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Structural Analyses of Polyelectrolyte Sequence Domains within 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 between layered aragonite mineral plates (i.e., nacre layers) in mollusk shell. These proteins exhibit elastomeric and adhesive behavior within the mineralized matrix. One member of the lustrin superfamily, Lustrin A, has been sequenced; the protein is organized into defined, modular sequence domains that are hypothesized to perform separate functions (e.g., force unfolding, mineral interaction, intermolecular binding) within the Lustrin A protein. Using NMR, CD spectrometry, and model peptides, we investigated the solution structure of two Lustrin A polyelectrolyte modular domains that represent putative sites for Lustrin A-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 exhibiting structural features of an extended conformer, and D4, a more labile, random-coil conformation. These results suggest that the Lustrin A protein possesses open, unfolded regions that could act as putative sites for Lustrin A-mineral or Lustrin A-macromolecular interactions that lead to the observed adhesive properties of the nacre organic matrix.
Introduction Fracture toughness in biocomposite materials such as the abalone shell arises from the presence of proteins in close approximation with the aragonite mineral phase.1 Members of one superfamily, the lustrins, are localized within the interstitial organic layer of the nacre of the red abalone, Haliotis rufescens.1a This interstitial organic layer exhibits elastic behavior (i.e., reversible extension and recovery).1b In addition, this same layer possesses adhesive interactions with the underlying aragonite mineral tablets.1b It has been hypothesized that the observed adhesive interactions may arise from a number of interactions involving the lustrin proteins and their environment (e.g., protein-mineral and/or protein-macromolecular). If true, then the lustrin proteins would most likely possess one or more domains that would foster interactions with these components, thereby promoting adhesion and allowing the lustrin proteins to “anchor” and resist removal from the nacre layer.1b This, in turn, may help to explain how proteins convey fracture resistance to mineralized biocomposites and could serve as an interesting molecular paradigm for developing fracture-resistant materials. As a starting point in identifying putative adhesive domains within lustrin proteins, our initial focus is on defined sequence regions that are polyelectrolyte in nature (i.e., a significant presence of cationic or anionic amino acid residues within a given sequence block). The significance of polyelectrolyte sequences within lustrin * To whom correspondence should be addressed. E-mail:
[email protected]. † 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.
proteins is two-fold. First, the molecular interaction of “anionic” polyelectrolyte sequences with mineral interfaces has been documented,2,3 and thus polyelectrolyte domain(s) could represent hypothetical site(s) for lustrin-mineral adhesion. Second, given the presence of “basic”4-6 and anionic2,3,7-10 polyelectrolyte domains in a number of biomineralization proteins, it is plausible that lustrin polyelectrolyte domains could participate in complementary protein-protein electrostatic interactions within the matrix. In either instance, Lustrin A polyelectrolyte domain interactions could, in principle, provide a plausible mechanism for lustrin-matrix adhesion. Additionally, the fact that polyelectrolyte biomineralization polypeptide domains adopt open, unfolded structures in solution11,12 would presumably facilitate side-chain access to complementary mineral or polypeptide surfaces within the matrix. Interestingly, one particular lustrin protein, Lustrin A (pacific red abalone H. rufescens, 116 kDa),1a possesses two modular, polyelectrolyte domains. The first is a 24AA Asp-containing domain, GKGASYDTDADSGSDNR(2) Lowenstam, H. A.; Weiner, S. In On Biomineralization; Oxford University 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, 534541.
10.1021/la025927m CCC: $22.00 © 2002 American Chemical Society Published on Web 10/29/2002
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SPGYLPQ (residues 1251-1264, designated as “D4”, charged residues underlined), which is localized between the Ser, Gly-rich domain and the C10 domain within the Lustrin A sequence. The second is a 30-residue basic Arg-, Lys-, Ser-, Tyr-containing domain, YRGPIARPRSSRYLAKYLKQGRSGKRLQKP (residues 1354-1383, designated as “RKSY”, charged residues underlined). What makes these sequences even more interesting is the presence of hydrogen bonding donor/acceptor amino acids (Asn, Gln, Arg, Thr, Ser, Tyr) within each domain. These amino acids may represent additional putative sites for Lustrin A-mineral or Lustrin A-macromolecule interactions via hydrogen-bonding mechanisms. To date, because of the difficulty in obtaining purified Lustrin A protein in quantity, no studies have been performed to assess the structure of either domain.1a To investigate these domains further, we determined the structural preferences for Lustrin A D4 and RKSY in solution using NMR spectroscopy, circular dichroism (CD) spectrometry, and N-acetyl-, C-amide-“capped” model peptides representing the D4 and RKSY domains in toto. We find that both polyelectrolyte sequences adopt open conformations in solution, with a subtle difference: RKSY adopts a more extended conformation, whereas D4 adopts a “random-coil” conformation. These findings are discussed in light of the possible functions for each domain within Lustrin A. Materials and Methods Peptide Synthesis, Purification, and Sample Preparation. Purified, N-acetyl-, C-amide-capped RKSY and D4 polypeptides were synthesized by Dr. Janet Crawford, Yale University HHMI Biopolymer/Keck Biotechnology Resource Laboratory, using an Applied Biosystems 431A Peptide Synthesizer and NRL-FMOC amino acids. Typical peptide synthesis runs were carried out at the 100-µmol level using the Applied Biosystems FastMoc 0.25 HBTU/HOBt/NMP protocol [HBTU ) 2-(1H-benzotriazole1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOBt ) N-hydroxybenzotriazole; NMP ) n-methylpyrrolidone; Applied Biosystems Technical Notes, November 1993) and MBHA resin (Novabiochem). To avoid possible side-chain modification during synthesis, NR-Trityl-Nγ-FMOC-Asn (abbreviated as FMOC-AsnTrt) 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 peptides were deprotected and cleaved (reaction time ) 3 h at 25 °C) from the resin using a cleavage cocktail (15 mL/g resin) containing 90% v/v TFA, 2.5% v/v water, 2.5% v/v ethanedithiol, 2.5% v/v phenol, and 2.5% v/v thioanisole;13-16 the thiol scavengers were utilized to enhance trityl removal. The reaction mixture was filtered under reduced pressure. The crude peptides were separately dissolved in deionized distilled water, extracted three times with diethyl ether, and then concentrated and lyophilized. Peptide purification involved a C18 reverse-phase high-performance 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 at 230 nm. Individual HPLC fractions were analyzed using a custommade MALDI/TOF delayed extraction mass spectrometer. Typically, a 100-µmol synthesis of each peptide yielded >90 mg dry mass of purified peptide that was >96% in purity and free of side-chain protection. The experimental molecular masses for the capped RKSY and D4 polypeptides were determined to be 3575.6 and 2499.96 Da, in agreement with the theoretical molecular masses of 3572.1 and 2499.55 Da respectively. For NMR and CD studies, both peptides were dissolved in 1 mM Na2HPO4 in deionized distilled water, pH 7.4. NMR samples (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.
Wustman et al. contained 10% v/v deuterium oxide (99.9% atom D, Cambridge Isotope Labs) and 10 µM d4-TSP. Final peptide concentrations were 2 mM for NMR and 2 µM for CD. At the 2 mM concentration, turbidity measurements at 380 nm revealed no evidence of peptide aggregation; this also was confirmed by analyses of NMR proton chemical shifts and line widths and the absence of longrange dsc-sc NOE connectivities.13,14 CD Spectrometry. CD spectra were obtained at pH 7.4 using an AVIV 62DS CD spectrometer running 60DS software version 4.1t and quartz cells with a 0.1-cm path length. The samples were scanned from 190 to 260 nm at 5 °C using a 1-nm bandwidth and a scan rate of 1 nm/s. The spectrometer was previously calibrated with d-(+)-10-camphorsulfphonic acid. A total of eight scans were acquired. Mean residue ellipticity [θM] is expressed in units of deg cm2 dmol-1 per mol of peptide.13,14 Thermal titration of the RKSY polypeptide was performed over the temperature range of 2 to 80 °C with monitoring of the 220-nm ellipticity intensity as a function of temperature.17 NMR Spectroscopy. NMR experiments were performed on a Varian UNITY 500 spectrometer equipped with a variable temperature controller and a three-channel (13C/15N/1H) z-axis PFG 5-mm solution probehead. The probehead sample temperature was maintained using an ethylene glycol cooling apparatus with filtered airflow. The temperature was maintained within ( 0.1 °C. With the exception of the proton amide temperature shift experiments, all reported NMR experiments were conducted at 278 K in order to slow the conformational exchange within the peptide. Proton scalar coupling assignments were obtained using “excitation sculpting” 2-D PFG- “clean” TOCSY experiments.13,14,18,19 Proton sequential assignments and NOEs were obtained using z-PFG-ROESY experiments13,14,20,21 using a range of mixing times from 50 to 200 ms; spectra were jointly analyzed to 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-relaxation crosstalk artifacts were detected in the z-PFG P. E. COSY J-coupling spectra; in addition, we found that the cross-peak line widths in the P. E. COSY spectra were not significantly affected by linebroadening via comparisons of spectra obtained at 5 and 20 °C (data not shown). NMR data were processed using FELIX95 software (MSI/Biosym Technologies, Inc). Relevant NMR acquisition and processing parameters are provided in the figure legends Using PFG TOCSY or NOESY experiments at 278, 283, 288, 293, and 298 K, amide proton temperature coefficients were determined from the slope of the temperature versus amide proton chemical shift curves for each residue.24 Temperature gradients are expressed in units of ppb/K with a negative sign indicating an upfield shift upon warming.24 Temperature calibration of the VT unit was determined prior to experimentation using neat methanol over a temperature range of 273 to 320 K.
Results CD Spectrometry. We first examine the conformational states of RKSY and D4 to determine qualitatively if either polypeptide sequence exhibits specific secondary structure 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 200 nm, consistent with the random-coil state.14,17,25 Unusu(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, 127134. (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, 43144325. (b) Butcher, D. J.; Nedved, M. L.; Neiss, T. G.; Moe, G. R. Biochemistry 1996, 35, 698-703.
Analyses of Polyelectrolyte Sequence Domains
Figure 1. CD spectra of Lustrin A RKSY (black, 2 µM) and D4 (gray, 2 µM) polypeptides at pH 7.4, 1 mM Na2HPO4, 5° C.
Figure 2. RKSY thermal CD titration plot. Thermal titration was monitored by continuous measurements of the CD value at 220 nm from 2 to 80 °C using 2 µM RKSY polypeptide in 1 mM Na2HPO4, pH 7.4. Linear regression analysis was utilized for line fitting.
ally, the RKSY polypeptide also exhibits a minor positive adsorption band (n-π* transition) observed near 220 nm, which is also consistent with the presence of the polyproline Type II structure.17,25 However, monitoring the 220nm band over the temperature range of 2-80 °C (i.e., thermal titration) did not reveal a pronounced biphasic 220-nm transition near 50 °C that is characteristic of polyproline Type II (Figure 2).17,25 Thus, the CD data for both polypeptides are consistent with the presence of unfolded, open structures in both sequences. This is consistent with the polyelectrolyte nature of both sequences, wherein charge repulsion between similarly charged side chains (i.e., Asp-Asp in D4; Arg-Arg, LysLys, and Lys-Arg in RKSY) would lead to unfolded, open conformations.11,12 NMR Spectroscopy. The findings obtained from CD spectrometry are supported by NMR experiments (PFG clean TOCSY, ROESY, and DQF-COSY) conducted on both polypeptides at pH 7.4, 1 mM Na2HPO4. 1H chemical shifts, intraresidue and interresidue NOEs, and 3JNH-CHR coupling constants were compiled for both polypeptides (Tables 1 and 2; Figures 3-5). Proton conformational shifts (∆δHR), which can be utilized to determine the presence or absence of the random-coil conformation within proteins,13,14,17,26 were calculated for both peptides (Figure 6). As shown in Figure 6, residues G3, A4, Y6, and G13 possess ∆δHR values >0.1 ppm, indicating significant deviation from random-coil database values;26 however, the remaining residues in D4 possess ∆δHR values that do not exceed the random-coil threshold of 0.1 ppm, suggesting that the majority of the D4 sequence is conformationally similar to a random-coil state.13,14,17,26 A similar trend is noted for RKSY (i.e., residues G3, A6, (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.
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R12, Y17, and G24 possess ∆δHRa values >0.1 ppm, with the remaining residues having ∆δHRa values 1 Hz are considered to be significant; positive ∆J values (+∆J) are indicative of the β-strand conformation, and negative ∆J (-∆J) values are representative of the R-helix conformation. ∆J values for the β-turn can be either (+) or (-).27 For D4, we note that G3, A4, D7, D11, S12, G13, S14, N16, and R17 possess (-) ∆J values >1 Hz. Furthermore, G1, A10, and G20 possess (+) ∆J values >1 Hz. The remaining residues possess ∆J values 1 Hz for R2, I5, A6, R7, R9, S10, Y13, Y17, L18, Q20, R22, S23, Q28, and K29, with the remaining residues possessing ∆J values 5.5 Å)14,17,28 and/or the presence of conformational exchange involving randomcoil states.14,17,28 The NOE intensity ratio, RN(i, i + 1)/ RN(i, i), can be used a guide for discriminating between extended and random-coil states;14,17,28 the value of 2.3 represents the population-weighted random-coil model, whereas values >4 are predicted for the β-strand.28 For those regions where nonoverlapping NOEs can be quantitated, we find that the RKSY polypeptide possesses resolvable NOE ratio values of 9.9, 5.1, 4.1, 3.3, and 35.6 for I5-A6, Y13-L14, L14-A15, K19-Q20, and R22-S23, respectively (Figure 5). Hence, these sequence regions in RKSY are similar to the β-strand and are not random coil in nature. These results support the findings obtained from RKSY ∆J calculations (Figure 7). At this time, it is not known whether the remaining regions of the RKSY sequence exist in an extended conformation similar to the β-strand. In comparison, we find that, with the exception of Y21-G20 (ratio ) 4.9), the remaining resolvable RN(i, i + 1)/RN(i, i) ratios in D4 are -6.0 ppb/K) obtained for polypeptides (Figures 4 and 5). These values are typical (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.
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Table 1. Proton Chemical Shiftsa (ppm, at 278 K) for Lustrin A RKSY Polypeptide, pH 7.4, 90% Water/10% D2O residue
NHR
CHR
CHβ
Y1 R2 G3 P4 I5 A6 R7 P8 R9 S10 S11 R12 Y13 L14 A15 K16 Y17 L18 K19 Q20 G21 R22 S23 G24 K25 R26 L27 Q28 K29 P30
8.49 8.72 8.56
4.42 4.29 3.98 4.47 4.12 4.30 4.59 4.42 4.33 4.47 4.43 4.22 4.51 4.26 4.14 4.15 4.54 4.29 4.22 4.32 3.98 4.39 4.47 3.98 4.29 4.32 4.34 4.33 4.59 4.39
3.05, 2.86 1.81
8.42 8.54 8.48 8.35 8.50 8.62 8.39 8.20 8.06 8.23 8.17 8.13 8.10 8.30 8.49 8.56 8.43 8.57 8.56 8.30 8.55 8.51 8.54 8.60
CHγ
2.27 1.83 1.35 1.80 2.27 1.81 3.96, 3.88 3.93, 3.87 1.69 3.06, 2.96 1.59, 1.56 1.37 1.64 3.05, 2.92 1.62, 1.60 1.68 2.11, 2.02
CHδ
CH
ring H
NH
2,6H: 6.80; 3,5H: 7.11 1.68
3.15
6.50, 6.95
1.96 CH2 1.49, 1.22; CH3: 0.90
3.62, 3.82 0.87
1.69 2.01 1.56
3.19 3.60, 3.82 3.15
1.47
3.11
6.50, 6.95 6.50, 6.95 6.50, 6.95 2,6H, 3,5H: 6.80
1.53
0.90, 0.86
1.24
1.67
2.95
N/O 2,6H: 6.80; 3,5H: 7.11
1.54 1.42 2.38
0.90, 0.86 1.68
1.80 3.95, 3.89
1.66
3.20
1.68 1.77 1.63, 1.61 2.05, 1.97 1.74 2.31, 2.02
1.42 1.63 1.55 2.41, 2.39 1.49 1.90, 1.77
1.68 3.20 0.93, 0.86
2.97
1.74 3.65, 3.85
2.97
2.97
N/O 7.62, 7.00 6.50, 6.95
6.50, 6.95 7.62, 7.00 N/O
a 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 ) not observed.
Table 2. Proton Chemical Shiftsa (ppm, at 278 K) for Lustrin A D4 Polypeptide, pH 7.4, 90% Water/10% D2O residue G1 K2 G3 A4 S5 Y6 D7 T8 D9 A10 D11 S12 G13 S14 D15 N16 R17 S18 P19 G20 Y21 L22 P23 Q24
NHR
CHR
8.28 8.38 8.38 8.19 8.24 8.11 8.31 8.09 8.37 8.16 8.30 8.24 8.51 8.17 8.42 8.31 8.20 8.31 --8.43 7.87 7.99 --8.44
3.93 4.34 4.34 4.34 4.42 4.62 4.66 4.32 4.63 4.27 4.66 4.42 4.03 4.45 4.60 4.58 4.33 4.71 4.34 3.90 4.51 4.61 4.34 4.28
CHβ
CHγ
1.84, 1.76
1.46, 1.40
1.33 3.91, 3.87 3.10, 2.96 2.69, 2.61 4.29 2.72, 2.68 1.35 2.76 3.92, 3.80
CHδ
CH
1.65
ring H
3.01, 2.96
NH N/O
2,6H: 7.10; 3,5H: 6.83 1.22
3.68 2.72,2.67 2.75 1.85,1.78 3.88 2.29
1.61
3.13,3.09
1.87, 1.72
3.55, 3.71
3.03,2.98 1.68,1.62 2.29 2.14,1.99
1.50 1.90, 1.77 2.42,2.39
0.89 3.55, 3.71
7.54, 6.86 7.54, 6.86
2,6H: 7.10; 3,5H: 6.83 7.54, 6.86
a
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 ) not observed.
of polypeptides that possess rapidly exchanging backbone amide protons and indicate that neither peptide possesses intrastrand backbone hydrogen bonding (as observed for R-helix, β-hairpin, and β-turn) or folded structures that would afford solvent shielding to amide NH sites along the polypeptide backbone.14,17,29,30 These findings clearly (29) Wustman, B. A.; Santos, R.; Zhang, B.; Evans, J. S. Biopolymers, in press.
indicate that both polypeptides adopt open conformations that are solvent-accessible and by extension should feature extended side-chain conformations that would allow significant polypeptide side chain-surface interactions. Thus, our NMR data support the qualitative findings of the CD experiments, namely, that both polyelectrolyte polypeptides do not adopt folded structures in solution; (30) Zhang, B.; Xu, G.; Evans, J. S. Biopolymers 2000, 54, 464-475.
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Figure 3. PFG-ROESY spectra of D4 and RKSY model polypeptides in 1 mM Na2HPO4, pH 7.4, 90% H2O/10% D2O, 278 K. Spectra of the NH-CHR fingerprint regions are given. Note that PFG-NOESY spectra are identical to the ROESY spectra. As noted in the text, no interresidue dNN NOE connectivities were observed for D4 or RKSY under the conditions described in this paper. For all NOESY 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 512 complex points were collected in t1 and t2, respectively. The carrier is centered on the water resonance. The tm or mixing time for NOESY and ROESY (compensated spinlock 180° pulse train) experiments was 200 ms. z-Gradient parameters: G1 ) 14 G/cm and G2 ) 6 G/cm, each with a duration of 1 ms with 500 µs of stabilization time. The hypercomplex phase-sensitive method was utilized for 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. The summary includes interresidue sequential RN(i, i + 1) and intraresidue RN(i, i) NOEs, RN(i, i + 1)/RN (i, i) ratios [RN ratio], 3J couplings (Hz), and amide temperature-shift coefficients (ATC, in negative ppb/K). For S5, S12, and N16, ATC values could not be unambiguously determined; these residues are represented by a blank space. 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.
rather, RKSY apparently exists in a partially extended conformation (Figures 1, 5, and 7), and D4 exists predominantly in a random-coil conformation (Figures 1, 4, and 7). Discussion As detailed in this report, our results indicate that D4 and RKSY possess structural features that are consistent with polyelectrolyte polypeptides (i.e., the presence of extended or random-coil conformations).11,12,17,29 These unfolded conformer types would presumably minimize electrostatic side-chain charge repulsion11,12 and permit excellent side-chain accessibility for charged residues (Asp, Lys), a prerequisite for polypeptide-surface interactions. The fact that RKSY adopts a more extended structure than D4 most likely arises from the significant charge
repulsion created by six Arg and four Lys amino acids within the RKSY 30-AA sequence.1a At this time, it is not known whether the Ser and Thr residues in D4 and RKSY of the Lustrin A sequence are phosphorylated.1a If this were to be the case, then phosphorylation of either domain would certainly increase the net charge on either sequence and likewise increase its polyelectrolyte behavior,11,12 making either sequence an excellent candidate for ionpairing electrostatic interactions within the nacre matrix. Given the presence of organic matrix components such as β-chitin polysaccharide and β-silk fibroin protein2 as well as nacre-specific polyanionic proteins and the aragonite mineral-phase itself,1a there exist numerous nacrespecific substrate surfaces for Lustrin A to interact with. These interactions, in turn, could lead to “tethering” of the Lustrin A protein to either mineral or macromolecular
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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 determination of 3J couplings for R26 and ATC values for G3, G21, and G24 could not be made; hence, these residues are represented by a blank space. 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 model polypeptides in 1 mM Na2HPO4, pH 7.4, 90% H2O/10% D2O, 278 K. Negative values represent upfield shifts, and positive values represent downfield shifts. No corrections have been made for terminal residue effects; however, corrected CHR chemical-shift values were utilized for all Pro and Xaa-Pro nearest neighbors. Proton chemical shifts were referenced from internal d4-TSP.
Figure 7. Calculated difference (∆J, Hz) between experimental and random-coil 3JNH-CHR values for the Lustrin A D4 and RKSY model polypeptides in 1 mM Na2HPO4, pH 7.4, 90% H2O/10% D2O, 278 K. For RKSY, 3J couplings could not be unambiguously determined for R26; hence, this residue is represented by a blank space.
surfaces, which would allow the protein to resist force extension, in accord with AFM observations.1b At present, the specific functions of D4 and RKSY vis-a`-vis Lustrin A elasticity and macromolecular adhesion are unknown. However, on the basis of their amino acid compositions,
both RKSY and D4 represent potential sites for Lustrin A adhesion to either charged or hydrogen-bonding donor/ acceptor surfaces. Experiments involving D4 and RKSY interactions with other matrix components (β-chitin, β-silk fibroin-like protein, polyanionic matrix proteins, and aragonite mineral) will be required to differentiate which molecular surfaces D4 and RKSY may interact with. Moreover, the reader should be aware that structural findings detailed in this report are subject to reinterpretation pending solution and solid-state NMR studies of recombinant Lustrin A protein. In recent biophysical studies of polypeptides representing the titin PEVK domain,17 the consensus repeat-loop domains of Lustrin A14 and PM27,28 as well as the “extended-turn” motif within the Pro, Asn-rich repeat sequence of SM50,29 progress has been made in discerning polypeptide structures that adopt open conformations (i.e., extended,14,29 polyproline Type II,17 and loop14,28) yet are not truly random coil in structure. The use of ∆δHR conformational shifts, ∆J random-coil coupling deviations, as well as intra- and interresidue NOE ratios has broadened the palette of solution-state NMR parameters that can be utilized for interpreting polypeptide structure. In doing so, we have become aware that polypeptide secondary structure extends beyond the traditional classifications of R-helix, β-sheet, β-turn, and random coil, particularly for nonglobular proteins that are involved in fascinating processes such as elasticity14,17,28 and biocomposite formation.11,12 It is hoped that this awareness will enable us to reexamine previously documented randomcoil sequences for additional structural information. Acknowledgment. This work was supported by the National Science Foundation (DMR 99-01356, MCB 9816703 to N.Y.U. and DMR 96-32716 to U.C.S.B.) and the Army Research Office (MURI DAAH04-96-1-0443 to U.C.S.B.). This paper represents contribution number 18 from the Laboratory for Chemical Physics, New York University. LA025927M
