Model Peptide Studies ofSequence Regions in theElastomeric BiomineralizationProtein, Lustrin A. I. The C-Domain Consensus—PG—,—NVNCT—Motif

Bo Zhang1

Brandon A. Wustman1

Daniel Morse2

John Spencer Evans1

1 Laboratory for ChemicalPhysics,

New York University,345 E. 24th Street,

New York, NY 10010

2 Department of Molecular,Cellular, and Developmental

Biology,and Materials Research

Laboratory,University of California,

Santa Barbara, CA 93106

Received 10 September 2001;accepted 8 November 2001

Published online ●●● in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bip.10069

Abstract: The lustrin superfamily represents a unique group of biomineralization proteins local-ized between layered aragonite mineral plates (i.e., nacre layer) in mollusk shell. Recent atomicforce microscopy (AFM) pulling studies have demonstrated that the lustrin-containing organicnacre layer in the abalone, Haliotis rufescens, exhibits a typical sawtooth force-extension curve withhysteretic recovery. This force extension behavior is reminiscent of reversible unfolding andrefolding in elastomeric proteins such as titin and tenascin. Since secondary structure plays animportant role in force-induced protein unfolding and refolding, the question is, What secondarystructure(s) exist within the major domains of Lustrin A? Using a model peptide (FPGKN-VNCTSGE) representing the 12-residue consensus sequence found near the N-termini of the firsteight cysteine-rich domains (C-domains) within the Lustrin A protein, we employed CD, NMRspectroscopy, and simulated annealing/minimization to determine the secondary structure prefer-ences for this sequence. At pH 7.4, we find that the 12-mer sequence adopts a loop conformation,consisting of a “bend” or “turn” involving residues G3–K4 and N7–C8–T9, with extendedconformations arising at F1–G3; K4–V6; T9–S10–G11 in the sequence. Minor pH-dependentconformational effects were noted for this peptide; however, there is no evidence for a salt-bridgeinteraction between the K4 and E12 side chains. The presence of a loop conformation within thehighly conserved —PG—, —NVNCT— sequence of C1–C8 domains may have important structural

Correspondence to: John Spencer Evans; email: jse@dave-edmunds.dental.nyu.edu

Contract grant sponsor: National Science Foundation (NSF) andArmy Research Office (ARO).

Contract grant number: DMR 99-01356 and MCB 98-16703(NSF); DAA19-99-1-0225 and MURI DAAH04-96-1-0443 (ARO).Biopolymers, Vol. 63, 358–369 (2002)© 2002 Wiley Periodicals, Inc.

358

and mechanistic implications for the Lustrin A protein with regard to elastic behavior. © 2002Wiley Periodicals, Inc. Biopolymers 63: 358–369, 2002

Keywords: lustrin; biomineralization proteins; aragonite mineral plates; mollusk shell; atomicforce microscopy; nacre layer; abalone; sawtooth force-extension; hysteretic recovery; reversibleunfolding and refolding; elastomeric proteins; secondary structure

INTRODUCTION

Molecular elasticity is associated with a select numberof polypeptides and proteins.1–6 As an example, thelustrins, a protein superfamily1,2 localized within lay-ered aragonite mineral plates (i.e., nacre layers) inmollusk shell, have been postulated to play a role inthe enhancement of nacre layer fracture toughness.2

Recent atomic force microscopy (AFM) pulling stud-ies have demonstrated that the organic layer of thenacre in the abalone, Haliotis rufescens, exhibits atypical sawtooth force-extension curve with hystereticrecovery,2 reminiscent of reversible unfolding andrefolding in elastomeric proteins such as titin3–5 andtenascin.6 One particular lustrin protein, Lustrin A (H.rufescens, 116 kDa) has a modular organization con-sisting of the following unique domains: (1) Tenhighly conserved cysteine-rich domains (C1–C10),that contain Val, Asn, Lys, Arg, Pro, Glu, and Aspresidues. (2) Eight Pro-rich domains (P1–P8), whichare comprised of Pro–Pro, Pro–X–Pro, and Pro–X–X–Pro sequence repeats. Each of the eight Pro-richdomains are flanked upstream and downstream byC-type domains. (3) A 250-residue Gly, Ser-rich do-main comprised of (GS)x and (GSSS)y repeats (i.e.,“serine loops”). Thus, the Lustrin A protein combinesseveral structural motifs within a single polypeptidechain. Consequently, there is considerable interest indefining the structure–function relationships withinthe lustrin superfamily and establishing their partici-pation in the molecular basis of nacre shell elasticity.

We pose the following question: What are thesecondary structures found within the major domainsof Lustrin A, and can domain secondary structureprovide an explanation for the observed elastic re-sponse in the nacre? Clearly, the molecular weightand sequence repetition of Lustrin A present us witha number of challenges with regard to secondarystructure determination. In previous studies of repeatmotifs in biomineralization proteins, a model peptidestrategy was successfully utilized to determine sec-ondary structure preferences11,12 and probe polypep-tide divalent cation binding sites13 within repetitivesequence domains. In addition, model peptide ap-proaches have advanced our understanding of proteinfolding and the relationship between primary se-quence and secondary structure.14–21

In this first of a series of reports, we employ NMRspectroscopy to define the secondary structure prefer-ences for sequence repeats in the Lustrin A protein.Our initial focus is on a 12-residue consensus se-quence found near the N-terminal end of the C1–C8domains within the Lustrin A protein1:

—�E/F/T/(PG)L/K/V(NVNCT)A/S/T/K(G)E/T/V�—

This 12-mer sequence is considered to be one ofseveral putative elastic motifs within Lustrin A, due toits sequence similarity with the extensible coil—PEVK— domain of titin.3–5 It also is believed thatthis consensus repeat may participate in interdomaindisulfide bond formation within the protein.1 Interest-ingly, the conserved —PG— and —VN— dipeptideregions (within the parentheses) have been shown toparticipate in �-turn and �-hairpin formation in otherprotein sequences,19–26 and, Glu and Lys side chainshave been demonstrated to participate in ion-pairinginteractions within �-hairpins.27 We created a repre-sentative model peptide of the above-mentionedLustrin A C-domain consensus repeat, FPGKN-VNCTSGE (sequence position 142–153, C2 domain,boldface residues indicating conserved consensus se-quence), and performed NMR structural studies onthe unblocked peptide.16,24,25,27 Because Lys–Gluside chain or termini salt bridge stabilization of�-hairpin and �-turn structures have been reported inpeptide models,27,28 we analyzed the peptide confor-mation at pH 7.4 and pH 2.0 to examine possiblesalt-bridging stabilization involving the K4/E12 resi-dues and/or the N,C-termini. We have deduced that, atpH 7.4, the 12-mer sequence adopts a loop conforma-tion, consisting of a “bend” or “turn” involving resi-dues G3–K4 and N7–C8–T9. These bend or turnregions are interspersed with extended conformationsarising at F1–G3; K4–V6–N7; T9–S10–G11 in thesequence. Thus the loop motif is formed by an alter-nating “extended-turn-extended-turn-extended” re-peat structure. We did not detect the presence of asalt-bridging interaction involving the K4 and E14side chains; however, our modeling studies indicatethat there may be electrostatic interactions betweenthe �-NH3

� and �-COO� termini. As discussedherein, the existence of loop regions within Lustrin A

Model Peptide Studies. I 359

are consistent with the proposed elastomeric functionof this unique protein.

MATERIALS AND METHODS

Purified unprotected 12-mer peptide was synthesized usingan Applied Biosystems 431A Peptide Synthesizer and N�-L-Fmoc-amino acids (Fmoc � 9-flourenylmethyoxycar-bony) (Genemed Synthesis, Inc., South San Francisco, CA94080). Typical peptide synthesis runs were carried out atthe 100 �mole level using the Applied Biosystems “Fast-Moc 0.25” HBTU/HOBt/NMP protocol [HBTU � 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-phosphate; HOBt � N-hydroxybenzotriazole; NMP � n-methylpyrrolidone; Applied Biosystems Technical Notes,November, 1993, 4-hydroxymethylphenoxyacetyl polyeth-yleneglycol acrylamide (HMPA-PEGA) resin (Novabio-chem, San Diego, CA] and a double coupling procedure.For C�-amide protected peptides, the synthesis protocolutilized methylbenzyhydrylamine (MBHA) resin (Novabio-chem). To avoid possible side-chain modification duringsynthesis, NY-Trityl-N�-Fmoc-Asn (abbreviated as Fmoc–Asn–Trt) was utilized in 12-mer synthesis runs.29 For“capped” peptides, the C�-amide peptides were treated withacetic anhydride in N-methyl pyrrolidone/diisopropylethyl-amine (NMP/DIEA) to create the N�-acetyl derivative. Thecompleted peptides were deprotected and cleaved (reactiontime � 3 h, at 25°C) from the resin using a cleavage cocktail(15 mL/g resin) containing 90% v/v triflouroacetic acid(TFA), 2.5% v/v water, 2.5% v/v ethanedithiol, 2.5% v/vphenol, and 2.5% v/v thioanisole.11,12 The thiol scavengerswere utilized to prevent Met and Cys oxidation and enhancetrityl removal.29,30 The reaction mixture was filtered underreduced pressure. The crude peptides were separately dis-solved in deionized distilled water, extracted three timeswith diethyl ether, and then concentrated and lyophilized.Peptide purification involved C18 reverse-phase high per-formance liquid chromatography (HPLC) column, using0.1% TFA/water mobile phase and eluting with a 80%acetonitrile/0.1% TFA/water linear gradient. Peptide elutionwas monitored at 230 nm. Individual HPLC fractions wereanalyzed using a custom-made matrix-assisted laser desorp-tion/deionization time of flight (MALDI/TOF) delayed ex-traction mass spectrometer. Typically, a 100 �mole synthe-sis of the 12-mer yielded � 70 mg dry mass of purifiedpeptide that is �96% in purity and free of side-chain pro-tection. The experimental molecular weight for the unpro-tected form was determined to be 1252 Da, in agreementwith the theoretical molecular weight of 1251 Da. For NMRstudies, purified peptide were dissolved in 1 mM Na2HPO4

in deionized distilled water, pH 7.4, containing 10% v/vdeuterium oxide (99.9% atom D, Cambridge Isotope Labs)and 10 micromolar d4-trimethylsilapentanoic acid (TSP);final peptide concentration was 8 mM in a volume of 450microliters. A parallel sample was prepared at pH 2.0. Atthese concentrations, turbidity measurements at 380 nmrevealed no evidence of peptide aggregation for all samples;this also was confirmed by analyses of NMR proton chem-

ical shifts and linewidths, and the absence of long-rangedsc-sc nuclear Overhauser effect (NOE) connectivities.

CD ExperimentsCD spectra were obtained at pH 2 and 7.4 using an AVIV62DS CD spectrometer, running 60DS software version 4.1tand quartz cells with a 0.1 cm pathlength. The samples werescanned from 190 to 260 nm at 5°C, using 1 nm bandwidthand a scan rate of 1 nm/s. The spectrometer was previouslycalibrated with d-(�)-10-camphorsulfphonic acid. A total of5 scans were acquired for each peptide. Mean residue ellip-ticity [�M] is expressed in deg cm2 dmol�1 per mole pep-tide.12

NMR ExperimentsAll NMR experiments were performed on a Varian UNITY500 spectrometer equipped with linear amplifiers, variabletemperature controller, and, a three-channel (13C/15N/1H)z-axis pulsed-field gradient (PFG) 5 mm solution probe-head.11,12 Probehead sample temperature was maintainedusing a ethylene glycol cooling apparatus with filtered air-flow. Temperature was maintained within �0.1°C. With theexception of the proton amide temperature shift experi-ments, all reported NMR experiments were conducted at278 K in order to slow down conformational exchangewithin the peptide.11,12 Proton scalar coupling assignmentswere obtained using “excitation sculpting”31 two-dimen-sional (2D) PFG-“clean” total correlated spectroscopy(TOCSY) experiments.11,12,32 Proton sequential assign-ments and NOEs were obtained using (z-axis)-PFG-NOEspectroscopy (NOESY) and rotating frame NOESY(ROESY) experiments,11,12,31 using a range of mixing timesfrom 50 to 200 ms; spectra were jointly analyzed to excludeartifactual NOEs arising from spin diffusion.19 3JNH-CH�

values were determined using z-PFG phase enhanced (P.E.)correlated spectroscopy (COSY) experiments.11,12,33 Noobservable cross-relaxation cross-talk artifacts were de-tected in the z-PFG P.E. COSY J-coupling spectra34; inaddition, we found that the cross-peak linewidths in the P.E.COSY spectra were not significantly affected by linebroad-ening, via comparisons of spectra obtained at 5 and 20°C(data not shown). NMR data were processed using FE-LIX2.30 software (MSI/Biosym Technologies, Inc.). Rele-vant NMR acquisition and processing parameters are pro-vided in the figure legends. Using TOCSY or NOESYexperiments at 278, 283, 288, 293, and 298 K, 12-meramide proton temperature coefficients were determinedfrom the slope of the temperature vs amide proton chemicalshift curves for each residue.11,12,35 Temperature gradientsare expressed in units of ppb/K with a negative sign indi-cating an upfield shift upon warming. Temperature calibra-tion of the VT unit was determined prior to experimentationusing neat methanol over a temperature range of 273–320 K.

Model BuildingTo construct conformations of the Lustrin A 12-mer at pH7.4, we performed simulated annealing molecular dynamics

360 Zhang et al.

using the MSI/Biosym InsightII/Discover 3.0 softwarepackage (MSI/Biosym Technologies, San Diego, CA).Starting peptide structures were constructed using theBIOPOLYMER module; all peptides initially are in theextended configuration (�,� � 0°, 180°). Pseudo-atomswere utilized for non-stereospecific protons. For pH 7.4simulations, the terminal �-NH3

�, K4 �-NH3� groups were

each assigned a net charge of �1, and the terminal�-COO�, E12 �-COO— groups were each assigned a netcharge value of �1. The overall net charge of the peptidewas zero. Once initial structures were completed, distanceand torsion angle restraints were applied; dihedral angleswere calculated for coupling constants of �8 Hz (errorvalue � �2 Hz) using the Karplus equation with parame-terization as described elsewhere,36 and inter- and intraresi-due interproton distances were calculated using a referencedistance of 2.47 Å for the -CH and �-CH aryl protons.37

With regard to interproton distances, all NOEs were classi-fied as either strong (1.8–2.5 Å), medium (2.5–3.5 Å), orweak (3.5–5.0 Å) interactions21,37; these distances wereemployed using flat-bottomed restraints with force con-stants of 200 and a maximum force value of 1000. The totalnumber of NMR restraints were 42 (33 NOEs, 9 J-cou-plings). Once NMR restraints were applied to the peptides,the extended peptide structures were minimized using theNewton–Raphson algorithm to convergence.

Parallel simulated annealing molecular dynamic simula-tion runs were performed using either the Verlet “leapfrog”(1 fs/step) or RATTLE (2 fs/step) algorithms and the Con-sistent Valence Force Field (CVFF).38 An implicit solventmodel was employed for both minimization and moleculardynamics simulations using a distance-dependent dielectricand no cutoffs for electrostatic interactions. Subsequently,the minimized structures were then subjected to 30 ps ofrestrained dynamics at 300 K (equilibration phase), heatedto 650 K over 30 ps, then cooled to 300 K over 30 ps for atotal simulation time of 90 ps. Upon termination of theannealing phase, a final minimization was performed usingconjugate gradient minimization to convergence. Finalstructures were generated by this protocol for each pHvalue; these structures were then compared to pH-specifictarget NMR proton–proton distance constraints and torsionangles to determine which conformers exhibit the bestmatch to experimental data.

RESULTS

CD Spectrometry

The far-uv CD spectra for the Lustrin A 12-mer at pH2 and pH 7.4 (Figure 1) indicates that at either pH, thepeptide exhibits a negative ellipticity centered at 234nm. Although atypical for �-helix or �-sheet, thisband is similar to the amide group n* negativetransition band observed near 224 nm for �-turn pep-tides39–42; however, in the 12-mer peptide, this bandappears to be red shifted.39 At pH 2, we note only a

small decrease in the 234 nm band intensity relative tomeasurements taken at pH 7.4, indicating that thisregion of the peptide does not experience significantconformational change upon protonation of the C-terminal and E12 side-chain carboxylate groups. Aweak negative ellipticity is observed below 198 nm atboth pH values, indicating the presence of randomcoil conformation.21,37,42 The presence of the aro-matic Phe residue in the sequence introduces a degreeof uncertainty into the interpretation of the CD spec-trum,19,37,39,40,42 and thus an unambiguous determi-nation of secondary structure classification is not pos-sible from the CD data. However, it is clear from theCD spectra the peptide does exhibit evidence of non-random coil conformation at both pH 2 and 7.4.

NMR Spectroscopy

Tables I and II list the amino acid spin assignmentsfor the 12-mer Lustrin A model peptide at 278 K in90% water/10% deuterium oxide, pH 7.4, and pH 2.0,respectively. The pH-dependent deviation in protonchemical shifts are within �0.1 ppm, with the follow-ing exceptions: F1 CH� and NH�; E12 �-CH2, CH�,and NH�; K4 �-CH2; G3 NH�; and S10 NH� reso-nances (Tables I and II). This is consistent with theCD data (Figure 1), where we note small changes inellipticity values for pH 2 vs pH 7.4. We also noteimproved resolution of diastereotopic side-chain pro-tons at pH 2.0 as compared to pH 7.4, particularlyfor P2.

Of particular interest is the potential for intrapep-tide Lys–Glu side-chain salt bridging interaction atpH 7.4. This type of interaction is usually observedvia chemical shift perturbation in the �-CH and side-chain methylene resonances of the ionizing residuesas a function of pH below and above the sidechainpKas.28 Other NMR parameters, such as residue-spe-cific 3JNH-CH�-couplings, would also be affected bypH change.28 In the 12-mer, E12 would be expected

FIGURE 1 Far-uv CD spectra of Lustrin A 12-mer pep-tide, 5°C. Sample pH is indicated in the Figure.

Model Peptide Studies. I 361

to have a pKa for the -COOH around pH 3.0–4.0.28

Hence, if a salt bridging interaction exists betweenE12 and K4, we would expect to see appropriate

chemical shift perturbations as the pH is varied from2.0 to 7.4. As shown in Tables I (pH 7.4) and II (pH2.0), we observe changes in the E12 �-CH2 diaste-

Table I Proton Chemical Shift Assignments for Lustrin A 12-mer, pH 7.4, in 90% water/10% D2O, 278 Ka

Residue�-

NH�-CH �-CH �-CH

-CH �-CH NH2 Ring H

Phe1 8.23 4.62 3.32; 3.11 — — — — 2,6H: 7.293,5H: 7.45

4H: 7.35Pro2 N/A 4.52 2.33 2.03; 2.00 3.42 — — —Gly3 8.29 3.93 — — — — — —Lys4 8.22 4.35 1.83 1.73; 1.71 1.44 3.10 — —

(pH 7.4: 2.99)(pH 12.0: 2.78)

Asn8 8.57 4.74 2.89; 2.79 — — — 7.72; 7.05 —Val6 8.16 4.14 2.14 0.94 — — — —Asn7 8.47 4.77 2.89; 2.79 — — — 7.72; 7.04Cys8 8.23 4.80 3.22; 3.09 — — — — —Thr9 8.37 4.47 4.33 1.22 — — — —Ser10 8.14 4.52 3.78 — — — — —Gly11 8.39 3.96 — — — — — —Glu12 8.04 4.17 1.89 2.21; 2.10 — — — —

(pH 7.4: 2.28, 2.10)(pH 12.0: 2.28, 2.10)

a The proton assignments were obtained from our analyses of the PFG-“clean” TOCSY, PFG DQF-COSY and PFG NOESY/ROESYexperiments. Diastereotopic protons are separated by a semicolon. Proton chemical shifts are referenced from internal TSP. For K4 and E12,chemical shift values obtained for �-CH and �-CH protons in the N�-acetyl, C�-amide capped 12-mer (pH 7.4, 12.0) are given in parentheses.

Table II Proton Chemical Shift Assignments for Lustrin A 12-mer, pH 2.0, in 90% Water/10% D2O, 278 Ka

Residue�-

NH�-CH �-CH �-CH -CH �-CH NH2 Ring H

Phe1 8.15 4.59 3.35; 3.11 — — — — 2,6H: 7.293,5H: 7.44

4H: 7.38Pro2 N/A 4.52 2.34; 1.96 2.02; 1.94 3.79; 3.43 — — —Gly3 8.50 3.88 — — — — — —Lys4 8.26 4.35 1.82; 1.74 1.68; 1.66 1.43 2.99 — —

(2.99)Asn8 8.72 4.76 2.87; 2.76 — — — 7.71; 7.04 —Val6 8.37 4.13 2.12 0.93 — — — —Asn7 8.65 4.78 2.87; 2.76 — — — 7.71; 7.04 —Cys8 8.45 4.80 3.25; 3.03 — — — — —Thr9 8.49 4.44 4.29 1.22 — — — —Ser10 8.42 4.56 4.01; 3.91 — — — — —Gly11 8.55 3.95 — — — — — —Glu12 8.37 4.47 2.00 2.47; 2.24 — — — —

(2.49; 2.17)

a The proton assignments were obtained from our analyses of the PFG-“clean” TOCSY, PFG DQF-COSY and PFG NOESY/ROESYexperiments. Diastereotopic protons are separated by a semicolon. Proton chemical shifts are referenced from internal TSP. For K4 and E12,chemical shift values obtained for �-CH and �-CH protons in the N�-acetyl, C�-amide capped 12-mer (at pH 2.0) are given in parentheses.

362 Zhang et al.

reotopic resonances (�0.26 and �0.14 ppm shift) andthe �-CH resonance (�0.3 ppm shift). Simulta-neously, the K4 �-CH2 experiences a small perturba-tion (�0.11 ppm shift), but the K4 �-CH resonancedoes not exhibit any pH-dependent shift (Tables I andII). To determine if these methylene proton shift ef-fects are due to intrapeptide K4–E12 interactions, weperformed parallel NMR experiments on a N�-acetyl,C�-amide “capped” 12-mer, at pH 2, 7.4, and 12.0,and measured the 1H chemical shifts for E12 �-CH2

and K4 �-CH2 protons. As shown in Table I, we donot observe any perturbation in the K4 �-CH2 reso-nances within the pH range of 2.0–7.4 (i.e., E12 �-and -COOH deprotonation), nor do we observe anyperturbation for E12 �-CH2 within the pH range of7.4–12.0, where we would expect the deprotonationof the K4 �NH3. Based upon these observations, weconclude the following: (1) E12 in the unprotectedpeptide is affected by pH change, most likely due toelectrostatic interactions between E12 �-COO— and-COO— groups, or, between the �-COO— and�-NH3 termini. (2) K4 appears to be unaffected by theprotonation state of E12; (3) from (1) and (2), itfollows that there are no significant intrapeptide K4–E12 side-chain interactions within the 12-mer se-quence. This finding is further confirmed by the ab-sence of a significant pH-dependent effect on K4�H� and �J values (see below).

CH� Conformational Shifts. Proton CH� chemicalshifts are sensitive to peptide backbone conformation,and the deviation of these chemical shifts from “ran-dom coil” values (referred to as CH� conformationalshifts or �H�, where �H� � H�,observed �H�,“random coil”)

43,44 have been used to qualitativelydeduce secondary structure in peptides and pro-teins.11,12,21,22,43,44 It is generally observed that �-turnand �-helical residues are upfield shifted relative tothe random coil state (��H�); �-strand residues aredownfield shifted (��H�).11,12,21,22,43,44 Using theWishart et al. CH� chemical shift index (database of70 proteins)43 and implementing corrections for Proand the Phe residue preceding Pro in the sequence,44

we calculated the �H� values for the 12-mer at pH7.4 and 2.0 (Figure 2). At pH 7.4, we find that the�H� values for F1, G3, and G11 are (�) and �0.1ppm. Most importantly, �H� is (�) and �0.1 ppmfor K4, N5, N7, C8, T9, and S10. It should be notedthat the presence of three or more sequential 1H NMRCH� values that are perturbed in the same directionare usually indicative of a nonrandom coil conforma-tion.43,44 In general, similar �H� findings were alsonoted at pH 2.0 (Figure 2), with the following excep-tions: (1) For T9, �H� 0.1 ppm; (2) for E12,

�H� is (�) and �0.1 ppm. Interestingly, K4 �H�

values do not exhibit significant variation as a func-tion of pH, again underscoring the absence of saltbridge interaction between E12 and K4. However, thepH-dependent effect observed for the E12 �-CH pro-ton, reflecting either protonation or other electrostaticinteractions at the �-COO— group. Thus, we con-clude that at pH 7.4, the 12-mer �H values suggestthe presence of a structural motif that incorporatesthe —NCTS— sequence region.

JNH-CH�-Coupling Constants. Coupling constantshave been widely utilized for the estimation of localconformational preferences in peptides. The 3JNH-

CH�-coupling constants are linear averages over allconformers; they are independent of the particularchemical environment and measure only the � anglepopulation at each position in the main chain.11,12,21,45

The 3JNH-CH�-couplings for the 12-mer are presentedin Figure 3. The deviation of the 12-mer 3JNH-CH�-coupling constants from random coil values (�J)were also calculated (Figure 2). In general, 3JNH-CH�

couplings larger than random coil values (��J) areindicative of �-strand conformation, whereas 3JNH-

CH� couplings smaller than random coil values (��J)represent �-helix conformation.11,12,21,45 The �-turnvalues can be either ��J or ��J.21,32,45 For the12-mer at pH 7.4, the most significant finding is thepresence of �1 Hz (�) �J for F1, G3, K4, N5, V6,N7, C8, T9, S10, and G11. Again, as observed for the�H� values, the contiguous —NCTS— region exhib-its significant directional deviations in J-couplingsfrom random coil values.45 At pH 2, similar trends arenoted, with the following exceptions: for V6, S10,E12, �J is (�) and 1 Hz; for N7, �J is (�) and 1Hz. We do not observe significant pH variation (i.e.,�1 Hz) in either the K4 or E12 �J value, whichcorrelates with the �H� measurements (Figure 2)and confirms the absence of salt-bridge interactionbetween E12 and K4. From these findings, we con-clude that the �J values obtained for the Lustrin A12-mer at pH 7.4 suggest the presence of a structuralmotif that incorporates the —NCTS— sequence re-gion.

Sequential ROE Connectivities. We now focus ourattention to determining the conformational prefer-ences of the Lustrin A 12-mer at pH 7.4. UsingPFG-NOESY and ROESY experiments, sequentialdNN(i,i�1) NOE magnetization transfer is observed forthe following residues: K43 N5, V63 N7, and G113 E12 (Figures 3 and 4). It has been established thatthe d�N(i,i�1)/dNN(i,i�1) intensity ratio can be used toevaluate the presence of random coil or structured

Model Peptide Studies. I 363

domains,11,12,21,46 since the NOE intensity ratios de-pend on the dihedral angles � and �. Typically,d�N(i,i�1)/dNN(i,i�1) intensity ratios �1.4 are indica-tive of random coil regions.21,46 As shown in Figure3, the d�N(i,i�1)/dNN(i,i�1) intensity ratios for K4 3N5, V63 N7, and G11–E12 exceed the random coilthreshold ratio by at least a factor of two or greater.Thus, the existence of nonrandom coil d�N(i,i�1)/dN-

N(i,i�1) NOE ratios and the presence of significant �Jand �H� values (Figure 2) indicate that the LustrinA peptide conformation is not random and unordered.

We should note that sequential dNN(i,i�1) NOEmagnetization transfer is not consistently observedthroughout the 12-mer; in particular, G3, T9, S10, andG11 do not exhibit sequential dNN(i,i�1) NOEs withother residues (Figure 4). This could be interpreted asbeing representative of a random coil state in theseregions. However, in unordered structures, we wouldexpect that a conformer distribution exists for eachresidue,47 and if the interconversion between con-formers is fast on the NMR time scale, then confor-mational averaging will occur but not the complete

cancellation of NOEs.48,49 In fact, the random coilmodel predicts that all sequential dNN(i,i�1) NOEs aswell as a number of medium-range NOEs should beobservable.46 Thus, a more accurate interpretationregarding the absence of sequential dNN(i,i�1) NOEsin the Lustrin A 12-mer would be the presence ofextended conformations (e.g., �-strand, polyprolineII) that possess backbone interproton distances �5.5Å. We note that similar extended structures exist inPro,Glu,Val,Lys-containing extended-turn consensusrepeats derived from the elastomeric protein, titin.50

Thus, the absence of sequential dNN(i,i�1) NOEs in theLustrin A 12-mer indicates the presence of extendedregions involving P2–G3, and T9–S10–G11.

Long-Range NOE Connectivities. As reported inprevious studies, the presence of dNN(i,i�2) NOE re-laxation transfer and sequential dNN(i,i�1) NOEs arecharacteristic of a turn conformation.11,12,14–22 Thus,to further define the conformational preference of the—KNVNC— region, we analyzed the PFG-NOESYand ROESY NH—NH fingerprint region for evidence

FIGURE 2 Top: Calculated difference (�J) between experimental and random coil 3JNH-CH�

values for the Lustrin A 12-mer model peptide in 90% water, 10% D2O at 278 K, pH 7.4 and pH2.0. Bottom: �H� values for the Lustrin A 12-mer model peptide in 90% water, 10% D2O, pH 7.4and pH 2.0, 278 K. Negative values represent upfield shifts, positive values represent downfieldshifts. No corrections for terminal residues effects have been made.44 Corrected CH� chemical shiftvalues were utilized for all Pro and Xaa–Pro nearest neighbors (where Xaa � Phe).44 Protonchemical shifts were referenced from internal d4-TSP.

364 Zhang et al.

of long-range NOE cross-relaxation transfer. Medi-um- and long-range d���(i, j), dsc-sc(i, j) NOE crosspeaks were not observed in our ROESY or NOESYspectra. However, we do observe long-range dNN(i,i�2)

NOE cross-relaxation transfer for N7 3 T9 (Figure4). This suggests that the N7 NH group is positioned5 Å from the NH group of T9, which can only occurif the polypeptide backbone possesses a turn or bendin this region of the sequence. Not surprisingly,Asn17,20,21 residues are often located within turn se-quences.

Combining these observations with the sequentialdNN(i,i�1) NOE transfer data, we conclude that theLustrin A 12-mer possesses turn-like or bend regionsthat involve K4, N5, V6, N7, and T9, the conservedresidues of the C-domain consensus repeat. ResiduesF1, P2, G3, S10, and G11 appear to adopt an ex-tended, nonrandom coil conformation as evidenced bythe absence of sequential dNN(i,i�1) NOE transfer andthe presence of nonrandom coil �J and �H� values(Figure 2).

Amide Temperature Coefficients and Solvent Ex-change. It has been shown that “random coil” orextended peptides exhibit amide temperature shift co-efficients in the range of �6.6 to �9.0 ppb/K inaqueous solution.35 Values of �4.5 to �5.3 ppb/Kwere found at NH hydrogen-bonding sites in tetramer

and pentamer �-turn peptides11,16,51,52 and �-turn mi-metics.20 For the 12-mer at pH 7.4, we found that thetemperature coefficients varied from �8.7 to �5.9ppb/K (Figure 3), with G3 and C8 possessing thesmallest amide temperature shift coefficients. To con-firm these findings, we estimated the presence orabsence of solvent exchange between H2O and thebackbone amide protons in the FPGKNVNCTSGEsequence, using PFG-NOESY experiments (150 msmixing time).11,12,32 The exchange regime that we canmeasure is on the NMR timescale (milliseconds toseconds); any exchange processes that occur on aslower timescale will not be observed. As shown inFigure 5, we observe solvent exchange for all �-NHprotons, indicating that there is proton exchange oc-curring at these sites. Hence, we conclude that there isno evidence for intrastrand hydrogen bonding withinthe 12-mer peptide. As observed in our earlier modelpeptide studies,12 it is likely that the small amidetemperature shift coefficients observed for G3 and C8arise from other shielding effects (e.g., folding), andare not due to intrastrand hydrogen bonding.

Model Building

Due to the presence of extended conformations (i.e.,NH—NH interproton distances �5.5 Å) for F1, P2,G3, S10, and G11, only 41 NMR restraints are avail-

FIGURE 3 Summary of NMR parameters for Lustrin A 12-mer, FPGKNVNCSTGE, in 90%water, 10% D2O at 278 at pH 7.4. The summary includes interresidue sequential [�N(i, i � 1),NN(i, i � 1)] and long-range NOEs [NN(i, i � 2)]; intraresidue NOEs [�N(i, i)]; d�N(i,i�1)/dNN(i,i�1) ratios (�N/NN); 3J-couplings (Hz); and amide temperature shift coefficients (ATC, inppb/K). The relative NOE intensities are reflected by the height of the lines. N/A � not available.

Model Peptide Studies. I 365

able for structure refinement. As shown in Pro-richpeptide models derived from the titin PEVK domain,when a limited number of intraresidue and sequentialinterresidue NOEs and 3J-coupling constraints areavailable, the overall convergence of the calculated structures is low, and the backbone superposition and

root mean square deviation are high.50 Hence, thesimulation results which are described below shouldbe considered as semiquantitative, and subject to re-interpretation pending further studies.

The two lowest energy simulated annealing molec-ular dynamics/minimized structures for the deproto-nated carboxylate state (pH 7.4, backbone RMSD� 4.31 Å) are presented in Figure 6. At pH 7.4, the12-mer adopts a loop structure, wherein the N- andC-termini are observed to be in close proximity (5Å). Given that we have represented the termini ascharged moieties (see Materials and Methods), thepairwise interactions between the complementarycharged species is not unexpected, and would contrib-

FIGURE 4 NH� fingerprint regions of 1H-1H z-PFG-ROESY spectrum of Lustrin A 12-mer model peptide. Thecarrier is centered on the water resonance. Acquisition pa-rameters included a relaxation delay of 1 s, spectral window� 5200 Hz, “hard” 90° � 10 �s, “hard” 180° � 20 �s;“soft” selective 180° � 3.5 ms; 256 transients per experi-ment were acquired; 2048 and 256 complex points werecollected in t1 and t2, respectively. Mixing time: 150 ms.Z-gradient parameters: G1 � 5.6 G/cm, G2 � 2.8 G/cm,each with a duration of 1 ms with 500 �s stabilization time.The hypercomplex phase-sensitive method was utilized forprocessing both 2D spectra, with zero-filling in the F2dimension. Proton chemical shifts are referenced from in-ternal d4-TSP. Note that identical results were obtainedusing PFG-NOESY experiments.

FIGURE 5 NH–water solvent exchange PFG-T-ROESYspectra for Lustrin A 12 mer at pH 7.4. Spectra obtainedusing acquisition and processing parameters presented inFigure 4.

FIGURE 6 Lowest energy structures (top) and corre-sponding Ramachandran plot (bottom) obtained from CVFFsimulated annealing molecular dynamics/minimization ofLustrin A 12-mer, pH 7.4. The backbone traces representthe nonhydrogen atom positions for each conformer; fittedto the mean structure. Sequence positions are labeled. Forthe Ramachandran plots, the �,� values obtained for eachconformer pair are shown superimposed over �,� regions.The �,� values for F1, P2, and E12 are not displayed on thisplot. Backbone structures, fitting, and �,� plots were gen-erated using the MOLMOL program.53

366 Zhang et al.

ute to the overall stability of the conformers. How-ever, due to the sequence separation between the K4and E12 residues, the lowest energy conformers donot possess pairwise interactions between thesidechains of K4 or E12, consistent with our NMRobservations (Tables I and II). The loop structureappears to consist of a “bend” or “turn” at G3–K4 andN7–C8–T9 (labeled on the structures). These bend orturn regions are interspersed with extended conforma-tions arising at F1–G3; K4–V6–N7; T9–S10–G11 inthe sequence (labeled on the structures). Thus the loopmotif is formed by an alternating “extended-turn-extended-turn-extended” repeat structure.

We note that the �,� distribution more closelyresembles that obtained for extended structures asopposed to true random coil,50 i.e., the �,� torsionsfor random coil conformers are normally broadly dis-tributed, whereas the distribution for extended peptidestructures (e.g., �-strand, polyproline type II) fallwithin the range of � � �45° to �180°, � � 90°–180°.50 Using the classifications provided in the liter-ature,11,12,53 the �,� dihedral distribution for bothlowest energy conformers is approximately 41%�-strand or extended, 22% �-turn, 37% unclassified(Figure 6). In both conformers, note that G3 possesses�,� values that are located in the IV quadrant of theRamachandran map, and N7 possesses �,� values thatare close to the �-turn region (Figure 6). Thus, G3 andN7 are key residues in the “bend” or “turn” regions ofthis peptide.

Overall, we find that these refined structures are inagreement with our NMR observations, viz: (i) N7exhibits long-range dNN(i,i�2) NOEs to T9, indicatingthat these residue pairs must be positioned close to-gether to permit interresidue NH—NH dipolar mag-netization transfer. Coupling this observation to thepresence of sequential dNN(i,i�1) NOEs for K43 N5,V6 3 N7 (Figures 3 and 4) and N5 3 V6, it isevident that the —NVN— sequence adopts a turn-likeor bend structure where the interproton distances be-tween adjacent NH—NH protons are 5 Å (Figure6). (ii) The FP— and —SGE sequence regions pos-sess �J and �H� values that are uncharacteristic ofa random coil conformation, and do not exhibitdNN(i,i�1) NOEs, indicating the presence of an ex-tended conformation (NH—NH interproton distance�5.5 Å). (iii) There is evidence that the —PG—sequence within the 12-mer is another likely site for aturn or “bend,” as evidenced by the presence ofsequential dNN(i,i�1) NOEs for G3–K4 (Figures 3and 4). Earlier studies have demonstrated that Proresidues induce kinks or bends within a sequencestretch.12,54–56 (iv) The presence of turn or extendedconformations is also consistent with the CD spectra

for the Lustrin A 12-mer (Figure 1), which is incon-sistent with a random coil conformation. (v) The pHdependence of the Lustrin A 12-mer conformation issmall (Figures 1 and 2) and does not appear to involvethe participation of K4 and E12 side chains in salt-bridging interactions (Figure 2, Tables I and II).

DISCUSSION

The C-domains of Lustrin A are organized in analternating fashion with the Pro-rich domains, in amanner similar to the domain organization found inthe diatom cell wall-specific glycoprotein family, thefrustulins.1 With their high degree of sequence simi-larity, C-domains have been postulated to fold intoCys–Cys linked structures which do not contain sig-nificant percentages of �-helix or �-sheet.2 We nowhave evidence that this assessment is true, at least forthe N-terminal region of the canonical C-domain.Using NMR spectroscopy and a model peptide, wehave identified a turn-like conformation within thehighly conserved —NVNCT— sequence block of theC2-domain sequence. There is also evidence for a“kink” or “turn” structure involving the conserved—PG— sequence. Globally, the 12-mer sequence atpH 7.4 is observed to adopt a “loop” structure thatconsists of the two turn-like sequence regions inter-spersed with extended domains (Figure 6). Obviously,further structural studies on larger C-domain modelpeptide sequences will be required to evaluate theconformational preferences throughout the C-do-mains.

The modular structure and use of repetitive se-quence motifs within the Lustrin A protein1 are rem-iniscent of similar motif organizations found withinother elastic proteins.3–5,6,9–11 Moreover, several elas-tic proteins possess turn regions that are believed toparticipate in the force extension and hysteretic re-covery processes.3–5,6,9,10 Thus, the presence of a—PG—, —NVNCT— loop conformation within theN-terminal region of domains C1–C8 may have im-portant structural and mechanistic implications for theLustrin A protein, viz: (1) Protein loops are believedto behave as entropic springs,57 and are capable ofreversible motion or flexibility within proteins.58,59

When the protein experiences force extension, non-bonding interactions within loop or turn regions couldbe disrupted, and possibly reformed once the exten-sion force is removed. With regard to Lustrin A, thisreversible unfolding and refolding of loop regions,such as the —PG—, —NVNCT— and the putativeelastic (GS)x and (GSSS)y “serine loop” domains,1,7

could possibly preserve the integrity of the nacre layer

Model Peptide Studies. I 367

and protect the mineral phase from fracture or sepa-ration.2 (2) Another consideration is the orientation ofthe P- and C-domains relative to one another withinthe block copolymer structure of Lustrin A.1 TheP-domains are Pro rich, and they are assumed to existin an extended or unfolded state.1 Thus, it is plausiblethat the P–C-domain junction for domains C1–C8may contain a “kink” or “turn” at the —NVNCT—region, which might lead to a repeated, specific align-ment of the preceding P-domain relative to the corre-sponding Cys-rich C-domain that follows it. This ar-rangement may allow P- and C-domains to “unfold”relative to one another about the turn region duringforce extension, which, in part, might explain theforce peak curves seen in the nacre layer AFM exper-iment.2 Obviously, these are hypothetical scenariosthat will require further experimentation and simula-tion in order to verify or refute them.

Support for this study has been made possible by grantsfrom the National Science Foundation (DMR 99-01356;MCB 98-16703 to NYU) and the Army Research Office(Young Investigator Award DAA19-99-1-0225 to JSE;MURI DAAH04-96-1-0443 to UCSB). This paper repre-sents contribution number 16 from the Laboratory forChemical Physics, New York University.

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