold increase in ThTfluorescence only in acetate buffer supplemented with 20%(v/v) TFE (Fig. 1). Such an increase follows apparent singleexponential kinetics and was previously shown to be related toamyloid-like aggregation, particularly protofibril formation (8).The observed rate constant k in these conditions is (1.4 0.2)�

10�3 s�1. Under the other three conditions, namely acetatebuffer without TFE and phosphate buffer with andwithout 20%(v/v) TFE, Sso AcP does not induce any significant increase inThT fluorescence, confirming that no amyloid-like aggregatesform in these conditions on the studied time scale (Fig. 1).Sso AcP Retains a Native-like Structure in the Initial Small

Aggregates as Revealed by the Combination of Pulse Field Gra-dient NMR Diffusion and Heterocorrelated NMR SpectraAnalysis—Under conditions promoting amyloid-like aggrega-tion, that is, acetate bufferwith 20% (v/v) TFE, samples contain-ing 0.3 mM Sso AcP prepared for NMR analysis showed a mas-sive precipitation. Therefore, a crucial point was to clarify theactual aggregation state of the fraction of Sso AcP that was stillpresent in solution after precipitation and that gave rise to theNMR signals. A series of pulse field gradient diffusion experi-ments were carried out in four different experimental condi-tions, acetate and phosphate buffers, both with and without20% (v/v) TFE (experimental conditions A–D, as describedunder “Materials and Methods”). Fig. 2 shows two representa-tive DOSY spectra obtained in acetate buffer and 20% (v/v) TFE(blue) and in phosphate buffer without TFE (black). From thediffusion coefficient values (D) obtained in each of these exper-iments, a value of apparent hydrodynamic radius (r) wasobtained according to the Stokes-Einstein equation r �kT/6�D, where k is the Boltzmann constant, T is the absolutetemperature, and � is the viscosity coefficient (26, 27). r valuesof 15.3 0.3 and 15.8 0.5 Å were obtained in phosphatebuffer without TFE andwith 20% (v/v) TFE, respectively. Thesevalues are in agreement with that of monomeric, native SsoAcP, as determined previously by NMR (7). A higher value of18.0 0.3 Å was determined for r in acetate buffer in the

absence of TFE, still in agreementwith that of amonomeric, native SsoAcP molecule. The DOSY spectraobtained in acetate buffer and 15%(v/v) TFE revealed a wide range ofDvalues (Fig. 2). Application of theStokes-Einstein equation to theobserved range ofD values leads to rvalues spanning from 24 to 75 Å.In these experiments the TFE con-centration was kept to 15% (v/v),rather than 20% (v/v), to avoid aprohibitive loss of signal. Precipita-tion of Sso AcP prevented the use ofstandard two-dimensional DOSYexperiments and required the defi-nition of a specific protocoldescribed under “Materials andMethods.” Such DOSY experimentsindicate the concomitant occur-rence of species with different oligo-meric states, ranging from lowmolecular weight species to aggre-gates composed of many molecules.Fig. 3 shows [1H,15N]HSQC spec-

tra acquired in acetate buffer, inwhich aggregation does not occur

FIGURE 1. Aggregation time courses, monitored using ThT fluorescence,of four samples containing 0.4 mg ml�1 Sso AcP and different experi-mental conditions. The conditions were: 50 mM NaCl, 50 mM acetate buffer,20% (v/v) TFE, pH 5.7 (F); 50 mM NaCl, 50 mM acetate buffer, pH 5.7 (E); 50 mM

NaCl, 50 mM phosphate buffer, 20% (v/v) TFE, pH 5.7 (f); and 50 mM NaCl, 50mM phosphate buffer, pH 5.7 (�). The continuous line represents the best fit toEquation 1 (see “Materials and Methods”) of the data collected in acetatebuffer with TFE.

FIGURE 2. Diffusion coefficient values for Sso AcP in aggregating and nonaggregating conditions. DOSYsignals for Sso AcP in phosphate without TFE (black) and in acetate with 15% (v/v) TFE (blue). A very noisy tracewas obtained for the latter condition because of a low signal to noise ratio. The paucity of the signal preventeda fine analysis by means of deconvolution techniques, and each column of the DOSY spectrum was processedwith a single exponential function. It is, however, evident that different oligomeric components are present inacetate and TFE.




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on the time investigated in this work, and in acetate supple-mentedwith 20% (v/v) TFE, where aggregation does occur. Thespectra indicate that in both conditions Sso AcP adopts a fullyfolded state. The resonance peaks obtained in aggregating con-ditions originate from the low molecular weight aggregates, inexchangewith the larger oligomers present in solution.Overall,the native-like appearance of theHSQC spectrum and the largeD values in the DOSY spectra indicate that, in acetate and TFE,the individual protein molecules adopt a native-like conforma-tion and are in intermolecular exchange within the aggregates.Changes of Structure and Dynamics of Sso AcP under Aggre-

gating Conditions:Methodology Set-up—TheThT fluorescenceanddiffusionNMRdata described above show that the additionof 20% (v/v) TFE to a sample containing Sso AcP in acetatebuffer triggers aggregation. On the other hand the same datashow that Sso AcP remains soluble in phosphate buffer, inde-pendently of the presence of TFE in solution. The persistentsolubility of Sso AcP in phosphate buffer upon the addition ofTFE is due to the fact that phosphate acts as a competitiveinhibitor of acylphosphatases (28) with a Kd of 1.12 0.10 mM

in the case of Sso AcP at pH 5.5 (12). The binding of the phos-phate ion to Sso AcP causes a dramatic increase in conforma-tional stability and structural rigidity of the native state, inhib-iting aggregation, which by contrast requires structuralfluctuations within the native state (6). This scenario is ideal toinvestigate the effect of TFE on the monomeric state of SsoAcP. Indeed, by comparing the dynamics and structure ofSso AcP in phosphate buffer with and without TFE, the effectof the fluoroalcohol on the monomeric state of Sso AcP canbe investigated.A further conditionwas also investigated inwhich phosphate

ions are in 6-fold molar excess with respect to Sso AcP in anacetate buffer. Under these conditions, whichwill be referred toas “pseudoacetate” buffer, Sso AcP aggregation is partiallyinhibited, and phosphate ions are present in the active site of

SsoAcP. Therefore, a comparison between the consequences ofadding TFE in phosphate and pseudoacetate buffer will give thepossibility of distinguishing between the chaotropic and localeffects of phosphate. On the other hand, the comparisonbetween the dynamics and structure of Sso AcP in acetatebuffer with and without TFE allows the consequences of theaggregation process to be determined directly.Structure and Dynamics of Monomeric Sso AcP in Phosphate

and Phosphate-TFE—As a first analysis we titrated the proteinin phosphate buffer with TFE concentrations ranging from 0 to20% (v/v) and acquiring [1H,15N]HSQC spectra at 25 °C foreach of the progressive TFE additions. Supplemental Fig. S1depicts the HSQC spectra acquired at 0, 10, and 20% (v/v) TFE.The 1H and 15N chemical shifts of the amide peaks are consis-tent with the maintenance of a structured protein up to 20%(v/v) TFE. The comparison of the combined 1H and 15N chem-ical shift changes in the presence and absence of 20% (v/v) TFEis reported in Fig. 4A. The major differences are in the N-ter-minal segment (residues Phe10–Met12), in loop L1 (residueLeu23), in helix H1 (residues Phe32–Gln34 and Arg39), in strandS2 (residue Gly44), in strand S3 (residues Ser53 and Val54), inloop L4 (residue Glu62), in helix H2 (residues Glu70–Ile72),in loop L5 (residue Ala78), in strand S4 (residue Lys83), in thelong loop L6 (residue Glu90), and in strand S5 (residue Tyr101).The cosolvent addition causes a widespread but moderateeffect in terms of chemical shift deviations.As a second analysis the rates of H/D exchange of the amide

hydrogen atoms were compared in phosphate buffer in thepresence and absence of 20% (v/v) TFE. Supplemental Fig. S2Areports the PF measured in the two conditions for all the non-proline amides. The high stability of Sso AcP, ultimately causedby its hyperthermophilic origin, determines a high structuralrigidity where the hydrogen atoms belonging to the core of theprotein (�1⁄3 of the total NHs) do not exchange in a time frameof a few days. On the other hand, another third of NHs are

FIGURE 3. Two-dimensional [1H,15N]HSQC spectra of Sso AcP acquired in acetate in the absence (A) and presence (B) of 20% (v/v) TFE. The assignmentsof backbone amide groups are labeled. The sc labels indicate side chain peaks from tryptophan, asparagine, glutamine, or arginine residues. The latter ones arefolded downfield by 33.55 ppm. Unassigned peaks are indicated by asterisks.




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solvent-exposed and exchange before our measurements arestarted. Nevertheless it was possible to qualitatively describethe differences in protein dynamics in the six experimental con-ditions studied here.We can first focus on the condition in the absence of TFE and

Fig. 5A depicts the structure of Sso AcP color-coded to showthe differences of protection factors among the various residuesin these conditions. 33 of 100 non-proline amide peaks did notexchange during the 71 h of experimental acquisition. This per-centage of slow exchange amides is much higher than usuallyobserved for mesophilic proteins but seems to be consistentwith the hyperthermophilic nature of Sso AcP. The overallbehavior highlights that the paired strands S1, S3, and S2,located in the central part of the sheet, do not exchange evenafter 3 days in deuterated solution except for Tyr45 located inthe center of S2 strand, which is in the fast exchange regime.The two edge strands S4 and S5 show amore “patchy” behavior;in particular in strand S4 all amides pointing outward (Val84,Tyr86, and Phe88) are in the fast exchange regime, whereasthose pointing inward and forming hydrogen bondswith strandS1 exchange at intermediate (Lys83, Asp85, and Ser89) or slow(Ser87) rates. Strand S5 is characterized by intermediateexchange rate except for the fast Phe98 amide, which pointsoutward. Amides of residues Thr99 and Arg101 are hydrogen-bonded to residues of strand S2, whereas residue Thr100 pointstoward the bulk. It is thus striking to observe an intermediateexchange rate for the latter.In both �-helices all residues of the protein core exchange

slowly except for the two N-terminal residues Phe29 and Arg30

in helix H1 that are in the fast exchange mode. The residuesexposed to the solvent show intermediate exchange rates,except for the fast exchange of Glu62, Glu63, and Ala64 at the Nterminus of helix H2 and the slow exchange of Ile35 in helix H1.As expected, the N-terminal segment of the protein and allloops are in fast or intermediate exchange regime except for theslow amides of residues Ile42 (loop L2) andTyr61 (loop L4), bothlocated at the N-terminal apex of the �-sheet.The presence of 20% (v/v) TFE in phosphate buffer induces a

general decrease in protection factor values, except for strandS1, remaining in the slow exchange regime, and for the facingLys92 in L6 and Gly41 in L2 loops, which instead increase theirPF values (Fig. 5B). The most remarkable variations are instrands S2 and S3 and in the facing helixH1. Residues Leu69 andLys73 in helix H2 and Ser87 in strand S4 are also affected. Tofacilitate the comparison between the two sets of conditions inphosphate buffer (with and without 20% (v/v) TFE), Fig. 6Areports the differences of protection factor regimes, viewed onboth the structure and along the sequence of Sso AcP.

Structure and Dynamics of Monomeric Sso AcP in Pseudoac-etate and Pseudoacetate-TFE—We next titrated the protein inpseudoacetate buffer with TFE concentration ranging from 0%(v/v) to 20% (v/v) by acquiring [1H,15N]HSQC spectra at 25 °Cfor each of the progressiveTFE additions. The combined chem-ical shift changes of the amide peaks, shown in Fig. 4B, aresimilar to those determined in phosphate buffer: N-terminalsegment (residues Phe10, Glu11, and Leu13), strand S1 (residueArg15), loop L1 (residue Leu23), helix H1 (residues Phe32, Val33,Ile35, and Arg39), loop L4 (residue Glu62), helix H2 (residueGlu63), strand S4 (residue Tyr86), and strand S5 (residueTyr101). Hence, similarly to the previously described analysis inthe phosphate buffer, in the pseudoacetate buffer the cosolventaddition causes a moderate effect involving the entire protein.The rates of H/D exchange of the amide hydrogen atoms

were then determined in pseudoacetate buffer in both the pres-ence and absence of 20% (v/v) TFE (supplemental Fig. S2B).Similarly to with the phosphate condition, in pseudoacetatebuffer without TFE the protein dynamics is characterized by avery rigid core, comprising the central part of the �-sheet andthe inner portion of the facing helices (Fig. 5C).A striking loosening of the conformational rigidity of Sso

AcP occurs upon the addition of 20% (v/v) TFE in pseudoac-etate buffer (Fig. 5D). No slow exchanging amide survives evenin the central strands. In contrast, the amides of eight residues,located on the sheet surface (residues Leu13, Gly44, Asn48,Gly60, and Thr100) and in loop L1 (residue Val24) and loop L6(residues Tyr91 and Lys92), switch from fast to intermediateexchange regime (Fig. 6B). It is worth noting that themolecularflexibility in phosphate and pseudoacetate are rather similar,whereas TFE increases internal motions more effectively inpseudoacetate than in phosphate, indicating a protective chao-tropic effect of phosphate (Fig. 5, compare D with B).Structure and Dynamics of Sso AcP in Acetate and

Acetate-TFE—We finally titrated the protein in acetate bufferwith increasing TFE concentrations. As described above, thecomparison between the NMR data obtained in acetate bufferwith and without TFE allows the consequences of amyloid-likeaggregation to be determined directly. The combined chemicalshift changes of the amide peaks following the addition of 20%(v/v) TFE indicate that TFE produces amore specific structuraleffect (Fig. 4C). Robust deviations are observed for residuesPhe10, Glu11, Leu13 (N-terminal segment); Gln25 (backbone andside chain amides, loop L1); Glu63 (helix H2); Tyr86 (strand S4);Lys92 and Gly93 (loop L6); and Phe98 (strand S5). It is also evi-dent that the regions of the sequence encompassing approxi-mately the proximal portion of the N-terminal segment (resi-dues Glu8–Glu11) and the regions involving the strand S4, loopL6, and strand S5 (residues Ala79–Phe98) contain the highest

FIGURE 4. Combined HN and N chemical shift changes determined for the various residues according to Mulder et al. (30) (where �� is the squareroot of (��HN)2 � (��N/6.51)2) between 0 and 20% (v/v) TFE, in phosphate buffer (A), in pseudoacetate buffer (B), and in acetate buffer (C).Charts color code: aquamarine for N-HN ��; blue for the Arg, Asn, and Gln side chains ��. The mean combined chemical shift change (��) is indicated bythe horizontal tick line; the �� value, where � is the standard deviation for the �� value, is indicated by the dashed line; the �� 2� value is indicated bythe dotted line. In all panels, insets report the �� values on the native structure of Sso AcP according to the following color code: green for residues with �� , yellow for residues with �� , orange for residues with �� 2�, red for residues with �� 2�; and gray for proline andfor nondefined residues. Orange cylinders and blue arrows above each panel refer to �-helices and �-strands, respectively, and facilitate the associationbetween the �� values reported in the main panel and their localization in the native structure of Sso AcP.




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density of residues with �� valueshigher than the mean, indicatingthat such regions undergo statisti-cally significant structural perturba-tions. A further analysis was con-ducted by subtracting from thecombined chemical shift changes inacetate (acetate in the presence andin the absence of TFE) the com-bined chemical shift changes inphosphate (phosphate in the pres-ence and in the absence of TFE),thus removing the structural contri-bution caused by TFE addition onthe monomeric protein (supple-mental Fig. S4). The analysis showsthat the net structural changescaused by the aggregation eventinvolve the N-terminal segment(Trp4 and Glu8–Leu13), the N-ter-minal portion of helix H2 (Glu63),residue Tyr86 belonging to strandS4, loop L6 (Lys92 and Gly93), andPhe98 and Tyr101 of strand S5.A global increase of the H/D

exchange rate following TFE addi-tion was observed in acetate buffer,although not as marked as inpseudoacetate buffer (supplemen-tal Fig. S2C; Fig. 5, compare F withD). In fact, the central strand S1remains mainly slow exchanging.Moreover 12 residues increase theirprotection factors: Lys14 and Tyr21(strand S1), Val24 (loop L1), Arg39(helixH1),Gly41 (loopL2), Ala64 andSer66 (helixH2),Ala79(loopL5),Tyr91and Lys92 (loop L6), and Thr100 andTyr101 (strand S5) (Fig. 6C).The Phosphate Ion Modifies the

Cradle Conformation of the Sso AcPActive Site and Influences the Pro-tein Dynamics—Variations in struc-ture and dynamics are caused by dif-ferent phosphate concentrations asinferred from comparison of thedata collected in experimental con-ditions C (acetate) and E (pseu-doacetate) with those obtained incondition A (phosphate). Upon de-creasing the phosphate concentra-tion (experimental conditions A, C,and E), themain chemical shift devi-ations are located, as expected (28),in the active site cradle (residuesGln25–Phe32 and Lys47–Gly52;supplemental Fig. S3). In particular,the �� extent is much larger in ace-

FIGURE 5. Protection factors of amide hydrogen atoms in phosphate buffer in the absence (A) and in thepresence (B) of 20% (v/v) TFE; in pseudoacetate buffer in the absence (C) and in the presence (D) of 20%(v/v) TFE; and in acetate buffer in the absence (E) and in the presence (F) of 20% (v/v) TFE. The color codeindicates the exchange regime of the amide residues: fast (red), intermediate (green), and slow (blue).




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tate than in pseudoacetate. This is probably due to an active siteconformational variation and to the deshielding effect of thephosphate anion oxygen. The phosphate ions exchangebetween the free and bound position in the active site of SsoAcP in an intermediate exchange regime, with respect to theNMR time scale (data not shown) and interact preferably withGly26 and Gly28 backbone amides. A 5-ns molecular dynamicssimulation was performed starting from the AcP phosphate-free form (2.6 mM) (NMR solution structure 1Y9O) in the pres-ence of 309 mM phosphate ions, as described under “Materialsand Methods.” The snapshots at 0, 1.1, and 5 ns are shown inFig. 7A. Interestingly, a phosphate ion after 2.4 ns faces exactlyGly26, Gly28, and Arg30. The phosphate anion appears to bedriven by electrostatic forces from the bulk to the active site.This simulation may be regarded as a refinement of the NMRstructure in the L1 and L3 loops that contain the active sitecradle. In fact the geometry of Gly26 and Gly28 changes, and the

two amide hydrogens, highly shiftedin the acetate NMR spectra, rotateto improve their interaction withthe phosphate anion (Fig. 7B).The backbone dynamics varia-

tions of Sso AcP, occurring inpseudoacetate, can be highlightedby comparing panels C andA of Fig.5. The dynamics of the �-sheet por-tion formed by S4-S1-S3 strands donot change, with the exception ofresidueGly60 (C-terminal portion ofstrand S3). On the other edge morechanges occur; the amides of resi-dues Gly44, Asn48 (strand S2), andThr100 (strand S5) increase theirmobility, whereas the amide of resi-due Gly99 (strand S5) becomes slowexchanging. Four residues belong-ing to the helices, namely residuesGln34, Ile35, Arg39 (helix H1), andLeu65 (helix H2) untie. The onlychange in the loops is the looseningof Val24 (loop L1).

A mild overall protection factordecrease is observed in acetate (Fig.5,A and E), more pronounced at thebeginning of strand S3 (Val54) con-sistently with the absence of boundphosphate, in the following L4 loop(Tyr61) and in H2 helix (Leu65). Theabsence of the protective chaotropicphosphate has an effect limited tothe C-terminal edge of the protein

(S5, S2, and H2), whereas when the phosphate is completelyabsent the increased H/D exchange rate is spread throughoutthe whole molecule.

DISCUSSION

It has been reported that the addition of 15–25% (v/v) TFE tosolutions containing Sso AcP at pH 5.5 induces formation ofnative-like, enzymatically active aggregates, which subse-quently reorganize, with no need of dissolution and reaggre-gation, into amyloid-like protofibrils (8, 10–12). The aim ofour work was to investigate, at a molecular level using NMRspectroscopy, the structural changes induced by TFE on themonomeric state of Sso AcP, before aggregation occurs to anysignificant extent, and on the structure adopted by the proteinwithin the initial native-like aggregates. The first aim wasachieved by adding TFE to solutions of Sso AcP in buffers con-

FIGURE 6. Protection factors changes, reported as differences of Log(PF) values, between the absence and the presence of 20% (v/v) TFE in phosphate(A), pseudoacetate (B), and acetate (C) buffer. The �Log(PF) value is arbitrarily given as 1 when the residue changes its protection factor from slow tointermediate and from intermediate to fast, as 2 from slow to fast, and as �1 from fast to intermediate and from intermediate to slow. The color code reflectsthe exchange regime of the residue in the presence of 20% (v/v) TFE (red for residues in fast exchange regime, green for intermediate, and blue for slowexchange regime). Protection factors changes are also reported on the native structure of Sso AcP, with the following color code: red for residues going fromslow to fast regime; orange from intermediate to fast and from slow to intermediate regime; and blue from fast to intermediate regime. Orange cylinders andblue arrows below each panel refer to �-helices and �-strands, respectively, and facilitate the association between the �Log(PF) values reported in the mainpanel and their localization in the native structure of Sso AcP.

FIGURE 7. Molecular dynamics simulation in the presence of phosphate anions. A phosphate moleculereaches the Sso AcP catalytic cradle, which undergoes significant conformational changes. A, snapshots of theSso AcP molecular dynamics trajectory in the presence of phosphate anions at the beginning, after 1.1 and at5 ns. B, backbone superimposition of the initial (green) and final (light blue) structure: Gly26 and Gly28 backboneHN, and the side chain of the two catalytic residues (Arg30 and Asn48) are highlighted by sticks. The distances inÅ between the HN of the two glycines and a phosphate oxygen are shown.




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taining high or small concentrations of phosphate and ex-ploiting the capability of this ion to bind to the active site of theprotein, stabilize the native state, and thus inhibit aggregation.The second aimwas achieved by studying the protein directly inacetate buffer in the absence of phosphate, i.e. under conditionsin which aggregation occurs.In the presence of phosphate, the radius of gyration of the

protein, measured from DOSY experiments, remains compati-ble with that of native-like, monomeric Sso AcP when TFE isadded. This confirms the effective inhibition of Sso AcP aggre-gation under these conditions upon TFE addition. By contrast,in acetate buffer the measured radius of gyration is compatiblewith that of native, monomeric Sso AcP only in the absence ofTFE, with larger aggregated species forming in its presence.[1H,15N]HSQC spectra obtained in the latter conditions high-light an essentially native-like structure for Sso AcP (Fig. 3),indicating that (i) the protein retains a compact native-like stateand (ii) a highly dynamic character occurs within the aggre-gated species. It was just the dynamic nature of the intermolec-ular interactions within the early aggregates that allowed theprotein to be probed within the aggregates.In phosphate buffer, the addition of 20% (v/v) TFE does not

induce global structural changes on SsoAcP. The proteinmain-tains an essentially folded, native-like state, as determined fromthe analysis of [1H,15N]HSQC spectra. Changes of the com-bined 1H and 15N chemical shifts are detected only for someresidues and are generally small to moderate (Fig. 4A). Theobservation that such changes involve different portions of thesequence and of the three-dimensional structure of Sso AcPsuggests that the structural modification following TFE addi-tion, albeitmoderate, is widespread throughout the protein foldand also involves the unstructured N-terminal region. Theanalysis performed in pseudoacetate buffer, at a phosphate con-centration that is sufficient to inhibit aggregation but withphosphate exchanging very rapidly between the free and boundstate and leaving �30% of the Sso AcP molecules unbound tothe ion, confirms that native Sso AcP undergoes a very moder-ate distortion in different areas of the three-dimensional fold(Fig. 4B). The only significant difference between the structuraldistortions observed upon TFE addition in phosphate andpseudoacetate involves strand S3, which appears to undergo alower degree of variation in pseudoacetate.In phosphate buffer native Sso AcP becomes more dynamic

following TFE addition, as determined by measurements ofH/D exchange rates (Fig. 5, compareBwithA).With the excep-tion of strand S1, all other portions of the molecule undergoincreased exchange rates, indicating a loosening of the wholestructure. This behavior is more marked in pseudoacete buffer(Fig. 5, compareDwithC), where even strand S1 becomesmoredynamic upon TFE addition. Overall, the NMR analysis indi-cates that following the addition of TFE Sso AcP maintains, inits monomeric state, a native-like structure that appears onlymarginally distorted, but markedly less rigid. Nevertheless, thehigher structural lability does not suffice to induce fibrilformation.A different picture holds in acetate buffer, where TFE addi-

tion causes the conversion of the native protein into aggregates.[1H,15N]HSQC spectra obtained in the presence of TFE show

that Sso AcP retains an essentially native-like structure in theaggregates (Fig. 3B). The peak line width observed in[1H,15N]HSQC spectra obtained in acetate buffer in the pres-ence of TFE is considerably increased relative to all other non-aggregating conditions. This observation is consistent with thepresence of a molecular weight distribution of oligomers of SsoAcP molecules, in fast intermolecular exchange, ranging fromtetramers up to large oligomers of approximately a hundredmolecules, when a protein density of 1.42 � 104 kg/m3 is used(29). It can be stated that the aggregates observedwithNMRarethe native-like aggregated species detected early in the aggre-gation process rather than the protofibrils forming later.Indeed, the latter have high molecular weights and stable�-sheet packing leading to prohibitive relaxation rates forNMRobservation.The analysis carried out with the combined 1H and 15N

chemical shifts reveals important differences between themonomeric, native state in acetate buffer in the absence ofTFE and the native-like species in the protein aggregatespopulated upon TFE addition. Although the structuralchanges are globally of small entity, more pronounced per-turbations occur in the N-terminal region and in the nearbyapical region involving strand S4, strand S5, and loop L6connecting them and in the opposite active site region.These perturbations are possibly induced by intermolecularcontacts (supplemental Fig. S4).The H/D exchange rate data are more challenging to inter-

pret in this case. Indeed, the loosening of the native structurecaused by TFE addition brings about an increase of theexchange rates, whereas structure packing as a consequence ofprotein aggregation causes the opposite effect. The observedchange of the H/D exchange rate for a given residue consists inthe net result of these two opposite tendencies, which compli-cates the analysis. The fact that the intermolecular interactionsoccurring within the aggregates may be highly dynamic repre-sents a further complicating factor. Despite these difficultiesthe observation that residues from different portions of thestructure decrease their exchange rates as a consequence ofaggregation, particularly in the two apical regions of the SsoAcP native structure (Fig. 6C), indicates again the involvementof different regions of the sequence in the formation of thenative-like aggregates. Similar findings were also observed bysimulation experiments for other small amyloidogenic globularproteins (22).The six experimental conditions used here permit the filter-

ing of the effects of the cosolvents and the different buffers onthe structure and dynamics of Sso AcP. In particular, it is evi-dent that the protective effect of the phosphate ion againstaggregation is mainly due to its binding to the active site butalso by a widespread chaotropic effect.The experimental approach adopted here is applicable only

to amyloidogenic proteins that do not unfold when placed inamyloidogenic conditions. It is well known, in fact, that formany amyloidogenic proteins the process of fibril formationrequires misfolded monomeric intermediates. In such cases asimilar structural study in aggregating conditionswould be pre-vented by the unfolding and eventually precipitation of theprotein.




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CONCLUSIONS

The data presented here illustrate, for the first time at amolecular level, that Sso AcP maintains a native-like structurein its monomeric state under conditions that promote aggrega-tion rather than undergoing a process of partial unfolding. Thisconfirms previous observations obtained with kinetic analysisof folding and unfolding, enzymatic activity measurements andCD spectroscopy (8, 10). Our data also indicate that TFE-in-duced aggregation of Sso AcP does not arise from the potentialability of TFE to promote a partial or global unfolding of thecompact native state of the protein. Instead, the fluoroalcoholincreases the internal dynamics within the native state, thusfacilitating the structural fluctuations that are necessary toreach a proper intermolecular matching and initiate aggrega-tion. Structural flexibility seems a necessary prerequisite for SsoAcP aggregation and requires the total absence of phosphateions, which prevent aggregation by binding to the active siteand limiting the structural fluctuations. The structural pertur-bations of Sso AcP are only moderate and localized underaggregating conditions, whereas the increase of its internaldynamics is substantial and seems to involve the whole struc-ture. The inverse correlation found in a group of mutantsbetween the conformational stability of the native state and therate of formation of the native-like aggregates supports thisview, particularly when considering that the mutations werelocated in different regions of the Sso AcP structure (10).In addition to providing information on the structure and

dynamics of the monomeric state populated under conditionspromoting aggregation, the NMR analysis presented here alsoprovides information on the structure adopted by Sso AcP inthe initial aggregates. The data show that the native-like fold ismaintained in the initial aggregates, which seem to be charac-terized by loose and dynamic intermolecular interactionsbetween native-like Sso AcP molecules. The combined chemi-cal shift analysis suggests that the regions undergoing the mostsignificant changes upon formation of the native-like aggre-gates include the N-terminal unstructured segment, the apicalregion encompassing strand S4, loop L6, and strand S5, and theactive site region. The native-like and dynamic character ofsuch aggregates explains why such species possess enzymaticactivity, have a secondary structure content similar to that ofthe native state, and do not bind dyes diagnostic for amyloid,such as ThT andCongo red (11). The involvement of theN-ter-minal and apical region involving strands S4-S5 and the inter-connecting loop is also in agreement with the finding that theN-terminal region and strand S4 are solvent-exposed and flex-ible, as detected using limited proteolysis (10), and that muta-tions affecting such regions influence the conversion of the

native-like state into initial aggregates more effectively thanother mutations (10, 13).

REFERENCES1. Stefani, M., and Dobson, C. M. (2003) J. Mol. Med. 81, 678–6992. Uversky, V. N., and Fink, A. L. (2004) Biochim. Biophys. Acta 1698,

131–1533. Jahn, T. R., and Radford, S. E. (2005) FEBS J. 272, 5962–59704. Ventura, S., and Villaverde, A. (2006) Trends Biotechnol. 24, 179–1855. Chiti, F., and Dobson, C. M. (2006) Annu. Rev. Biochem. 75, 333–3666. Chiti, F., and Dobson, C. M. (2009) Nat. Chem. Biol. 5, 15–227. Corazza, A., Rosano, C., Pagano, K., Alverdi, V., Esposito, G., Capanni, C.,

Bemporad, F., Plakoutsi, G., Stefani, M., Chiti, F., Zuccotti, S., Bolognesi,M., and Viglino, P. (2006) Proteins 62, 64–79

8. Plakoutsi, G., Taddei, N., Stefani, M., and Chiti, F. (2004) J. Biol. Chem.279, 14111–14119

9. Soldi, G., Plakoutsi, G., Taddei, N., and Chiti, F. (2006) J. Med. Chem. 49,6057–6064

10. Plakoutsi, G., Bemporad, F., Monti, M., Pagnozzi, D., Pucci, P., and Chiti,F. (2006) Structure 14, 993–1001

11. Plakoutsi, G., Bemporad, F., Calamai, M., Taddei, N., Dobson, C. M., andChiti, F. (2005) J. Mol. Biol. 351, 910–922

12. Bemporad, F., Vannocci, T., Varela, L., Azuaga, A. I., and Chiti, F. (2008)Biochim. Biophys. Acta 1784, 1986–1996

13. Soldi, G., Bemporad, F., and Chiti, F. (2008) J. Am. Chem. Soc. 130,4295–4302

14. Modesti, A., Taddei, N., Bucciantini, M., Stefani, M., Colombini, B.,Raugei, G., and Ramponi, G. (1995) Protein Expr. Purif. 6, 799–805

15. Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A.(1995) J. Biomol. NMR 6, 277–293

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Funct. Genet. 17, 75–8618. Jerschow, A., and Muller, N. (1997) J. Magn Res. 125, 372–37519. Hwang, T. L., and Shaka, A. J. (1995) J. Magn Res. 112, 275–27920. Stejskal, E. O., and Tanner, J. E. (1965) J. Chem. Phys. 42, 288–29221. Baldwin, A. J., Anthony-Cahill, S. J., Knowles, T. P., Lippens, G.,

Christodoulou, J., Barker, P. D., and Dobson, C. M. (2008) Angew Chem.Int. Ed. Engl. 47, 3385–3387

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25. Koradi, R., Billeter, M., andWuthrich, K. (1996) J. Mol. Graph. 14, 51–5526. Einstein, A. (1956) Investigations on the Theory of Brownian Movement,

Dover Publication Inc., New York27. Poling, B. E., Prausnitz, J. M., and O’Connel, J. P. (2001) The Properties of

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Structural and dynamics characteristics of Acylphosphatase from Sulfolobus solfataricus in

the monomeric state and in the initial native-like aggregates

Katiuscia Pagano1, Francesco Bemporad

2,#, Federico Fogolari

1,3, Gennaro Esposito

1,3, Paolo

Viglino1,3

, Fabrizio Chiti2,3

and Alessandra Corazza1,3,§

1Department of Biomedical Sciences and Technologies, University of Udine, P.le Kolbe 4, 33100

Udine, Italy. 2Department of Biochemical Sciences, University of Firenze, Viale Morgagni 50,

50134, Firenze, Italy. 3Consorzio Interuniversitario “Istituto Nazionale di Biostrutture e Biosistemi”

(I.N.B.B.), Viale Medaglie d’Oro 305, 00136 Roma, Italy. §Corresponding author.

#Present address: Department of Chemistry, University of Cambridge,

Lensfield Road, Cambridge CB2 1EW, UK.

REFERENCES

1. Mulder, F. A., Schipper, D., Bott, R., and Boelens, R. (1999) J Mol Biol 292, 111-123

FIGURE LEGENDS

Fig. S1. Overlay of a series of [1H,

15N] HSQC spectra acquired for Sso AcP at 25 °C in phosphate

buffer in the absence of TFE (black), in the presence of 10% (v/v) TFE (blue) and in the presence

of 20% (v/v) TFE (red).

Fig. S2. Logarithm of the protection factor (Log(PF)) for the various residues in phosphate buffer

(A), pseudoacetate (B) and acetate buffer (C) in the absence (dashed bars) and in the presence

(continuous bars) of 20% (v/v) TFE. The color code reflects the exchange regime of the residues:

the blue and red bars represent lower and upper bounds of Log(PF) values for very slow and very

fast exchanging amides, respectively, whereas the green bars refer to the calculated Log(PF)

values.

Fig. S3. Combined HN and N chemical shifts changes determined for the various residues according

to Mulder et al. (1999) ( ( )2

2

51.6

∆+∆=∆ N

HN

δδδ ) (1) between A) phosphate and pseudoacetate

buffer, and B) phosphate and acetate buffer. Charts colour code: marine for N-HN δ∆ ; blue for the

Arg, Asn and Gln side chains δ∆ , and magenta for residues not assigned in acetate buffer. The

mean chemical shift change ( δ∆ ) is indicated by the horizontal tick line; the σδ +∆ value, where

σ is the standard deviation for the δ∆ value, is indicated by the dashed line; the σδ 2+∆ value is

indicated by the dotted line. Inset reports the ∆δ values on the native structure of Sso AcP

according to the following colour code: green for residues with δδ ∆≤∆ , yellow for residues with

σδδδ +∆≤∆<∆ , orange for residues with σδδσδ 2+∆≤∆<+∆ , red for residues with

σδδ 2+∆>∆ ; grey for proline and for non-defined residues.

Fig. S4. Structural changes due to the aggregation event: the structural contribution caused by TFE

addition on the monomeric protein was cleaned out subtracting from the combined chemical shift in

acetate - acetate-TFE, the combined chemical shift in phosphate - phosphate-TFE ( δ∆∆ ). Charts

colour code: marine for N-HN δ∆∆ ; blue for the Arg, Asn and Gln side chains δ∆∆ . The δ∆∆

mean value is indicated by the horizontal tick line; the σδ +∆∆ value, where σ is the standard

deviation for the δ∆∆ value, is indicated by the dashed line; the σδ 2+∆∆ value is indicated by

the dotted line. Inset reports the δ∆∆ values on the native structure of Sso AcP according to the

following colour code: green for residues with σδδ +∆∆≤∆∆ , orange for residues with

σδδσδ 2+∆∆≤∆∆<+∆∆ , red for residues with σδδ 2+∆∆>∆∆ .

Table S1 TrimethylSilylPropionic Acid Diffusion coefficients in the different experimental

conditions

Phosphate Pseudoacetate Acetate Phosphate +

20% (v/v)

TFE

Pseudoacetat

e +20% (v/v)

TFE

Acetate +

15% (v/v)

TFE

TSP

Diffusion

coefficient

10-10

(m2/s)

8.99 9.05 8.98 6.04 6.15 6.86

Figure S1

Figure S2

Figure S3

Figure S4

Viglino, Fabrizio Chiti and Alessandra CorazzaKatiuscia Pagano, Francesco Bemporad, Federico Fogolari, Gennaro Esposito, Paolo

in the Monomeric State and in the Initial Native-like AggregatessolfataricusSulfolobusStructural and Dynamics Characteristics of Acylphosphatase from

doi: 10.1074/jbc.M109.082156 originally published online March 11, 20102010, 285:14689-14700.J. Biol. Chem.

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