Structural Analyses of Polyelectrolyte Sequence Domainswithin the Adhesive Elastomeric Biomineralization

Protein Lustrin A

Brandon A. Wustman,† Daniel E. Morse,‡ and John Spencer Evans*,†

Laboratory for Chemical Physics, New York University, 345 E. 24th Street,New York, New York, 10010, and the Department of Molecular, Cellular, and

Developmental Biology and Materials Research Laboratory, University of California,Santa Barbara, California 93106

Received May 9, 2002. In Final Form: August 16, 2002

The lustrin superfamily represents a unique group of biomineralization proteins localized betweenlayered aragonite mineral plates (i.e., nacre layers) in mollusk shell. These proteins exhibit elastomericand adhesive behavior within the mineralized matrix. One member of the lustrin superfamily, LustrinA, has been sequenced; the protein is organized into defined, modular sequence domains that are hypothesizedto perform separate functions (e.g., force unfolding, mineral interaction, intermolecular binding) withinthe Lustrin A protein. Using NMR, CD spectrometry, and model peptides, we investigated the solutionstructure of two Lustrin A polyelectrolyte modular domains that represent putative sites for LustrinA-nacre component interaction: the 30-AA Arg, Lys, Tyr, Ser-rich (RKSY), and 24-AA Asp-rich (D4)domains. The results indicate that both sequences adopt open unfolded structures, with RKSY exhibitingstructural features of an extended conformer, and D4, a more labile, random-coil conformation. Theseresults suggest that the Lustrin A protein possesses open, unfolded regions that could act as putative sitesfor Lustrin A-mineral or Lustrin A-macromolecular interactions that lead to the observed adhesiveproperties of the nacre organic matrix.

Introduction

Fracture toughness in biocomposite materials such asthe abalone shell arises from the presence of proteins inclose approximation with the aragonite mineral phase.1Members of one superfamily, the lustrins, are localizedwithin the interstitial organic layer of the nacre of the redabalone, Haliotis rufescens.1a This interstitial organic layerexhibits elastic behavior (i.e., reversible extension andrecovery).1b In addition, this same layer possesses adhesiveinteractions with the underlying aragonite mineraltablets.1b It has been hypothesized that the observedadhesive interactions may arise from a number of inter-actions involving the lustrin proteins and their environ-ment (e.g., protein-mineral and/or protein-macromo-lecular). If true, then the lustrin proteins would most likelypossess one or more domains that would foster interactionswith these components, thereby promoting adhesion andallowing the lustrin proteins to “anchor” and resist removalfrom the nacre layer.1b This, in turn, may help to explainhow proteins convey fracture resistance to mineralizedbiocomposites and could serve as an interesting molecularparadigm for developing fracture-resistant materials.

As a starting point in identifying putative adhesivedomains within lustrin proteins, our initial focus is ondefined sequence regions that are polyelectrolyte in nature(i.e., a significant presence of cationic or anionic aminoacid residues within a given sequence block). The sig-nificance of polyelectrolyte sequences within lustrin

proteins is two-fold. First, the molecular interaction of“anionic”polyelectrolytesequenceswithmineral interfaceshas been documented,2,3 and thus polyelectrolyte domain-(s) could represent hypothetical site(s) for lustrin-mineraladhesion. Second, given the presence of “basic”4-6 andanionic2,3,7-10 polyelectrolyte domains in a number ofbiomineralization proteins, it is plausible that lustrinpolyelectrolyte domains could participate in complemen-tary protein-protein electrostatic interactions within thematrix. In either instance, Lustrin A polyelectrolytedomain interactions could, in principle, provide a plausiblemechanism for lustrin-matrix adhesion. Additionally, thefact that polyelectrolyte biomineralization polypeptidedomains adopt open, unfolded structures in solution11,12

would presumably facilitate side-chain access to comple-mentary mineral or polypeptide surfaces within thematrix.

Interestingly, one particular lustrin protein, Lustrin A(pacific red abalone H. rufescens, 116 kDa),1a possessestwo modular, polyelectrolyte domains. The first is a 24-AA Asp-containing domain, GKGASYDTDADSGSDNR-

* To whom correspondence should be addressed. E-mail:jse@dave-edmunds.dental.nyu.edu.

† New York University.‡ University of California.(1) (a) Shen, X.; Belcher, A. M.; Hansma, P. K.; Stucky, G. D.; Morse,

D. E. J. Biol. Chem. 1997, 272, 32472-32481. (b) Smith, B. L.; Schaffer,T. E.; Viani, M.; Thompson, J. B.; Frederick, N. A.; Kindt, J.; Belcher,A.; Stucky, G. D.; Morse, D. E.; Hansma, P. K. Nature (London) 1999,399, 761-763.

(2) Lowenstam, H. A.; Weiner, S. In On Biomineralization; OxfordUniversity Press: New York, 1989; pp 1-30.

(3) Gerbaud, V.; Pignol, D.; Loret, E.; Bertrand, J. A.; Berlandi, Y.;Fontecilla-Camps, J. C.; Canselier, J. P.; Gabas, N.; Verdier, J.-M J.Biol. Chem. 2001, 275, 1057-1064.

(4) Harkey, M. A.; Klueg, K.; Sheppard, P.; Raff, R. A. Dev. Biol.1995, 168, 549-566.

(5) Killian, C. E.; Wilt, F. H. J. Biol. Chem. 1996, 271, 9150-9159.(6) Wilt, F. H. J. Struct. Biol. 1999, 126, 216-226.(7) Sarashina, I.; Endo, K. Am. Mineral. 1998, 83, 1510-1515.(8) Samata, T.; Hayashi, N.; Kono, M.; Hasegawa, K.; Horita, C.;

Akera, S. FEBS Lett. 1999, 462, 225-229.(9) Kono, M.; Hayashi, N.; Samata, T. Biochem. Biophys. Res.

Commun. 2000, 269, 213-218.(10) Bedouet, L.; Schuller, M. J.; Marin, F.; Milet, C.; Lopez, E.;

Giraud, M. Comp. Biochem. Physiol., B 2001, 128, 389-400.(11) Evans, J. S.; Chan, S. I. Biopolymers 1994, 34, 534-541.(12) Evans, J. S.; Chiu, T.; Chan, S. I. Biopolymers 1994, 34, 534-

541.

9901Langmuir 2002, 18, 9901-9906

10.1021/la025927m CCC: $22.00 © 2002 American Chemical SocietyPublished on Web 10/29/2002

SPGYLPQ (residues 1251-1264, designated as “D4”,charged residues underlined), which is localized betweenthe Ser, Gly-rich domain and the C10 domain within theLustrin A sequence. The second is a 30-residue basic Arg-,Lys-, Ser-, Tyr-containing domain, YRGPIARPRSSRY-LAKYLKQGRSGKRLQKP (residues 1354-1383, desig-nated as “RKSY”, charged residues underlined). Whatmakes these sequences even more interesting is thepresence of hydrogen bonding donor/acceptor amino acids(Asn, Gln, Arg, Thr, Ser, Tyr) within each domain. Theseamino acids may represent additional putative sites forLustrin A-mineral or Lustrin A-macromolecule interac-tions via hydrogen-bonding mechanisms. To date, becauseof the difficulty in obtaining purified Lustrin A protein inquantity, no studies have been performed to assess thestructure of either domain.1a

To investigate these domains further, we determinedthe structural preferences for Lustrin A D4 and RKSY insolution using NMR spectroscopy, circular dichroism (CD)spectrometry, and N-acetyl-, C-amide-“capped” modelpeptides representing the D4 and RKSY domains in toto.We find that both polyelectrolyte sequences adopt openconformations in solution, with a subtle difference: RKSYadopts a more extended conformation, whereas D4 adoptsa “random-coil” conformation. These findings are discussedin light of the possible functions for each domain withinLustrin A.

Materials and MethodsPeptide Synthesis, Purification, and Sample Prepara-

tion. Purified, N-acetyl-, C-amide-capped RKSY and D4 polypep-tides were synthesized by Dr. Janet Crawford, Yale UniversityHHMI Biopolymer/Keck Biotechnology Resource Laboratory,using an Applied Biosystems 431A Peptide Synthesizer and NR-L-FMOC amino acids. Typical peptide synthesis runs were carriedout at the 100-µmol level using the Applied Biosystems FastMoc0.25 HBTU/HOBt/NMP protocol [HBTU ) 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOBt )N-hydroxybenzotriazole; NMP ) n-methylpyrrolidone; AppliedBiosystems Technical Notes, November 1993) and MBHA resin(Novabiochem). To avoid possible side-chain modification duringsynthesis, NR-Trityl-Nγ-FMOC-Asn (abbreviated as FMOC-Asn-Trt) was utilized in synthesis runs.13,14 To create a capped peptide,the CR-amide peptide was treated with acetic anhydride in NMP/DIEA to create the NR-acetyl derivative. The completed peptideswere deprotected and cleaved (reaction time ) 3 h at 25 °C) fromthe resin using a cleavage cocktail (15 mL/g resin) containing90% v/v TFA, 2.5% v/v water, 2.5% v/v ethanedithiol, 2.5% v/vphenol, and 2.5% v/v thioanisole;13-16 the thiol scavengers wereutilized to enhance trityl removal. The reaction mixture wasfiltered under reduced pressure. The crude peptides wereseparately dissolved in deionized distilled water, extracted threetimes with diethyl ether, and then concentrated and lyophilized.Peptide purification involved a C18 reverse-phase high-perfor-mance liquid chromatography (HPLC) column using a 0.1% TFA/water mobile phase and eluting with an 80% acetonitrile/0.1%TFA/water linear gradient. Peptide elution was monitored at230nm. IndividualHPLCfractions were analyzedusingacustom-made MALDI/TOF delayed extraction mass spectrometer. Typi-cally, a 100-µmol synthesis of each peptide yielded >90 mg drymass of purified peptide that was >96% in purity and free ofside-chain protection. The experimental molecular masses forthe capped RKSY and D4 polypeptides were determined to be3575.6 and 2499.96 Da, in agreement with the theoreticalmolecular masses of 3572.1 and 2499.55 Da respectively.

For NMR and CD studies, both peptides were dissolved in 1mM Na2HPO4 in deionized distilled water, pH 7.4. NMR samples

contained 10% v/v deuterium oxide (99.9% atom D, CambridgeIsotope Labs) and 10 µM d4-TSP. Final peptide concentrationswere 2 mM for NMR and 2 µM for CD. At the 2 mM concentration,turbidity measurements at 380 nm revealed no evidence ofpeptide aggregation; this also was confirmed by analyses of NMRproton chemical shifts and line widths and the absence of long-range dsc-sc NOE connectivities.13,14

CD Spectrometry. CD spectra were obtained at pH 7.4 usingan AVIV 62DS CD spectrometer running 60DS software version4.1t and quartz cells with a 0.1-cm path length. The sampleswere scanned from 190 to 260 nm at 5 °C using a 1-nm bandwidthand a scan rate of 1 nm/s. The spectrometer was previouslycalibrated with d-(+)-10-camphorsulfphonic acid. A total of eightscans were acquired. Mean residue ellipticity [θM] is expressedin units of deg cm2 dmol-1 per mol of peptide.13,14 Thermal titrationof the RKSY polypeptide was performed over the temperaturerange of 2 to 80 °C with monitoring of the 220-nm ellipticityintensity as a function of temperature.17

NMR Spectroscopy. NMR experiments were performed ona Varian UNITY 500 spectrometer equipped with a variabletemperature controller and a three-channel (13C/15N/1H) z-axisPFG 5-mm solution probehead. The probehead sample temper-ature was maintained using an ethylene glycol cooling apparatuswith filtered airflow. The temperature was maintained within( 0.1 °C. With the exception of the proton amide temperatureshift experiments, all reported NMR experiments were conductedat 278 K in order to slow the conformational exchange within thepeptide. Proton scalar coupling assignments were obtainedusing “excitation sculpting” 2-D PFG- “clean” TOCSY experi-ments.13,14,18,19 Proton sequential assignments and NOEs wereobtained using z-PFG-ROESY experiments13,14,20,21 using a rangeof mixing times from 50 to 200 ms; spectra were jointly analyzedto exclude artifactual NOEs arising from spin diffusion. 3JNH-CHR

values were determined using z-PFG DQF-COSY13,14 and P. E.COSY13,14,22,23 experiments. No observable cross-relaxationcrosstalk artifacts were detected in the z-PFG P. E. COSYJ-coupling spectra; in addition, we found that the cross-peakline widths in the P. E. COSY spectra were not significantlyaffected by linebroadening via comparisons of spectra obtainedat 5 and 20 °C (data not shown). NMR data were processed usingFELIX95 software (MSI/Biosym Technologies, Inc). RelevantNMR acquisition and processing parameters are provided in thefigure legends Using PFG TOCSY or NOESY experiments at278, 283, 288, 293, and 298 K, amide proton temperaturecoefficients were determined from the slope of the temperatureversus amide proton chemical shift curves for each residue.24

Temperature gradients are expressed in units of ppb/K with anegative sign indicating an upfield shift upon warming.24

Temperature calibration of the VT unit was determined prior toexperimentation using neat methanol over a temperature rangeof 273 to 320 K.

Results

CD Spectrometry. We first examine the conforma-tional states of RKSY and D4 to determine qualitativelyif either polypeptide sequence exhibits specific secondarystructure preferences. As shown in Figure 1, at pH 7.4,both polypeptides exhibit strong, broad negative bands(π-π* transition) that are centered between 195 and 200nm, consistent with the random-coil state.14,17,25 Unusu-

(13) Xu, G.; Evans, J. S. Biopolymers 1999, 49, 303-312.(14) Zhang, B.; Wustman, B.; Morse, D. E.; Evans, J. S. Biopolymers

2002, 63, 358-369.(15) Sieber, P.; Rinker, B. Tetrahedron Lett. 1991, 32, 739-745.(16) King, D. S.; Fields, C. G.; Fields, G. B. Int. J. Pept. Protein Res.

1990, 36, 255-266.

(17) Ma, K.; Kan, L. S.; Wang, S. Biochemistry 2001, 40, 3427-3438.(18) Hwang, T. L.; Shaka, A. J. J. Magn. Reson., Ser. A 1995, 112,

275-279.(19) Xu, G.; Evans, J. S. J. Magn. Reson., Ser. B 1996, 111, 183-185.(20) Callahan, D.; West, J.; Kumar, S.; Schweitzer, B. L.; Logan, T.

M. J. Magn. Reson., Ser. B 1996, 112, 82-85.(21) Stott, K.; Stonehouse, J.; Keeler, J.; Hwang, T.-L.; Shaka, A. J.

J. Am. Chem. Soc. 1995, 117, 4199-4200.(22) Muller, L. J. Magn. Reson. 1987, 72, 191-186.(23) Xu, G.; Zhang, B.; Evans, J. S. J. Magn. Reson. 1999, 138, 127-

134.(24) Andersen, N. H.; Neidigh, J. W.; Harris, S. M.; Lee, G. M.; Liu,

Z.; Tong, H. J. Am. Chem. Soc. 1997, 119, 8547-8561.(25) (a) Marcus, P. A.; Edgerton, E. M. Biochemistry 1996, 35, 4314-

4325. (b) Butcher, D. J.; Nedved, M. L.; Neiss, T. G.; Moe, G. R.Biochemistry 1996, 35, 698-703.

9902 Langmuir, Vol. 18, No. 25, 2002 Wustman et al.

ally, the RKSY polypeptide also exhibits a minor positiveadsorption band (n-π* transition) observed near 220 nm,which is also consistent with the presence of the polypro-line Type II structure.17,25 However, monitoring the 220-nm band over the temperature range of 2-80 °C (i.e.,thermal titration) did not reveal a pronounced biphasic220-nm transition near 50 °C that is characteristic ofpolyproline Type II (Figure 2).17,25 Thus, the CD data forboth polypeptides are consistent with the presence ofunfolded, open structures in both sequences. This isconsistent with the polyelectrolyte nature of both se-quences, wherein charge repulsion between similarlycharged side chains (i.e., Asp-Asp in D4; Arg-Arg, Lys-Lys, and Lys-Arg in RKSY) would lead to unfolded, openconformations.11,12

NMR Spectroscopy. The findings obtained from CDspectrometry are supported by NMR experiments (PFGclean TOCSY, ROESY, and DQF-COSY) conducted on bothpolypeptides at pH 7.4, 1 mM Na2HPO4. 1H chemical shifts,intraresidue and interresidue NOEs, and 3JNH-CHR cou-pling constants were compiled for both polypeptides(Tables 1 and 2; Figures 3-5). Proton conformational shifts(∆δHR), which can be utilized to determine the presenceor absence of the random-coil conformation within pro-teins,13,14,17,26 were calculated for both peptides (Figure6). As shown in Figure 6, residues G3, A4, Y6, and G13possess ∆δHR values >0.1 ppm, indicating significantdeviation from random-coil database values;26 however,the remaining residues in D4 possess ∆δHR values thatdo not exceed the random-coil threshold of 0.1 ppm,suggesting that the majority of the D4 sequence isconformationally similar to a random-coil state.13,14,17,26 Asimilar trend is noted for RKSY (i.e., residues G3, A6,

R12, Y17, and G24 possess ∆δHRa values >0.1 ppm, withthe remaining residues having ∆δHRa values <0.1 ppm).

The ∆J, or deviation of observed 3JNH-CHR from random-coil values,13,14,17,27 was also calculated for both polypep-tides (Figure7). Ingeneral, ∆Jvalues >1Hzareconsideredto be significant; positive ∆J values (+∆J) are indicativeof the â-strand conformation, and negative ∆J (-∆J)values are representative of the R-helix conformation. ∆Jvalues for the â-turn can be either (+) or (-).27 For D4,we note that G3, A4, D7, D11, S12, G13, S14, N16, andR17 possess (-) ∆J values >1 Hz. Furthermore, G1, A10,and G20 possess (+) ∆J values >1 Hz. The remainingresidues possess ∆J values <1 Hz. Note that the D4polypeptide does not exhibit consistent (+) or (-) ∆Jdeviations but instead possesses a combination of the two.In contrast, the RKSY polypeptide exhibits (+) ∆J values>1 Hz for R2, I5, A6, R7, R9, S10, Y13, Y17, L18, Q20,R22, S23, Q28, and K29, with the remaining residuespossessing ∆J values <1 Hz. What is interesting is thatall of the calculated RKSY ∆J values are consistently (+),indicating a trend toward the â-strand.27 From the ∆δHRand ∆J data, we conclude the following: (1) The D4polypeptide does not adopt a defined secondary structurein solution, in accord with the CD findings that indicatethe presence of a conformationally labile or random-coilstructure (Figure 1). (2) The RKSY polypeptide appearsto exhibit more deviation from the random-coil structurethan D4 does.

The qualitative NMR findings described above aresupported by quantitative NOE measurements. The mostrevealing finding was the total absence of sequentialdNN(i,i+1) NOEs for both polypeptides (Figures 4 and 5).The absence of interresidue NOEs together with theabsence of backbone hydrogen bonding indicates thatneither polypeptide adopts an R-helix, â-hairpin, or â-turnsecondary structure.14,28 However, the absence of sequen-tial NOEs is associated with extended polypeptide struc-tures (i.e., NH-NH distances >5.5 Å)14,17,28 and/or thepresence of conformational exchange involving random-coil states.14,17,28 The NOE intensity ratio, RN(i, i + 1)/RN(i, i), can be used a guide for discriminating betweenextended and random-coil states;14,17,28 the value of 2.3represents the population-weighted random-coil model,whereas values >4 are predicted for the â-strand.28 Forthose regions where nonoverlapping NOEs can be quan-titated, we find that the RKSY polypeptide possessesresolvable NOE ratio values of 9.9, 5.1, 4.1, 3.3, and 35.6for I5-A6, Y13-L14, L14-A15, K19-Q20, and R22-S23,respectively (Figure 5). Hence, these sequence regions inRKSY are similar to the â-strand and are not random coilin nature. These results support the findings obtainedfrom RKSY ∆J calculations (Figure 7). At this time, it isnot known whether the remaining regions of the RKSYsequence exist in an extended conformation similar to theâ-strand. In comparison, we find that, with the exceptionof Y21-G20 (ratio ) 4.9), the remaining resolvable RN(i,i + 1)/RN(i, i) ratios in D4 are <2.3 (Figure 4), indicatingthat D4 globally exists as a random-coil conformer atneutral pH, with no strong evidence of extended backbonestructure.

One final note should be made regarding the large,negative ATC values (i.e, ATC > -6.0 ppb/K) obtained forpolypeptides (Figures 4 and 5). These values are typical

(26) (a) Wishart, D. S.; Sykes, B. D.; Richards, F. M. J. Mol. Biol.1991, 222, 311-333. (b) Wishart, D. S.; Bigam, C. G.; Yao, J.; Abildgaard,F.; Dyson, H. J.; Oldfield, E.; Markley, J. L.; Sykes, B. D. J. Biomol.NMR 1995, 6, 135-140. (c) Wishart, D. S.; Bigam, C. G.; Holm, A.;Hodges, R. S.; Sykes, B. D. J. Biomol. NMR 1995, 5, 67-81.

(27) (a) Smith, L. J.; Bolin, K. A.; Schwalbe, H.; MacArthur, M. W.;Thornton, J. M.; Dobson, C. M. J. Mol. Biol. 1996, 255, 494-506. (b)Serrano, L. J. Mol. Biol. 1995, 254, 322-333.

(28) (a) Fiebig, K. M.; Schwalbe, H.; Buck, M.; Smith, L. J.; Dobson,C. M. J. Phys. Chem. 1996, 100, 2661-2666. (b) Smith, L. J.; Fiebig,K. M.; Schwalbe, H.; Dobson, C. M. Folding Des. 1996, 1, 95-106.

Figure 1. CD spectra of Lustrin A RKSY (black, 2 µM) andD4 (gray, 2 µM) polypeptides at pH 7.4, 1 mM Na2HPO4, 5° C.

Figure 2. RKSY thermal CD titration plot. Thermal titrationwas monitored by continuous measurements of the CD valueat 220 nm from 2 to 80 °C using 2 µM RKSY polypeptide in 1mM Na2HPO4, pH 7.4. Linear regression analysis was utilizedfor line fitting.

Analyses of Polyelectrolyte Sequence Domains Langmuir, Vol. 18, No. 25, 2002 9903

of polypeptides that possess rapidly exchanging backboneamide protons and indicate that neither peptide possessesintrastrand backbone hydrogen bonding (as observed forR-helix, â-hairpin, and â-turn) or folded structures thatwould afford solvent shielding to amide NH sites alongthe polypeptide backbone.14,17,29,30 These findings clearly

indicate that both polypeptides adopt open conformationsthat are solvent-accessible and by extension should featureextended side-chain conformations that would allowsignificant polypeptide side chain-surface interactions.Thus, our NMR data support the qualitative findings ofthe CD experiments, namely, that both polyelectrolytepolypeptides do not adopt folded structures in solution;

(29) Wustman, B. A.; Santos, R.; Zhang, B.; Evans, J. S. Biopolymers,in press. (30) Zhang, B.; Xu, G.; Evans, J. S. Biopolymers 2000, 54, 464-475.

Table 1. Proton Chemical Shiftsa (ppm, at 278 K) for Lustrin A RKSY Polypeptide, pH 7.4, 90% Water/10% D2O

residue NHR CHR CHâ CHγ CHδ CHε ring H NH

Y1 8.49 4.42 3.05, 2.86 2,6H: 6.80; 3,5H: 7.11R2 8.72 4.29 1.81 1.68 3.15 6.50, 6.95G3 8.56 3.98P4 4.47 2.27 1.96 3.62, 3.82I5 8.42 4.12 1.83 CH2 1.49, 1.22; CH3: 0.90 0.87A6 8.54 4.30 1.35R7 8.48 4.59 1.80 1.69 3.19 6.50, 6.95P8 4.42 2.27 2.01 3.60, 3.82R9 8.35 4.33 1.81 1.56 3.15 6.50, 6.95S10 8.50 4.47 3.96, 3.88S11 8.62 4.43 3.93, 3.87R12 8.39 4.22 1.69 1.47 3.11 6.50, 6.95Y13 8.20 4.51 3.06, 2.96 2,6H, 3,5H: 6.80L14 8.06 4.26 1.59, 1.56 1.53 0.90, 0.86A15 8.23 4.14 1.37K16 8.17 4.15 1.64 1.24 1.67 2.95 N/OY17 8.13 4.54 3.05, 2.92 2,6H: 6.80; 3,5H: 7.11L18 8.10 4.29 1.62, 1.60 1.54 0.90, 0.86K19 8.30 4.22 1.68 1.42 1.68 2.97 N/OQ20 8.49 4.32 2.11, 2.02 2.38 7.62, 7.00G21 8.56 3.98R22 8.43 4.39 1.80 1.66 3.20 6.50, 6.95S23 8.57 4.47 3.95, 3.89G24 8.56 3.98K25 8.30 4.29 1.68 1.42 1.68 2.97R26 8.55 4.32 1.77 1.63 3.20 6.50, 6.95L27 8.51 4.34 1.63, 1.61 1.55 0.93, 0.86Q28 8.54 4.33 2.05, 1.97 2.41, 2.39 7.62, 7.00K29 8.60 4.59 1.74 1.49 1.74 2.97 N/OP30 4.39 2.31, 2.02 1.90, 1.77 3.65, 3.85

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 comma. Proton chemical shifts are referenced from internal TSP. N/O ) notobserved.

Table 2. Proton Chemical Shiftsa (ppm, at 278 K) for Lustrin A D4 Polypeptide, pH 7.4, 90% Water/10% D2O

residue NHR CHR CHâ CHγ CHδ CHε ring H NH

G1 8.28 3.93K2 8.38 4.34 1.84, 1.76 1.46, 1.40 1.65 3.01, 2.96 N/OG3 8.38 4.34A4 8.19 4.34 1.33S5 8.24 4.42 3.91, 3.87Y6 8.11 4.62 3.10, 2.96 2,6H: 7.10; 3,5H: 6.83D7 8.31 4.66 2.69, 2.61T8 8.09 4.32 4.29 1.22D9 8.37 4.63 2.72, 2.68A10 8.16 4.27 1.35D11 8.30 4.66 2.76S12 8.24 4.42 3.92, 3.80G13 8.51 4.03S14 8.17 4.45 3.68D15 8.42 4.60 2.72,2.67N16 8.31 4.58 2.75 7.54, 6.86R17 8.20 4.33 1.85,1.78 1.61 3.13,3.09 7.54, 6.86S18 8.31 4.71 3.88P19 - - - 4.34 2.29 1.87, 1.72 3.55, 3.71G20 8.43 3.90Y21 7.87 4.51 3.03,2.98 2,6H: 7.10; 3,5H: 6.83L22 7.99 4.61 1.68,1.62 1.50 0.89P23 - - - 4.34 2.29 1.90, 1.77 3.55, 3.71Q24 8.44 4.28 2.14,1.99 2.42,2.39 7.54, 6.86a The proton assignments were obtained from our analyses of the PFG-clean TOCSY, PFG DQF-COSY, and PFG NOESY/ROESY

experiments. Diastereotopic protons are separated by a comma. Proton chemical shifts are referenced from internal TSP. N/O ) notobserved.

9904 Langmuir, Vol. 18, No. 25, 2002 Wustman et al.

rather, RKSY apparently exists in a partially extendedconformation (Figures 1, 5, and 7), and D4 exists pre-dominantly in a random-coil conformation (Figures 1, 4,and 7).

DiscussionAs detailed in this report, our results indicate that D4

and RKSY possess structural features that are consistentwith polyelectrolyte polypeptides (i.e., the presence ofextended or random-coil conformations).11,12,17,29 Theseunfolded conformer types would presumably minimizeelectrostatic side-chain charge repulsion11,12 and permitexcellent side-chain accessibility for charged residues (Asp,Lys), a prerequisite for polypeptide-surface interactions.The fact that RKSY adopts a more extended structurethan D4 most likely arises from the significant charge

repulsion created by six Arg and four Lys amino acidswithin the RKSY 30-AA sequence.1a At this time, it is notknown whether the Ser and Thr residues in D4 and RKSYof the Lustrin A sequence are phosphorylated.1a If thiswere to be the case, then phosphorylation of either domainwould certainly increase the net charge on either sequenceand likewise increase its polyelectrolyte behavior,11,12

making either sequence an excellent candidate for ion-pairing electrostatic interactions within the nacre matrix.

Given the presence of organic matrix components suchas â-chitin polysaccharide and â-silk fibroin protein2 aswell as nacre-specific polyanionic proteins and the ara-gonite mineral-phase itself,1a there exist numerous nacre-specific substrate surfaces for Lustrin A to interact with.These interactions, in turn, could lead to “tethering” ofthe Lustrin A protein to either mineral or macromolecular

Figure 3. PFG-ROESY spectra of D4 and RKSY model polypeptides in 1 mM Na2HPO4, pH 7.4, 90% H2O/10% D2O, 278 K. Spectraof the NH-CHR fingerprint regions are given. Note that PFG-NOESY spectra are identical to the ROESY spectra. As noted in thetext, no interresidue dNN NOE connectivities were observed for D4 or RKSY under the conditions described in this paper. For allNOESY and ROESY experiments, acquisition parameters included a relaxation delay of 1 s, spectral window ) 5200 Hz, “hard”90° ) 10.5 µs, “hard” 180° ) 21 µs, “soft” selective 180° ) 3.2 ms; 128 transients/experiment were acquired, and 4096 and 512complex points were collected in t1 and t2, respectively. The carrier is centered on the water resonance. The tm or mixing time forNOESY and ROESY (compensated spinlock 180° pulse train) experiments was 200 ms. z-Gradient parameters: G1 ) 14 G/cm andG2 ) 6 G/cm, each with a duration of 1 ms with 500 µs of stabilization time. The hypercomplex phase-sensitive method was utilizedfor processing both 2-D spectra, with zero filling in the F2 dimension. Proton chemical shifts are referenced from internal d4-TSP.

Figure 4. Summary of NMR parameters for Lustrin A D4 peptide in 1 mM Na2HPO4, 90% water/10% D2O, 278 K, pH 7.4. Thesummary includes interresidue sequential RN(i, i + 1) and intraresidue RN(i, i) NOEs, RN(i, i + 1)/RN (i, i) ratios [RN ratio], 3Jcouplings (Hz), and amide temperature-shift coefficients (ATC, in negative ppb/K). For S5, S12, and N16, ATC values could notbe unambiguously determined; these residues are represented by a blank space. The relative NOE intensities are reflected by theheight of the histograms. x ) observed, but because of cross-peak overlap, is not quantitated. N/A ) not available.

Analyses of Polyelectrolyte Sequence Domains Langmuir, Vol. 18, No. 25, 2002 9905

surfaces, which would allow the protein to resist forceextension, in accord with AFM observations.1b At present,the specific functions of D4 and RKSY vis-a-vis LustrinA elasticity and macromolecular adhesion are unknown.However, on the basis of their amino acid compositions,

both RKSY and D4 represent potential sites for LustrinA adhesion to either charged or hydrogen-bonding donor/acceptor surfaces. Experiments involving D4 and RKSYinteractions with other matrix components (â-chitin, â-silkfibroin-like protein, polyanionic matrix proteins, andaragonite mineral) will be required to differentiate whichmolecular surfaces D4 and RKSY may interact with.Moreover, the reader should be aware that structuralfindings detailed in this report are subject to reinterpre-tation pending solution and solid-state NMR studies ofrecombinant Lustrin A protein.

In recent biophysical studies of polypeptides represent-ing the titin PEVK domain,17 the consensus repeat-loopdomains of Lustrin A14 and PM27,28 as well as the“extended-turn” motif within the Pro, Asn-rich repeatsequence of SM50,29 progress has been made in discerningpolypeptide structures that adopt open conformations (i.e.,extended,14,29 polyproline Type II,17 and loop14,28) yet arenot truly random coil in structure. The use of ∆δHRconformational shifts, ∆J random-coil coupling deviations,as well as intra- and interresidue NOE ratios hasbroadened the palette of solution-state NMR parametersthat can be utilized for interpreting polypeptide structure.In doing so, we have become aware that polypeptidesecondary structure extends beyond the traditional clas-sifications of R-helix, â-sheet, â-turn, and random coil,particularly for nonglobular proteins that are involved infascinating processes such as elasticity14,17,28 and biocom-posite formation.11,12 It is hoped that this awareness willenable us to reexamine previously documented random-coil sequences for additional structural information.

Acknowledgment. This work was supported by theNational Science Foundation (DMR 99-01356, MCB 98-16703 to N.Y.U. and DMR 96-32716 to U.C.S.B.) and theArmy Research Office (MURI DAAH04-96-1-0443 toU.C.S.B.). This paper represents contribution number 18from the Laboratory for Chemical Physics, New YorkUniversity.

LA025927M

Figure 5. Summary of NMR parameters for Lustrin A RKSY peptide in 1 mM Na2HPO4, 90% water/10% D2O, 278 K, pH 7.4.The summary includes interresidue sequential RN(i, i + 1) and intraresidue RN(i, i) NOEs, RN(i, i + 1)/RN (i, i) ratios [RN(i +1)/RN(i)], 3J couplings (Hz), and amide temperature-shift coefficients (ATC, in negative ppb/K). An unambiguous determinationof 3J couplings for R26 and ATC values for G3, G21, and G24 could not be made; hence, these residues are represented by a blankspace. The relative NOE intensities are reflected by the height of the histograms. x ) observed, but because of cross-peak overlap,is not quantitated. N/A ) not available.

Figure 6. ∆δHR values obtained for D4 and RKSY modelpolypeptides in 1 mM Na2HPO4, pH 7.4, 90% H2O/10% D2O,278 K. Negative values represent upfield shifts, and positivevalues represent downfield shifts. No corrections have beenmade for terminal residue effects; however, corrected CHRchemical-shift values were utilized for all Pro and Xaa-Pronearest neighbors. Proton chemical shifts were referenced frominternal d4-TSP.

Figure 7. Calculated difference (∆J, Hz) between experimentaland random-coil 3JNH-CHR values for the Lustrin A D4 and RKSYmodel polypeptides in 1 mM Na2HPO4, pH 7.4, 90% H2O/10%D2O, 278 K. For RKSY, 3J couplings could not be unambiguouslydetermined for R26; hence, this residue is represented by ablank space.

9906 Langmuir, Vol. 18, No. 25, 2002 Wustman et al.