Structure and properties of a dimeric N-terminal fragment of human ubiquitin

doi:10.1006/jmbi.2001.5181 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 314, 773±787

Structure and Properties of a Dimeric N-terminalFragment of Human Ubiquitin

David Bolton, Philip A. Evans, Katherine Stottand R. William Broadhurst*

Cambridge Centre forMolecular RecognitionDepartment of BiochemistryUniversity of Cambridge, 80Tennis Court Road, CambridgeCB2 1GA, UK

E-mail address of the [email protected]

Abbreviations used: AUC, analytultracentrifugation; GST, glutathionHSQC, heteronuclear single quantuspectroscopy; MALDI, matrix-assistionisation; MG, molten globule; NOOverhauser effect; NOESY, NOE sproot-mean-square deviation; TOCSYspectroscopy.

0022-2836/01/040773±15 $35.00/0

Previous peptide dissection and kinetic experiments have indicated thatin vitro folding of ubiquitin may proceed via transient species in whichnative-like structure has been acquired in the ®rst 45 residues. A peptidefragment, UQ(1-51), encompassing residues 1 to 51 of ubiquitin was pro-duced in order to test whether this portion has propensity for indepen-dent self-assembly. Surprisingly, the construct formed a foldedsymmetrical dimer that was stabilised by 0.8 M sodium sulphate at298 K (the S state). The solution structure of the UQ(1-51) dimer wasdetermined by multinuclear NMR spectroscopy. Each subunit of UQ(1-51) consists of an N-terminal b-hairpin followed by an a-helix and a ®nalb-strand, with orientations similar to intact ubiquitin. The dimer isformed by the third b-strand of one subunit interleaving between thehairpin and third strand of the other to give a six-stranded b-sheet, withthe two a-helices sitting on top. The helix-helix and strand portions ofthe dimer interface also mimic related features in the structure of ubiqui-tin. The structural speci®city of the UQ(1-51) peptide is tuneable: as theconcentration of sodium sulphate is decreased, near-native alternativeconformations are populated in slow chemical exchange. Magnetizationtransfer experiments were performed to characterize the various speciespresent in 0.35 M sodium sulphate, namely the S state and two minorforms. Chemical shift differences suggest that one minor form is verysimilar to the S state, while the other experiences a signi®cant confor-mational change in the third strand. A segmental rearrangement of thethird strand in one subunit of the S state would render the dimer asym-metric, accounting for most of our results. Similar small-scale transitionsin proteins are often invoked to explain solvent exchange at backboneamide proton sites that have an intermediate level of protection.

# 2001 Academic Press

Keywords: ubiquitin; dimer; protein dissection; structural speci®city;NMR spectroscopy
*Corresponding author
Introduction

A complete analysis of protein folding1 requiresknowledge of each point on the energy landscapeof a polypeptide chain, from the dynamic ensemble

ing author:

icale-S-transferase;m correlationed laser desorptionE, nuclearectroscopy; RMSD,, total correlation

of conformations that constitute the unfolded stateto the uniquely de®ned native state.2 ± 5 X-ray crys-tallography and solution state NMR techniquescan provide detailed structures of proteins in theirnative states, so the ®nal network of stabilisingand destabilising inter-residue interactions can, inprinciple, be elucidated. Thanks to kinetic analysis,a picture of interactions that are crucial to the tran-sition state of folding can be de®ned in some case-s.6,7 However, for other regions of the energylandscape, we possess only limited experimentaldetail.

The native state of a protein has been de®ned asa unique, lowest-energy conformation that is separ-ated from all other states by an energy gap signi®-

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774 A Dimeric Fragment of Ubiquitin

cantly larger than the available thermal energy,kT.8 As a result, the overwhelming majority of pro-tein molecules in solution adopt the single nativeconformation and near-native alternative states arepopulated poorly. However, a growing number ofproteins that lack structural speci®city have beenidenti®ed, which adopt distinct ensembles of con-formations under different solution conditions.9 Atone extreme, molten globule (MG) states possessnative-like secondary structure but lack de®nedtertiary contacts.10 ± 14 The many conformationssampled by a typical MG state interconvert in theintermediate exchange regime on the chemicalshift timescale, making NMR spectra broad anddif®cult to interpret. In other proteins, such as thepheromone-binding protein from Bombyx mori,15

exchange between a more limited set of structuresoccurs at a rate slow enough for separate NMR sig-nals to be distinguished for each state. For such anequilibrium between two states to be detected byNMR, the minor conformation should be popu-lated to more than 5 %, giving an equilibrium con-stant greater than 0.05. The energy landscape ofthe protein would therefore possess multiple mini-ma that are separated in free energy by less than7.3 kJ molÿ1 at 298 K.

The folded conformation of the DrkN SH3domain was found to exist in equilibrium with apartially unfolded species, which has been termedthe physiologically unfolded state, Dphys.

16 ± 21

Detailed NMR studies have demonstrated that theDphys state is compact and samples a discrete set ofnear-native conformations that are in fast exchangewith each other on the chemical shift timescale.Similar conclusions have been drawn from spin-labelling experiments on a Dphys state formed bytruncation of staphyloccocal nulcease.22,23 Designedproteins also frequently lack structural speci®city.Alternative sub-populations were apparent in aredesigned core mutant of ubiquitin24,25 and moreMG-like states have been observed in arti®cialhelix bundles.26 ± 28 The MG state of a protein can-not be equated simply with the unfolded state. Forexample, all of the dimeric four-helix bundle MGstates studied by the DeGrado group were signi®-cantly more stable than the chemically denaturedstate.29 Furthermore, mutations that improved thestructural speci®city of the helix bundles also madethe proteins more susceptible to chemicaldenaturation.29 An understanding of the causes ofstructural speci®city is therefore relevant to funda-mental issues in protein folding, including the fac-tors that determine the energy difference betweenthe native and unfolded states, as well as to strat-egies for protein engineering and design.

Studies of protein folding have traditionallyrelied on tractable, well-de®ned model systemsthat do not require disulphide bonds, cis-prolineisomers, or co-factors to fold, such as the smalleukaryotic protein human ubiquitin (see Table 1).High-resolution structural studies30,31 of this 76residue protein show that it possesses a ®vestranded b-sheet (residues 1-7 (U1), 11-17 (U2), 40-

45 (U3), 48-50 (U4) and 64-72 (U5)), 3.5 turns of ana-helix (residues 23-34) and a 310-helix (residues56-59). The topology of the b-sheet is mixed, withU1 and U5 parallel and the other strands packingin an anti-parallel arrangement. The native state ofubiquitin is stable over wide ranges of pH andtemperature 32 and is extremely resistant to trypticdigestion33 or chemical denaturation by guanidi-nium hydrochloride, possessing a free energy ofunfolding of 30 kJ molÿ1.24 The folding propensi-ties of structural elements that contribute to thisstability have been assayed by conducting exper-iments in altered solution conditions34 or by study-ing short peptides derived from the nativesequence.35 In 60 % (v/v) methanol at pH 2, intactubiquitin adopts a partially folded state (the Astate) in which the N-terminal b-hairpin is largelynative-like and the rest of the protein is dynamic,with non-native helical structure.34,36,37 This workled to the suggestion that the local structure of theN-terminal hairpin of ubiquitin is strongly encodedby the primary sequence. Peptide dissection stu-dies have con®rmed that the N-terminal hairpin ispartially populated in aqueous solution.38,39

The folding of intact ubiquitin has been studiedextensively by a variety of methods, although it isstill a matter of debate whether the reaction pro-ceeds via a two-state or a three-state mechanism.Dead-time hydrogen exchange experiments andstopped-¯ow far-UV circular dichroism spec-troscopy failed to observe a burst-phase intermedi-ate, suggesting that folding can be described by atwo-state model.40 However, in high concen-trations of the structure-promoting Hoffmeister saltsodium sulphate, dead-time amide hydrogen pro-tection was evident with signs of retarded hydro-gen exchange at sites in the N-terminal hairpin andup to the third b-strand.41 A separate study used¯uorescence to follow the folding kinetics of aF45W mutant of ubiquitin, demonstrating that anintermediate could be detected in the ®rst fewmilliseconds of folding at 298 K.42 Analysis of thefolding kinetics of other mutants showed that theintermediate was avoided when the side-chain ofresidue Val26 was replaced by a smaller hydro-phobic group.43 In 0.4 M sodium sulphate, theVal26 mutants regained their ability to fold via anintermediate, indicating that formation of the inter-mediate may be driven by hydrophobic inter-actions. In contrast with these results, more recentexperiments on the F45W mutant failed to ®ndevidence for three-state kinetics.44

Peptide dissection and kinetic experiments there-fore indicate that the folding of ubiquitin may pro-ceed via transient species in which native-likestructure has been acquired in strands U1, U2

and U3 (residues 1-45). We therefore producedUQ(1-51), a peptide fragment encompassing the®rst 51 residues of ubiquitin, from the N terminusto the end of strand U4, in order to test whetherthis portion has any propensity for independentself-assembly or could act as a model of the Dphys

state. Surprisingly, the construct formed a speci®c,

A Dimeric Fragment of Ubiquitin 775

folded, dimeric structure that was stabilised by0.8 M sodium sulphate at 298 K. Here, we presentthe solution structure of the dimer determined bymultinuclear NMR spectroscopy. We show that thestructural speci®city of the UQ(1-51) peptide istuneable: as the concentration of salt wasdecreased, near-native alternative conformationswere populated. Exchange between the differentstates was found to be slow on the chemical shifttimescale, so we also describe magnetization trans-fer experiments that have enabled us to character-ize the species present in 0.35 M sodium sulphate.

Results and Discussion

Effect of solution conditions on NMR spectraof UQ(1-51)

Two-dimensional [1H,15N]-HSQC spectra of auniformly 15N-enriched sample of UQ(1-51) (seeTable 1) were recorded to monitor the chemicalshifts of backbone and side-chain amide signals invarious solution conditions, as shown in Figure 1.In 50 mM sodium phosphate at pH 3.0 and 298 K,linewidths are narrow and the chemical shift dis-persion of the 1H spectrum is severely limited,being less than 0.85 ppm for backbone amide sites(Figure 1(a)). Both of these features are character-istic of a dynamic unfolded state, which we callthe DA or acid-denatured state of UQ(1-51). Bycontrast, when the pH is raised to 7.0 (Figure 1(b)),

Table 1. Comparison of amino acid sequenfragments UQ(1-51) and UQ(1-17)

a Comparison of segments of the sequence ofa-helix and the 310-helix. Identical and similarbackgrounds, respectively.

the 1HN shift dispersion increases to 3.20 ppm. Thissuggests that folded conformations have beenadopted, introducing structure-dependent second-ary shifts. From the sequence of UQ(1-51), a totalof 49 backbone amide crosspeaks would beexpected, but many more signals than this areobserved at pH 7.0, indicating that several distinctspecies are present. However, according to SDS-PAGE, amino acid analysis and MALDI mass spec-trometry (not shown), the peptide in the NMRsample was intact and free of impurities. Thus, allof the signals in Figure 1(b) are caused by mol-ecules of UQ(1-51), which must adopt several dis-tinct conformations at pH 7.0. Addition of sodiumsulphate was found to change the position of theequilibrium between these different species. At0.8 M sodium sulphate (Figure 1(c)), one confor-mation is populated to more than 95 % and a singleamide crosspeak can be distinguished for each resi-due in the [1H,15N]-HSQC spectrum. This confor-mation, which we denote the S or stabilisable stateof UQ(1-51), remains highly structured with 1HN

chemical shifts widely dispersed between 6.2 and9.3 ppm. Below, we refer to the other species thatare populated at low salt and neutral pH as theminor forms.

Aggregation state of UQ(1-51)

The aggregation state of UQ(1-51) was assessedby analytical ultracentrifugation (AUC) in 50 mM

ces of intact ubiquitin and the peptide

ubiquitin spanning the interface between theresidues are indicated with black and grey

Figure 1. Two-dimensional [1H,15N]-HSQC spectra ofthe UQ(1-51) peptide in 50 mM sodium phosphate buf-fer at 298 K and: (a) pH 3.0; (b) pH 7.0; (c) pH 7.0 and0.8 M sodium sulphate.


sodium phosphate at pH 7.0 and 278 K (notshown), since slow precipitation at room tempera-ture made equilibrium sedimentation analysisunreliable. Assuming a common molecular massfor all peptide species present in the solution, thedata were best described by a dimeric model witha calculated molecular mass of 11(�1) kDa, nearlytwice the value of 5884.8 Da expected from thesequence of UQ(1-51).

Using 50 mM sodium phosphate, pH 7.0 and278 K as reference conditions, the apparent size ofUQ(1-51) was assayed in a variety of solutions bysize exclusion chromatography. In gel-®ltrationexperiments under reference conditions, UQ(1-51)emerged as a single peak with a retention time of12.11 minutes, shorter than the 13.05 minute delayobserved for intact ubiquitin (not shown). Thus, allof the UQ(1-51) species present under the referenceconditions are similar in size (giving a single peak)and apparently slightly larger than wild-typemonomeric ubiquitin. Using the method of Wilkinset al.45 to estimate protein size, folded monomericand dimeric UQ(1-51) species are expected to havehydrodynamic radii of 15.0 AÊ and 18.4 AÊ , respect-ively. For comparison, the radius of folded ubiqui-tin is predicted to be 16.7 AÊ , while that of unfoldedmonomeric UQ(1-51) should be 21.2 AÊ . The obser-vation from gel-®ltration studies, that UQ(1-51)appears to be slightly larger than monomeric ubi-quitin therefore agrees with the conclusions ofsedimentation analysis and supports the sugges-tion that the peptide species are folded and dimericat pH 7.0 and 278 K.

The retention time of UQ(1-51) was not affectedsigni®cantly by raising the temperature of the gel-®ltration experiments from 278 K to 298 K,suggesting that the apparent size of the peptide issimilar in both cases. In 0.8 M sodium sulphate at298 K, the retention times of UQ(1-51) and ubiqui-tin are both increased slightly to 12.72 minutes and13.10 minutes, respectively. The S state of UQ(1-51)is apparently slightly more compact than thedimeric species present at low salt, but is still a lit-tle larger than the ubiquitin monomer and there-fore must be both folded and dimeric. At pH 3.0,the sample of UQ(1-51) gradually formed a viscousgel, so size-exclusion chromatography experimentswere not attempted.

Chemical shift assignments of the DA and Sstates of UQ(1-51)

Both homonuclear and triple resonance strat-egies were used to obtain unambiguous assign-ments for 1HN, 1Ha, 15N, 13Ca and 13Cb nuclei inuniformly 15N and 13C-enriched samples of UQ(1-51) in the DA state at 278 K and the S state at298 K. For the S state, 3D 15N-TOCSY-HSQC,HCCH-TOCSY and HNCO spectra were acquiredto assign side-chain 1H, side-chain 13C and back-bone 13C0 chemical shifts, respectively. Only onesignal is apparent for each residue in the [1H,15N]-HSQC spectrum of the S state (Figure 1(c)), fromwhich it can be deduced that the UQ(1-51) dimer issymmetrical, with a 2-fold rotational axis.

Previous 1H NMR studies of a peptide fragmentspanning the N-terminal 17 residues of ubiquitin,UQ(1-17) (see Table 1), had indicated that the U1/U2 hairpin is partially populated in aqueous sol-ution at pH 3.8 and 275 K.38 The 1Ha chemicalshifts of the DA state of UQ(1-51) are very close tothose of the UQ(1-17) peptide, the only signi®cant

Figure 2. Summary of data usedin the determination of the second-ary structure of the UQ(1-51) Sstate monomer unit. The aminoacid sequence is indicated, plussequential NOEs and the consensuschemical shift index based on theHa, Ca and Cb shifts. For NOEs, theheight of the bar indicates thestrength of the NOE.


difference being observed for residue Met1, whichpossesses a charged terminal amine group inUQ(1-17). Despite the similarity to UQ(1-17), the1Ha,1HN and 15N chemical shifts of the DA state ofUQ(1-51) are still very different from those offolded native ubiquitin.46,47

By contrast, the chemical shifts of 1Ha nuclei inthe S state of UQ(1-51) are generally much closerto those of intact ubiquitin, in particular betweenresidues Gly8 and Arg42, which are located at theend of strand U1 and the start of strand U3,respectively, in the structure of ubiquitin. As thechemical shifts of 1Ha nuclei are related to second-ary structure,48 these observations suggest thatelements of the ubiquitin fold, including strand U2

and the following a-helix, are retained with onlyminor changes in the central sections of both sub-units of the UQ(1-51) dimer. The largest shiftdifferences are found in residues belonging to theN and C-terminal strands of ubiquitin, U1 and U3,indicating a degree of structural rearrangement inthese regions of the dimer.

Table 2. Structural statistics for the ®nal ensemble of re®ned

A. RMSD from restraints and idealized geometryNOE distances (AÊ )Dihedral angles (deg.)Bonds (AÊ )Angles (deg.)Impropers (deg.)Size of ensembleFinal energy EL-J (kJ molÿ1)

B. Assessment of backbone quality according to the Ramachandran plotMost favored regionAdditionally allowed region

a Represents the average RMSD for the ensemble.b Represents the value for the ®nal structure that is closest to the mc Represents the value for all the structures in the ensemble.

Solution structure of the S state of UQ(1-51)

Analysis of 3D 15N and 13C-separated NOESYspectra of the S state yielded 1973 inter-protonNOE distance restraints. In addition, 74 unambigu-ous inter-subunit NOE distance restraints wereidenti®ed in a 3D 13C-rejected, 13C-separatedNOESY experiment49,50 on a sample prepared froman equimolar mixture of unlabelled (12C/14N) andlabelled (13C/15N) UQ(1-51) peptides. Figure 2shows the elements of secondary structure inferredusing sequential NOE connectivities51 and 1Ha,13Ca, and 13Cb chemical shifts,48 indicating thateach subunit of the dimer has an a � b fold com-prising three b-strands (residues 1-7, 11-18, and 41-47) and one a-helix (residues 23-34). In addition, 74f and c backbone dihedral angle restraints weregenerated from the chemical shifts of 1Ha, 13Ca,13Cb, 13C0 and 15N nuclei by the TALOS program.52

Each of the three X-Pro peptide bonds in the Sstate exhibit a trans conformation, as evidenced by

structures of the UQ(1-51) dimer

hSAia (SA)clob

0.039 � 0.003 0.0370.36 � 0.09 0.26

0.0018 � 0.0001 0.00180.36 � 0.01 0.370.29 � 0.02 0.26

16/65ÿ241.9 � 32.9 ÿ178.4

89.0 %c 88.9 %b

9.0 %c 11.1 %b

ean.

Figure 3. (a) Stereo view of a superposition of backbone traces from the ®nal ensemble of 16 solution structures ofthe S state dimer of UQ(1-51), with one subunit in red and the other in blue. (b) A representation of the fold of the Sstate dimer, with b-strands labelled. (c) A representation of the fold of wild-type ubiquitin with b-strands labelled.


the presence of characteristic 1Ha-1Hd/d0 sequentialNOEs and 13Cg/d chemical shifts.

Preliminary structures were calculated by simu-lated annealing using the CNS program,53 initiallyby treating all 1H-1H distance restraints from the3D 15N and 13C- separated NOESY spectra asambiguous in terms of the subunit, but includingall of the explicitly de®ned restraints identi®ed inthe 13C-®ltered experiment.54 ± 56 In subsequent iter-ations, the NOE restraints were analysed in thelight of the re®ned structures with lowest energyin order to decrease the number of subunit-ambig-uous connections. Of the 65 structures calculated inthe last round, 16 structures with the lowestenergy, no NOE violations greater than 0.5 AÊ andno dihedral angle violations greater than 5 � wereselected for the ®nal ensemble. Each of these struc-tures had good covalent geometry according toPROCHECK,57 with 89 % of residues in the mostfavoured regions of the Ramachandran plot.Further structural statistics are summarised inTable 2. A superposition of the 16 backbone tracesof the ®nal ensemble of the dimeric S state of

UQ(1-51) is shown in Figure 3(a). The structure iswell de®ned except for the N-terminal Gly-Serextension and the C-terminal residues Lys48-Glu51; between residues Met1 and Gly47, themean RMSD is 0.48(�0.20) AÊ for the backbone Ca

atoms and 0.89(�0.52) AÊ for all heavy atoms.The fold of each UQ(1-51) subunit consists of a

b-hairpin (strands U1 and U2), followed by an a-helix and a ®nal b-strand (U3) (Figure 3(b)). Thethird b-strand of each subunit interleaves betweenthe N-terminal hairpin and third strand of theother subunit to form a six-stranded b-sheet, withthe two a-helices sitting on top. The selected cross-strand NOEs displayed in Figure 4 show that thethird b-strand of one subunit (U03) makes paralleland antiparallel contacts with the ®rst (U1) andthird (U3) strands of the other subunit, respect-ively, which imparts a substantial twist to the b-sheet. The dimer interface also involves packinginteractions between the N-terminal portions ofboth a-helices. The contact between the two sub-units is predominantly hydrophobic and extensive,with a total surface area of 1405 AÊ 2 that can be

Figure 4. Representation of intra-and intersubunit long-range back-bone-to-backbone NOE contactsobserved in the b-sheet of the Sstate dimer of UQ(1-51).


divided into two portions: 315 AÊ 2 for the helix-helix interaction and 1090 AÊ 2 for the four centralstrands of the b-sheet. A total of 14 hydrogenbonds link the subunits (four across the helix-helixinterface and ten between the U1 and U3 strands),

Figure 5. Comparison of details of the structures ofwild-type ubiquitin (left) and the S state of UQ(1-51)(right). (a) Interaction between strand U2 from the N-terminal hairpin and the a-helix. (b) Interface betweenthe a-helix and the 310-helix in ubiquitin and betweenthe two a-helices of UQ(1-51). (c) Arrangement ofstrands U1, U5 and U3 of ubiquitin and strands U3, U03and U1 of UQ(1-51).

consistent with the expected trend of one hydrogenbond for every 100 AÊ 2 of a dimer interface.58

Structural comparison of the S state ofUQ(1-51) with ubiquitin

A comparison of the structures of UQ(1-51)(Figure 3(b)) and intact ubiquitin (Figure 3(c)) isuseful for rationalising both the fold and the oligo-meric nature of the S state. Each subunit of UQ(1-51) contains a hairpin-helix-strand motif, as doesubiquitin. The orientation of several elements ofsecondary structure is similar in both cases. Forexample, the N-terminal hairpin and the a-helixpack against one another at approximately thesame angle and the facing side-chains adopt simi-lar conformations (Figure 5(a)). When the structurefrom the S state ensemble that is closest to themean is compared with the equivalent solutionstructure of intact ubiquitin,31 the largest differ-ences are found in the turn between strands U1

and U2. This region is, however, not well de®nedin either ensemble. Other notable deviations are atthe centre of the six-stranded b-sheet of UQ(1-51),particularly strand U3, which makes up much ofthe dimer interface. If the hairpin turn section isexcluded and the two structures are comparedbetween residues 1 to 6 and 11 to 45, the mean Ca

RMSD is 1.56 AÊ . Within this range, the region fromthe start of U2 to the end of the a-helix (residues 11to 45) is the most similar, giving a Ca RMSD of0.88 AÊ .

Portions of the helix-helix and U1, U03, U3 and U01strand interfaces are shown in Figure 5(b) and (c),respectively. The packing between the two helicesis remarkably similar for both ubiquitin andUQ(1-51), with the helix in the second subunit ofthe dimer taking the place of the 310-helix of ubi-quitin. When residues 18 to 27 and 51 to 60 of ubi-quitin are matched with residues 18 to 27 fromboth subunits of the S state dimer, the Ca RMSD is1.01 AÊ . This comparison highlights a self-similarityin the structure of wild-type ubiquitin previously


identi®ed by Vijay-Kumar et al.:30 residues 18 to 27(from the a-helix and the previous turn) overlaywith 51 to 60 (from the 310-helix and its precedingturn), giving a Ca RMSD of 0.71 AÊ . The sequencesof these two regions also resemble one another, asshown in Table 1. Four antiparallel hydrogenbonds link Asp52 O, Arg54 O, Leu56 N and Ser57N from the loop and the beginning of the 310-helixof ubiquitin with Glu24 N, Ile23 N, Asp21 O andPro19 O, respectively, at the start of the a-helix. Allfour of these hydrogen bonds are preserved in theUQ(1-51) dimer, with Pro190 O, Asp210 O, Ile230 Nand Glu240 N from the second subunit now joiningwith Glu24 N, Ile23 N, Asp21 O and Pro19 O,respectively, in the ®rst. As Figure 5(b) demon-strates, side-chain conformations at the two inter-faces are also very similar, with residues Thr220and Ile230 in the a-helix of the second subunit ofthe S state corresponding to Thr55 and Leu56 ofthe 310-helix of ubiquitin.

The main part of the dimer interface of the Sstate is formed by strand U3 (see Figures 3(b) and5(c)). In ubiquitin, U1 from the N-terminal hairpinis joined by hydrogen bonds in a parallel mannerto U5, which also makes an antiparallel packingarrangement with U3. The topology is similar inthe UQ(1-51) dimer, with U1 from one subunitmaking parallel contacts with U03 from the other,which in turn docks in an antiparallel fashion withU3 of the ®rst subunit. In the b-sheet of the S state,the third strand of the second subunit effectivelytakes the place of U5 in intact ubiquitin. Whenboth structures are compared over strands U1 (resi-dues 1 to 6) and U2 (11 to 17), along with a portionof the ubiquitin strand U5 (65 to 70) and U03 (43 to48) from the second subunit of UQ(1-51), the meanCa RMSD is 1.28 AÊ . However, Figure 3(c) alsoreveals some subtle differences between the twostructures. The four central strands in the b-sheetof the dimer are twisted differently to ubiquitin.Although the interactions between U03 and U3 ofUQ(1-51) and U5 and U3 of ubiquitin are parallel ineach case, the relative orientations of the elementshave changed and the strand registry has slippedby two residues. If a fourth strand is to be addedto the comparison described above, then a satisfac-tory overlay can be achieved only if residues 42 to45 of ubiquitin are matched with residues 40 to 43of UQ(1-51). In this case, the mean Ca RMSD is1.29 AÊ , which must be compared with the value of2.61 AÊ obtained if residues 42 to 45 are includedfor both structures.

In the UQ(1-51) dimer, the third strand U3

makes both parallel and antiparallel packing con-tacts with its adjacent b-strands, while in ubiquitinboth sides of U3 pack in an antiparallel manner(Figure 3(b) and (c)). Strand U3 is extended by tworesidues in the S state to include Ala46 and Gly47,which form part of the hairpin turn betweenstrands U3 and U4 in intact ubiquitin. The hydro-gen bonds between U1 and U5 (in ubiquitin) andU03 (in the dimer) are both parallel. In ubiquitin,residues Phe4 and Lys6 hydrogen bond to residues

Ser65, Leu67 and Leu69, while in UQ(1-51) thepartners are Leu430, Phe450 and Gly470 from thesecond subunit. Against these similarities, the car-bonyl group of residue Gln2 is not hydrogenbonded in UQ(1-51), while in ubiquitin it forms ahydrogen bond to the amide nitrogen atom of resi-due Glu64. The contacts between U3 and U5 (inubiquitin) and U03 (in the dimer) are antiparallel inboth systems, but the registry of the strands hasslipped back by two residues in the latter case.Additionally, the hydrogen bonding network link-ing the strands is more extensive in the dimer,with six hydrogen bonds connecting residuesArg420, Ile440 and Ala460 with Ala46, Ile44 andArg42, respectively, compared with three joiningThr66 and His68 to Ile44 and Arg42, respectively,in ubiquitin. The packing of the U5 and U3 strandsof ubiquitin is anchored by residue Leu67, which is¯anked by the side-chains of residues Phe45 andLeu43. However, because of the different strandregistry in the dimer, the side-chains of residuesLeu430 and Phe450 from U03 face residues Phe45and Leu43 symmetrically, as shown in Figure 5(c).In ubiquitin, U3 also packs against U4 in an anti-parallel arrangement, with hydrophobic inter-actions involving residue Leu50 packing againstLeu43 and Phe45 making a tertiary contact withIle61 in the loop that connects the 310-helix withU5. These contacts between U3 and U4 are lost inthe S state of UQ(1-51), despite the fact that Leu50is still present in the primary sequence. Instead,residues Leu43 and Phe45 make parallel contactswith Ile30 and Val50 from U01 in the other subunit.

The hydrophobic core of ubiquitin is formed byinteractions between the side-chains of three resi-dues from the a-helix and 11 from the b-sheet.30

Many of these contacts are retained in the S stateof UQ(1-51), but some modi®cations have beenmade to accommodate the dimer interface. In ubi-quitin, the side-chain of Leu43 reaches over fromstrand U3 to nest between those of Val26 and Ile30from the hydrophobic face of the a-helix. A similarpacking interaction is observed in the dimer, butthe place of the Leu43 side-chain is occupied byPhe450 from the adjacent U03 strand from the othersubunit. In the S state of UQ(1-51), the side-chainof Leu430 has replaced that of ubiquitin Leu67 inthe hydrophobic core (Figure 5(c)). The aromaticring of Phe45 is exposed to the solvent in ubiquitinbut in the dimer this residue plays a key role in theinterface between the two subunits, making contactwith Leu430.

NMR studies of the minor forms of UQ(1-51)

In 50 mM sodium phosphate at pH 7.0 manyresonances from the minor forms of UQ(1-51) pos-sess chemical shifts that are strongly perturbedfrom those of the largely unfolded DA state (seeFigure 1(a) and (b)). Several minor forms are pre-sent in addition to the S state and the large chemi-cal shift dispersion indicates that these alternativeconformations possess a high degree of structure.

Figure 6. Portions of two-dimensional [1H,15N] mag-netisation transfer spectra of the UQ(1-51) peptide at298 K and pH 7.0 in 0.35 M sodium sulphate. (a)Exchange delay of 1 ms. (b) Exchange delay of 100 ms.

Figure 7. Normalised changes in 1HN and 15N chemi-cal shifts between the minor forms of UQ(1-51) observedat 298 K and pH 7.0 in 0.35 M sodium sulphate withthose of the S state for each residue, with�d(1HN/15N) � ((�d(1HN))2 � (�d(15N)/10)2)0.5. (a)Differences between the M1 form and the S state. (b)Differences between the M2 form and the S state. Forresidues with only one minor form, the value of�d(1HN/15N) was included in both sets.


Some minor form resonances are weak and broad,while others are narrow and intense. In addition,under these conditions the peptide fragment bindsto the hydrophobic dye 8-anilino-1-naphthalenesulfonate (ANS), giving rise to a strong ¯uor-escence (not shown). Thus, in 50 mM sodiumphosphate at pH 7.0, UQ(1-51) has characteristicsin common with a molten globule state, consistentwith exposed hydrophobic patches and a lack ofconformational speci®city. However, in contrastwith most MG states, separate species can be dis-tinguished. Furthermore, the 3D 15N- and 13C-sep-arated NOESY spectra collected for structuralstudies of the S state contained a few weak cross-peaks due to magnetization transfer to minorforms that are still present at low levels in 0.8 Msodium sulphate. We therefore attempted tocharacterise the minor forms by magnetisationtransfer techniques using a 15N-labelled sample ofUQ(1-51) and a sodium sulphate concentration of0.35 M. The [1H,15N]-HSQC spectrum under theseconditions was crowded, so two 3D magnetisation

transfer experiments were developed to gain resol-ution by adding an extra 1H or 15N dimension.

Analysis of these spectra showed that nearly allof the resonances in the [1H,15N]-HSQC spectrumof the S state have at least one correspondingminor form in 0.35 M sodium sulphate, with mostpossessing two. For example, residue Ser20 hastwo extra resonances, both with 1HN and 15Nchemical shifts close to those of the S state(Figure 6(a)). The chemical environments of Ser20are therefore similar in all three cases, implyingthat in the vicinity of this residue any structuralchanges between the different forms are minimal.As Ser20 forms inter-subunit contacts in the helix-helix portion of the dimer interface of the S state, itis likely that this feature still exists in both minorforms. Two additional signals are also seen forIle36 and Gln41, but in one of the minor forms thechemical shifts of both residues are strongly per-turbed (Figure 6(b)). This observation suggests thatin the perturbed minor form, residues in the thirdstrand and the preceding turn experience a verydifferent chemical environment.

Crosspeaks from the two most signi®cant minorforms present in 0.35 M sodium sulphate were sep-arated into two sets, M1 and M2, according to thesmallest and largest values, respectively, of thenormalised difference in 1HN and 15N chemicalshifts between the minor form and the S state.Figure 7(a) clearly implies that all of the resonances


in the M1 set have chemical environments that areclose to those of the S state. The chemical shifts ofresidues Gln2-Gly35 in the M2 set are also similarto those of the S state dimer, but the signals fromresidues Met1, Ile36 and Gln41-Ala46 are stronglyperturbed (see Figure 7(b)). We have not demon-strated that each of the resonances allocated to setM1 or set M2 belong to the same species, but thesimplest interpretation of these chemical shiftchanges is that one of the minor forms possesses astructure similar to that of the S state. The otherminor form apparently also has a similar fold, butwith large changes in the environments of nucleiclustered in strands U1 and U3, which make uppart of the dimer interface at the centre of the b-sheet in the S state structure.

These conclusions agree with the results of CDspectroscopy on UQ(1-51), which indicated that thesecondary structure content did not change signi®-cantly between 0.35 M and 0.8 M sodium sulphate(not shown). Gel-®ltration experiments in 0.35 Msodium sulphate gave a single peak with retentiontime of 12.54 minutes for UQ(1-51) compared with12.97 minutes for ubiquitin (not shown). This resultsuggests that all three of the UQ(1-51) species pre-sent have the same size and that both of the minorforms are dimers. Furthermore, close examinationof 2D [1H, 15N]-HSQC spectra of UQ(1-51) in var-ious concentrations of sodium sulphate revealedthat the two minor forms are always populatedequally (e.g. Ser20 in Figure 6(a)), while magnetiza-tion transfer experiments showed that the minorforms are in exchange with each other as well aswith the S state (e.g. Ser20 in Figure 6(b)).

It therefore seems plausible to suggest that boththe M1 and the M2 signals are caused by a singledimeric species with a structure related to that ofthe S state but having lost its 2-fold centre of sym-metry, probably as a result of the movement of asingle b-strand. For each residue in the sequence ofUQ(1-51) this would produce two signals of equalintensity but with different chemical shifts. Theconformation of one of the dimer subunits wouldclosely resemble that of the S state, producingmost of the resonances that we have classedtogether as the M1 set. The structure of the othersubunit would be broadly similar, but with a seg-mental rearrangement in strand U3, causing chemi-cal shifts to be perturbed in this region. Largechanges in the chemical shifts of the M2 set werefound for residue Met1 and for sites in U3, whileonly small changes were observed for strand U3 inthe M1 set. Hence, we can speculate further thatthe symmetry of the dimer could be broken due toa disruption of the interaction between the ®rststrand of one subunit and the third strand of theother. The two minor conformers are able toexchange with one another and with the S state ona timescale of 100 ms (Figure 6(b)). According toour model, exchange between the asymmetricminor forms could occur via the S state. After arearrangement, the disrupted interactions betweenstrands U1 and U03 could be restored, producing

the symmetrical S state structure. Following this,the conformation could change again, with theb-sheet being ruptured either between U1 and U03,to reform the ®rst asymmetric dimer, or betweenU01 and U3, yielding the alternative form. It is per-haps not surprising that strand U3 is the most het-erogeneous portion of UQ(1-51) in low saltconditions, as this is the region that is most differ-ent from ubiquitin in our structure of the S state in0.8 M sodium sulphate.

Conclusions

We have demonstrated that the peptide frag-ment UQ(1-51), spanning the sequence of humanubiquitin from the N terminus to the end of thefourth b-strand, is unfolded in acid conditions (theDA state) but forms a variety of structured confor-mations at pH 7.0. In a 0.8 M solution of sodiumsulphate, a uniquely folded symmetrical dimericstructure is achieved (the S state), which is amen-able to studies by NMR. The solution structure ofthe S state offers several clues as to why the pep-tide fragment is able to fold successfully. First, theN-terminal hairpin-helix motif of the UQ(1-51)dimer is very similar to that of the intact protein.30

Second, there is a remarkable 2-fold pseudo-sym-metry in the structure of ubiquitin between resi-dues 18 to 27 and 51 to 60 at the turn and Ntermini of the a-helix and the 310-helix, respect-ively. When the protein is truncated, the a-helicesof the two subunits dock together in a way thatclosely mimics the a-helix:310-helix interface ofwild-type ubiquitin. Next, the dimer interface alsoinvolves an intercalation of the third strands (U3)of each monomer, which make antiparallel inter-actions with each other and parallel contacts withthe ®rst strand (U1) of the dimer partner. Thisinterface is topologically similar to the strandarrangement adopted in wild-type ubiquitin,where strands U1 and U5 interact in a parallelfashion. Last, the hydrophobic core of ubiquitin islargely preserved and extended in the UQ(1-51)dimer, although some side-chain conformations arealtered to accommodate the interface between thetwo subunits.

The topology of the UQ(1-51) dimer is reminis-cent of that found in oligomeric systems created bydomain swapping, in which a structural elementfrom one subunit substitutes for the same elementin a partner molecule but otherwise retains anidentical environment.59 ± 62 However, a differentphenomenon is occuring in the S state of UQ(1-51),as structural elements in the wild-type monomer(U5 and the 310-helix) are replaced by differentunits from the dimer partner (U3 and the a-helix).The substituted elements therefore experiencealtered rather than equivalent quaternary environ-ments.

As well as providing a rationale for the structureof UQ(1-51), NMR studies of the S state providedinsight into the properties of the various minor


forms populated in low salt conditions. In 50 mMsodium phosphate at pH 7.0, the peptide frag-ments associate as dimers but lack structural speci-®city, having characteristics in common with themolten globule states of some proteins.11 ± 13 Ananalysis of differences in chemical shift betweenresonances from the minor forms and the S state in0.35 M sodium sulphate identi®ed regions of thepeptide backbone that undergo the greatestchanges in chemical environment between the var-ious species. Of the two minor forms, one is appar-ently very similar to the S state, while the other issubject to large changes in the third strand. A seg-mental rearrangement of U3 in one subunit of the Sstate would render the dimer asymmetric, whichwould account for many of our experimentalobservations.

The turn between the helix and strand U3 in thestructure of the UQ(1-51) dimer contains two pro-line residues, isomerisation of which could beresponsible for the differences in structure betweenthe S state and the minor forms. However,exchange between the various conformations ofUQ(1-51) is very rapid, occurring on a timescale of100 ms. By contrast, the intrinsic rate of proline iso-merisation has been measured as 0.03-0.2 minÿ1.63

A previous case of structural heterogeneity occur-ring on the 100 ms timescale that was initiallyinterpreted as due to proline isomerisation64,}65

was later found to be the result of a movement ofhydrophobic side-chains.66 In experiments on CI267

and staphylococcal nuclease68 that followed proteinfolding from the denatured state, proline isomeri-sation occurred with relaxation times of 2.5 sÿ1

and 17 sÿ1, respectively. It is likely that these iso-merisations occur so rapidly because there arelocal and tertiary interactions that stabilise the for-mation of native proline isomers, facilitated by thestructural freedom of the unfolded state. In UQ(1-51) the dimer is already structured, so a degree ofunfolding would be required for such isomerisa-tion to occur. Furthermore, in the S state both pro-line residues are in the most favoured trans isomer,as found in wild-type ubiquitin. The heterogeneityobserved in UQ(1-51) is therefore more likely dueto a loss of structural speci®city, similar to thatseen in redesigned forms of ubiquitin25 and othersystems.16 ± 21,63,69

The behaviour of UQ(1-51) in low salt is intri-guing because it throws light on transitions thatcan occur between low-lying states on the energylandscape of a protein. As a result of the highcooperativity of protein folding, highly populatedalternative states are seldom observed in the slow-exchange regime, as we ®nd here. A natural pro-tein with a unique native conformation possesses asigni®cant energy gap between the folded stateand the next accessible partially folded state.8 Incontrast to wild-type ubiquitin, the energy gap ofthe arti®cial S state dimer has not been optimisedby evolutionary pressures, so alternative confor-mations are populated under low salt conditions.Small-scale conformational motions of the third

strand appear to be responsible for transitionsbetween these different low-lying states. Suchrearrangements are similar to the local unfoldingevents that have been invoked to provide a mech-anism for solvent exchange at amide proton sitesthat have an intermediate level of protection.70,71

An understanding of small-scale unfolding eventslike these could be used to benchmark moleculardynamics simulations of protein unfolding. Accu-rate simulations of such structural ¯uctuationsmay be useful in modelling the motions of proteinsin diverse processes, including protein foldingand unfolding, enzyme catalysis and molecularrecognition.

Low-lying excited state conformations haverecently been invoked to account for thetemperature72 and pressure73 dependence of back-bone chemical shifts in a variety of proteins. Highpressure can increase the population of rare confor-mers that possess smaller partial volumes than thenative state. Neutron diffraction studies havedemonstrated that high concentrations of sodiumsulphate act to decrease the extent of hydrogenbonding in bulk water in a manner similar to thatdetected in pure water under high pressure.74 It islikely that the symmetrical S state of UQ(1-51) ismore compact than the partially unfolded minorforms observed at low salt. This suggests that themechanism by which the population of the S stateincreases with increasing concentrations of sodiumsulphate could be similar to that operating underhigh-pressure conditions.

Materials and Methods

DNA manipulation, protein expressionand purification

Standard methods were used for DNA manipulationsand cloning,75 using reagents from Sigma-Aldrich Co.The gene encoding the ®rst 51 residues of ubiquitin(UQ(1-51)) was generated by PCR and cloned into theplasmid pGEX-4T3 using BamHI and EcoRI restrictionsites. UQ(1-51) as a fusion to glutathione-S-transferase(GST)76 was expressed in BL21 Escherichia coli, grown at37 �C, by induction of mid log phase cells with 0.1 mMisopropyl-b-D-thiogalactoside (IPTG). For NMR samplepreparation, bacteria were grown in a modi®ed minimalmedium containing [13C]glucose, 15NH4Cl and 15N or15N/13C-labelled Celtone (Martek Biosciences Corp.) asthe sole sources of carbon and/or nitrogen. The cellswere harvested by centrifugation ®ve hours after induc-tion and lysed by sonication. The cell lysate was clari®edby centrifugation (35,000 g) and the GST-fusion was pur-i®ed from the supernatant by standard procedures usinga glutathione-agarose column (Pharmacia). Puri®ed GST-fusion protein was digested with thrombin proteaseovernight at 4 �C and the peptide fragment puri®ed on a15 ml self-poured RPC-15 reverse phase column with alinear gradient of acetonitrile 0.1 % (v/v) tri¯uoraceticacid (TFA) in acetonitrile/0.1 % aqueous TFA using anHP1090 liquid chromatograph HPLC system (Hewlett-Packard). Samples were then lyophilised and stored at4 �C. The UQ(1-51) peptide has an N-terminal Gly-Serextension relative to the ubiquitin sequence (see Table 1),


but the amino acid residues are numbered as in theparent molecule for clarity.

Equilibrium analytical ultracentrifugation

Solutions, 0.5, 1 and 2 mg mlÿ1, of UQ(1-51) weremade up in 50 mM sodium phosphate buffer at pH 7.0.The samples were spun in a Beckman XL-I AnalyticalUltracentrifuge in an An60 Ti rotor at speeds of17,000 rpm, 23,000 rpm and 30,000 rpm at 278 K untilequilibrium had been reached.77 Interference optics wereused to determine the equilibrium concentration pro®leof UQ(1-51), which was analysed according to equation(1) of McRorie & Voelker78 using the program NonLinv.2 (Beckman), assuming a common molecular mass forall peptide species present in the solution. Data for all ofthe concentrations and rotor speeds were consideredsimultaneously using a non-linear least-squares method,treating the reduced molecular mass, the concentrationoffset of the ®rst data point, and the initial monomerconcentration of each run as ¯oating variables.

Analytical gel-filtration

Gel-®ltration was carried out with a TSK-gel G2000SWXL (TosoHaas, Japan) column, at 0.8 ml minÿ1, usinga HP1090 liquid chromatograph HPLC system (Hewlett-Packard). The UQ(1-51) peptide was prepared unfoldedat pH 3.0 by dissolution in ice-cold water. Oligomericcomplexes were refolded by addition of sodium phos-phate solution at pH 7.0 to a ®nal concentration of50 mM. Sodium sulphate was added to the running buf-fer as required prior to the gel-®ltration.

NMR spectroscopy and assignment of spectra

All samples were prepared containing 1 mM UQ(1-51), 50 mM sodium phosphate, 0.05 % (w/v) sodiumazide, 20 mM 3,3,3-trimethylsilylpropionate (TSP) and10 % 2H2O, to a ®nal volume of 550 ml in 5 mm Ultra-Imperial grade NMR tubes (Wilmad Glass Co.). All spec-tra were recorded at a 1H frequency of 500.01 MHz on aBruker DRX spectrometer equipped with a z-shieldedgradient triple resonance probe. The data were collectedwith 128* and 1024* pairs of complex (*) points andacquisition times (tmax) of 64 ms and 102 ms in the 15Nand (1HN) dimensions, respectively ([1H,15N]-HSQC);128* � 639* points and tmax of 34 ms and 64 ms inthe 13C and 1H dimensions ([1H,13C]-HSQC);32* � 92* � 1024* points and tmax of 16 ms, 18 ms and102 ms in the 15N, 1H and 1HN dimensions (15N-TOCSY-HSQC and 15N-NOESY-HSQC); 26* � 64* � 640* pointsand tmax of 13 ms, 6 ms and 64 ms in the 15N, 13C and1HN dimensions (HNCA and HN(CO)CA);32* � 80* � 639 points and tmax of 16 ms, 14 ms and64 ms in the 15N, 13C and 1HN dimensions (HNCACBand CBCA(CO)NH); 26* � 64* � 639* points and tmax of13 ms, 12 ms and 64 ms in the 15N, 1H and 1HN dimen-sions (HBHA(CBCACO)NH); 26* � 64* � 640* pointsand tmax of 13 ms, 6 ms and 64 ms in the 15N, 13C0 and1HN dimensions (HNCO); 40* � 100* � 640* points andtmax of 4 ms, 18 ms and 64 ms in the 13C, 1H and 1Hdimensions (HCCH-TOCSY); 36* � 100* � 1024* pointsand tmax of 7.2 ms, 18 ms and 64 ms in the 13C, 1H and1H dimensions (13C-NOESY-HSQC); 24* � 100* � 640*points and tmax of 5 ms, 18 ms and 64 ms in the 13C, 1Hand 1H dimensions (13C-rejected, 13C-separated NOESY-HSQC). Water suppression was achieved by ``¯ip-back''

methods, using shaped selective pulses to return waterto the z-axis prior to acquisition. Pulsed ®eld gradientswere used to suppress undesired coherence pathwaysand the residual water signal.79 Data processing andspectral analysis were carried out on a Silicon GraphicsO2 workstation using the programs AZARA (W. Bou-cher, unpublished work) and ANSIG 3.3,80 respectively.

For the DA state of UQ(1-51) at pH 3.0 and 278 K,backbone assignments were obtained from 3DHNCACB, CBCA(CO)NH81 and 15N-separated TOCSY82

spectra on a 15N/13C-labelled sample, analysed withreference to a 2D [1H,15N]-HSQC83 experiment.

Backbone assignments for the S state of UQ(1-51) atpH 7.0 and 298 K were derived initially from 3D15N-separated TOCSY and NOESY83 experiments on a15N-labelled sample. These results were later con®rmedwith a 15N/13C-labelled sample using unambiguousthrough-bond connections from 3D HNCA, HN(CO)CA,HNCO, HNCACB, and HBHA(CBCACO)NH experi-ments.84 The assignments were extended to side-chainsites using 3D CBCA(CO)NH, HBHA(CBCACO)NH andHCCH-TOCSY84 spectra, analysed with reference to a2D constant time [1H,13C]-HSQC experiment.85 NOEdata were collected from 3D 15N and 13C-separatedNOESY84 experiments on the 15N and 15N/13C-labelledsamples, respectively, both with mixing times of 100 ms.Inter-subunit NOEs were identi®ed from a 3D13C-rejected, 13C-separated NOESY experiment on asample prepared from an equimolar mixture ofunlabelled (12C/14N) and labelled (13C/15N) UQ(1-51)peptides.49,56

An experiment based on a [1H,15N]-HSQC, but with2HzNz periods on either side of the t1 period, was usedto observe magnetisation transfer between 1HN sites ofthe different UQ(1-51) species present at 298 K andpH 7.0 in 0.35 M sodium sulphate. A delay of the orderof 100 ms during the second of these periods allowstransfer of 2HzNz two-spin order between the species,giving rise to exchange crosspeaks in the ®nal spectrumin a similar way to the Nz-based experiment reportedby Zhang et al.16 In order to gain resolution, the exper-iment was extended into a third dimension. Two ver-sions were performed, which may be summarised asF1(

1H)-F2(15N)-tm-F3(

1H) and F1(15N)-tm-F2(

15N)-F3(1H),

using 24* � 32* � 639* points and tmax of 24 ms, 32 msand 64 ms in the 1HN, 15N and 1HN dimensions and32* � 32* � 639* points and tmax of 32 ms, 32 ms and64 ms in the 15N, 15N and 1HN dimensions, respectively.

Restraints and structure calculations for the S stateof UQ(1-51)

NOE crosspeak intensities were classi®ed as strong,medium and weak and, using secondary structureelements for calibration, were converted into distancerestraints by grouping them into three ranges, <2.8 AÊ ,<3.5 AÊ and <5.0 AÊ , respectively. Additional backbonedihedral angle restraints were obtained from analysis of1Ha, 13Ca, 13Cb, 13C0 and 15N chemical shifts with the pro-gram TALOS,52 but with the chemical shift and structur-al data for wild-type ubiquitin excluded from thedatabase in order to remove possible bias.

Structures were calculated with the program CNS0.9,53 using the PARALLHDG v5.1 force-®eld inPROLSQ mode86 and ``sum'' averaging with an rÿ6

potential for all atoms. The C2 symmetry of the dimersubunits was maintained as described by Brasher et al.,87

using the non-crystallographic symmetry (NCS) term


and pseudo-NOE distance restraints.56 An initial ensem-ble of structures was generated using a simulatedannealing protocol in Cartesian space; subsequent iter-ations were performed, decreasing the number of ambig-uous intra- or intersubunit NOE distance restraints witha distance ®lter.55 Structures with the lowest total energyand with no distance violation greater than 0.5 AÊ wereselected for the ®nal ensemble and then analysed usingCNS53 and PROCHECK.57 Figures were generated usingMOLSCRIPT88 and Raster3D.89

Protein and Biomolecular Magnetic Resonance DataBank accession numbers

The atomic coordinates of the ®nal ensemble of 16conformers of the S state of UQ(1-51), together withthe experimental distance and angle constraints, havebeen deposited in the RCSB Protein Data Bank(www.rscb.org; accession code 1GJZ). NMR assignmentshave been deposited at the Biomolecular MagneticResonance Databank (www.bmrb.wisc.edu; accessionnumber 5101).

Acknowledgements

We thank Dr M. Deacon for help with the AUC exper-iments; the PNAC facility in the Department of Biochem-istry for DNA, protein and peptide analysis; and Drs R.Fogh, B. Smith, and H. Mott for help with the structurecalculations. The core facilities of the Cambridge Centrefor Molecular Recognition are funded by the BBSRC andthe Wellcome Trust. D.B. was supported by a researchstudentship from the MRC.

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Edited by R. Huber

(Received 9 August 2001; received in revised form 5 October 2001; accepted 10 October 2001)

Structure and properties of a dimeric N-terminal fragment of human ubiquitin

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