Solid-State NMR Comparison of Various Spiders' Dragline Silk Fiber

Jul 1, 2010 - Major ampullate (dragline) spider silk is a coveted biopolymer due to its ... The dragline silk of different spiders have distinct mecha...
0 downloads 0 Views 655KB Size
Biomacromolecules 2010, 11, 2039–2043

2039

Solid-State NMR Comparison of Various Spiders’ Dragline Silk Fiber Melinda S. Creager,† Janelle E. Jenkins,‡ Leigh A. Thagard-Yeaman,‡ Amanda E. Brooks,† Justin A. Jones,§ Randolph V. Lewis,† Gregory P. Holland,‡ and Jeffery L. Yarger*,‡ Department of Molecular Biology and Macromolecular Core Facility, University of Wyoming, Laramie, Wyoming 82071, and Department of Chemistry and Biochemistry, Magnetic Resonance Research Center, Arizona State University, Tempe, Arizona 85287-1604 Received April 14, 2010; Revised Manuscript Received May 24, 2010

Major ampullate (dragline) spider silk is a coveted biopolymer due to its combination of strength and extensibility. The dragline silk of different spiders have distinct mechanical properties that can be qualitatively correlated to the protein sequence. This study uses amino acid analysis and carbon-13 solid-state NMR to compare the molecular composition, structure, and dynamics of major ampullate dragline silk of four orb-web spider species (Nephila claVipes, Araneus gemmoides, Argiope aurantia, and Argiope argentata) and one cobweb species (Latrodectus hesperus). The mobility of the protein backbone and amino acid side chains in water exposed silk fibers is shown to correlate to the proline content. This implies that regions of major ampullate spidroin 2 protein, which is the only dragline silk protein with any significant proline content, become significantly hydrated in dragline spider silk.

Introduction Spiders have evolved over hundreds of millions of years. The Araenoidea superfamily diverged into the Araneidae and the “derived araneoids” around 125 million years ago.1 This split, which defines araneidae as orb weavers, includes Araneus gemmoides, Argiope argentata, and Argiope aurantia, and groups other species such as orb weaver Nephila claVipes and cobweb weavers Latrodectus hesperus into “derived araneoids” (see Figure 1). All five species listed above have evolved to make six different types of silk fibers and an aqueous glue.2 These silks generally have the same function, including web structure (major ampullate, minor ampullate, flagelliform, pyriform, and aqueous glue), prey immobilization (aciniform), and egg case (aciniform and tubuliform).3 Although the silks of various species serve the same general purposes, the mechanical properties differ slightly for each silk of a given species, allowing them to adapt to their unique ecosystems. Of all the different silk fibers, dragline silk (major ampullate silk) has shown the greatest mechanical variation between individual species.4,5 The mechanical property variation in dragline silks can be partially attributed to the nanostructure composite nature of major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2) proteins that make up dragline fibers.6,7 Both MaSp1 and MaSp2 have evolutionarily conserved highly repetitive motif structures found in a large class of web building spiders.8 Repetitive amino acid motifs make up the majority of the major ampullate spidrion proteins and have been the focus of numerous molecular-level structural investigations. Solid-state NMR (ssNMR) has been instrumental in elucidating secondary structure within the highly repetitive amino acid motifs of spider and silkworm silk. For example, NMR was * To whom correspondence should be addressed. Tel.: (480) 965-0673. E-mail: [email protected]. † Department of Molecular Biology, University of Wyoming. ‡ Arizona State University. § Macromolecular Core Facility, University of Wyoming.

Figure 1. Cladogram showing the relationship between Araneus gemmoides (A.g.), Argiope argentata (A.ar), Argiope aurantia (A.au), Latrodectus hesperus (L.h.), and Nephila clavipes (N.c.) produced using sequenced genes indexed in Pubmed. All five spiders (order: Araneae) are in the superfamily Araneoidea and produce either orb webs (Nephilidae and Araneidae) or cob webs (Theridiidae).

used to determine the amino acid repetitive motifs responsible for β-sheet crystalline domains in orb-weaving dragline spider silk9-11 and cocoon silk from Bombyx mori.12,13 Furthermore, in spider dragline silk, ssNMR has been integral in characterizing the molecular structure of glycine-rich regions (GGX and GPGXX repetitive motifs)14,15 and providing molecular structure and dynamic elucidation of supercontraction and the plasticizing effect of water.16-20 To date, however, very few papers have performed NMR studies on spider silks on any genus of spider other than Nephila.21-24 In this work, we compare dragline fibers from three arachnid families (four different genuses). The similarities and differences in the cross-polarization and direct 13 C detection NMR spectra of all five species are discussed.

Materials and Methods Spider Dragline Silks. Araneus gemmoides, Argiope argentata, Argiope aurantia, Latrodectus hesperus, and Nephila claVipes major silk were collected by forcibly silking adult female spiders at 2 cm/ s.25 The spiders were anesthetized using CO2, which was done to reduce the stress of capture. Spiders were restrained and typically gained function within 2-5 min. Silking occurred after a spider was able to drink 20 µL of water to ensure that it was awake. The silking process was monitored

10.1021/bm100399x  2010 American Chemical Society Published on Web 07/01/2010

2040

Biomacromolecules, Vol. 11, No. 8, 2010

Creager et al.

Table 1. Amino Acid Analysis of Major Ampullate Silk from Araneus gemmoides, Argiope argentata, Argiope aurantia, Latrodectus hesperus, and Nephila clavipes, Giving Molar Percent of the Most Abundant Amino Acidsa

Araneus gemmoides Argiope argentata Argiope aurantia Latrodectus hesperus Nephila clavipes

Gly

Ala

Pro

Glx

Ser

Tyr

Leu

Val

Thr

Asx

Arg

Phe

42.8 44.6 46.4 45.7 47.1

19.4 19.3 17.9 31.1 26.5

11.1 10.2 9.5 1.5 1.2

8.2 9.2 9.4 8.7 8.8

7.1 6.8 5.9 1.1 3.0

5.2 5.4 4.8 4.5 3.6

1.1 1.0 1.5 0.7 4.2

1.1 0.5 0.7 0.6 1.1

1.0 0.5 0.5 0.7 0.6

0.9 0.4 0.5 0.7 0.6

0.6 1.1 1.2 1.5 1.2

0.5 0.8 0.9 0.4 0.3

a Asx ) aspartate and aspartic acid, Glx ) glutamine and glutamate. The variability in composition within each spider silk fiber sample was observed to be large (up to 50%), and the values presented are the average of five analysis runs on five different samples of each spider silk.

under a dissection microscope to ensure that only major ampullate silk was collected (no minor ampullate silk was mixed with the fiber). The spiders were fed one small cricket per silking and webs were misted with water twice daily. All silk samples have the natural abundance of 13C and 15 N; no enrichment was performed on these samples. The amount of silk collected from each type of spider species was 9.1 mg of Araneus gemmoides silk, 11.5 mg of Argiope argentata silk, 6.4 mg of Argiope aurantia silk, 8.2 mg of Latrodectus hesperus silk for the dry experiments, 14.1 mg of Latrodectus hesperus silk for the wet experiments, 13.1 mg of Nephila claVipes silk for the dry experiments, and 10.6 mg of Nephila claVipes dragline silk for the wet experiments. Amino Acid Analysis (AAA). Amino acid analysis was done using the Acquity Ultra Performance LC from Waters according to manufacturer protocols for the AccQ-Tag system. A small sample of natural silk fiber (40% fwhm) compared to the Nephila claVipes and Latrodectus hesperus silk. This indicates that MaSp2-rich spider silks (silks high in proline content) become significantly more plasticized compared to MaSp 1-rich spider silks. The enhanced resolution afforded by the fast-repetition direct 13 C MAS spectra of wetted silks can be used to identify proline and glutamine (Gln) resonances in Argiope and Araneus dragline silk. Also, we see a significant increased resolution of the carbonyl region in these spider silks and can resolve the Gln Cδ resonance. In combination with INADEQUATE ssNMR,45 the increased resolution and ability to identify Pro residues in

Through evolution, spider species have produced unique properties in silk by changing the MaSp1 to MaSp2 ratio, with minor changes to the amino acid sequence. 13C CP-MAS and DD-MAS have allowed a more detailed examination into the similarities and differences of five spider species’ major ampullate dragline silk. Four orb-web spider species (Nephila claVipes, Araneus gemmoides, Argiope aurantia, and Argiope argentata) and one cobweb species (Latrodectus hesperus) were studied and shown to have proline content ranging from 0.9 to 11.1 mol percent. The mobility of the protein backbone and amino acid side chains in water exposed silk fibers is shown to correlate to the proline content. This implies that regions of major ampullate spidroin 2 protein, which is the only dragline silk protein with any significant proline content, become significantly hydrated in dragline spider silk. Also, it is clear that various solid state NMR techniques can be used to discern various types of spider silk and characterize their structure and hydration dynamics. Acknowledgment. This work was supported by grants from the U.S. National Science Foundation (NSF-DMR 0805197), the U.S. National Institutes of Health (NIH-EB000490), and the DOD Air Force Office of Scientific Research (DOD-AFOSR FA9550-10-1-0275). We would like to thank Dr. Brian Cherry for his help with NMR experiments, and the Magnetic Resonance Research Center at Arizona State University and the Macromolecular Core Facility at the University of Wyoming for the use of NMR and AAA instrumentation, respectively.

References and Notes (1) Gatesy, J.; Hayashi, C.; Motriuk, D.; Woods, J.; Lewis, R. Science 2001, 291 (5513), 2603–5. (2) Peters, H. M. Z. Naturforsch. 1955, 10, 95. (3) Vollrath, F. Sci. Am. 1992, 266, 70–76. (4) Brooks, A. E.; Steinkraus, H. B.; Nelson, S. R.; Lewis, R. V. Biomacromolecules 2005, 6 (6), 3095–9. (5) Blackledge, T. A.; Summers, A. P.; Hayashi, C. Y. Zoology (Jena) 2005, 108 (1), 41–6. (6) Xu, M.; Lewis, R. V. Proc. Natl. Acad. Sci. U.S.A. 1990, 87 (18), 7120–4. (7) Hinman, M.; Lewis, R. V. J. Biol. Chem. 1992, 267 (27), 19320– 19324. (8) Hayashi, C. Y.; Shipley, N. H.; Lewis, R. V. Int. J. Biol. Macromol. 1999, 24 (2-3), 271–5. (9) Simmons, A.; Ray, E.; Jelinski, L. W. Macromolecules 1994, 27 (18), 5235–5237. (10) Simmons, A. H.; Michal, C. A.; Jelinski, L. W. Science 1996, 271 (5245), 84–87. (11) Parkhe, A. D.; Seeley, S. K.; Gardner, K.; Thompson, L.; Lewis, R. V. J. Mol. Recognit. 1997, 10 (1), 1–6. (12) Asakura, T.; Ashida, J.; Yamane, T.; Kameda, T.; Nakazawa, Y.; Ohgo, K.; Komatsu, K. J. Mol. Biol. 2001, 306 (2), 291–305. (13) Asakura, T.; Yang, M.; Kawase, T.; Nakazawa, Y. Macromolecules 2005, 38, 3356–3363. (14) van Beek, J. D.; Hess, S.; Vollrath, F.; Meier, B. H. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (16), 10266–71. (15) Eles, P. T.; Michal, C. A. Biomacromolecules 2004, 5 (3), 661–5. (16) Eles, P.; Michal, C. A. Macromolecules 2004, 37, 1342–1345. (17) Yang, Z.; Liivak, O.; Seidel, A.; LaVerde, G.; Zax, D. B.; Jelinski, L. W. J. Am. Chem. Soc. 2000, 122 (37), 9019–9025. (18) Holland, G. P.; Lewis, R. V.; Yarger, J. L. J. Am. Chem. Soc. 2004, 126 (18), 5867–72. (19) Holland, G. P.; Jenkins, J. E.; Creager, M. S.; Lewis, R. V.; Yarger, J. L. Biomacromolecules 2008, 130 (30), 9871–77.

Solid-State NMR of Spiders’ Dragline Silk (20) Sapede, D.; Seydel, T.; Forsyth, V. T.; Koza, M. M.; Schweins, R.; Vollrath, F.; Riekel, C. Macromolecules 2005, 38, 8447–8453. (21) Bonthrone, K. M.; Vollrath, F.; Hunter, B. K.; Sanders, J. K. M. Proc. R. Soc. London, Ser. B 1992, 248 (1322), 141–144. (22) Hu, X. Y.; Lawrence, B.; Kohler, K.; Falick, A. M.; Moore, A. M. F.; McMullen, E.; Jones, P. R.; Vierra, C. Biochemistry 2005, 44 (30), 10020–10027. (23) Lawrence, B. A.; Vierra, C. A.; Moore, A. M. Biomacromolecules 2004, 5 (3), 689–95. (24) Bonev, B.; Grieve, S.; Herberstein, M. E.; Kishore, A. I.; Watts, A.; Separovic, F. Biopolymers 2006, 82 (2), 134–143. (25) Work, R. W.; Emerson, P. D. J. Arachn. 1982, 10 (1), 1–10. (26) Kaplan, D. L. Polym. Degrad. Stab. 1998, 59 (1-3), 25–32. (27) Bennet, A. E.; Rienstra, C. M.; Auger, M.; Lakshmi, K. V.; Griffin, R. G. J. Chem. Phys. 1995, 103 (16), 6951–6958. (28) Liu, Y.; Sponner, A.; Porter, D.; Vollrath, F. Biomacromolecules 2008, 9 (1), 116–21. (29) Gosline, J. M.; Guerette, P. A.; Ortlepp, C. S.; Savage, K. N. J. Exp. Biol. 1999, 202 (Pt 23), 3295–303. (30) Lombardi, S. J.; Kaplan, D. L. J. Arachn. 1990, 18 (3), 297–306. (31) Work, R. W.; Young, C. T. J. Arachn. 1987, 15, 65–80. (32) Andersen, S. O. Comp. Biochem. Phys. 1970, 35 (3), 705–711. (33) Holland, G. P.; Creager, M. S.; Jenkins, J. E.; Lewis, R. V.; Yarger, J. L. J. Am. Chem. Soc. 2008, 130 (30), 9871–7. (34) Izdebski, T.; Akhenblit, P.; Jenkins, J. E.; Yarger, J. L.; Holland, G. P. Biomacromolecules 2010, 11 (1), 168–174. (35) Jenkins, J. E.; Creager, M. S.; Lewis, R. V.; Holland, G. P.; Yarger, J. L. Biomacromolecules 2010, 11 (1), 192–200. (36) Jelinski, L. W.; Blye, A.; Liivak, O.; Michal, C.; LaVerde, G.; Seidel, A.; Shah, N.; Yang, Z. Int. J. Biol. Macromol. 1999, 24 (2-3), 197– 201. (37) Michal, C. A.; Jelinski, L. W. J. Biomol. NMR 1998, 12 (2), 231–41. (38) Taki, T.; Yamashita, S.; Satoh, M.; Shibata, A.; Yamashita, T.; Tabeta, R.; Hazime, S. Chem. Lett. 1981, 10 (12), 1803–1806. (39) Jaroniec, C. P.; MacPhee, C. E.; Astrof, N. S.; Dobson, C. M.; Griffin, R. G. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (26), 16748–53. (40) Kricheldorf, H.; Muller, D. Int. J. Biol. Macromol. 1984, 6 (3), 145– 51. (41) Ohgo, K.; Kawase, T.; Ashida, J.; Asakura, T. Biomacromolecules 2006, 7 (4), 1210–1214.

Biomacromolecules, Vol. 11, No. 8, 2010

2043

(42) Saitoˆ, H.; Tabeta, R.; Shoji, A.; Ozaki, T.; Ando, I.; Miyata, T. Biopolymers 1984, 23 (11), 2279–2297. (43) Liivak, O.; Flores, A.; Lewis, R. V.; Jelinski, L. W. Macromolecules 1997, 30, 7127–7130. (44) Asakura, T.; Yang, M.; Kawase, T. Polym. J. 2004, 36 (12), 999– 1003. (45) Holland, G. P.; Jenkins, J. E.; Creager, M. S.; Lewis, R. V.; Yarger, J. L. Chem. Commun. 2008, (43), 5568–5570. (46) Rathore, O.; Sogah, D. J. Am. Chem. Soc. 2001, 123, 5231–5239. (47) Marcotte, I.; van Beek, J.; Meier, B. Macromolecules 2007, 40, 1995– 2001. (48) Bell, F. I.; McEwen, I. J.; Viney, C. Nature 2002, 416 (6876), 37. (49) Bonthrone, K. M.; Vollrath, F.; Hunter, B. K.; Sanders, J. K. M. Proc. R. Soc. London, Ser. B 1992, 248 (1322), 141–144. (50) Dunford, H. B.; Morrison, J. L. Can. J. Chem. 1955, 33, 904–12. (51) Hirai, Y.; Ishikuro, J.; Nakajima, T. Polymer 2001, 42 (12), 5495– 5499. (52) Jelinski, L. W.; Blye, A.; Liivak, O.; Michal, C.; LaVerde, G.; Seidel, A.; Shah, N.; Yang, Z. Int. J. Biol. Macromol. 1999, 24 (2-3), 197– 201. (53) Savage, K. N.; Guerette, P. A.; Gosline, J. M. Biomacromolecules 2004, 5 (3), 675–9. (54) Sohn, S.; Strey, H. H.; Gido, S. P. Biomacromolecules 2004, 5 (3), 751–7. (55) van Beek, J. D.; Kummerlen, J.; Vollrath, F.; Meier, B. H. Int. J. Biol. Macromol. 1999, 24 (2-3), 173–8. (56) Grubb, D. T.; Jackrel, D.; Jelinski, L. W. Polym. Prepr. (Am. Chem. Soc., DiV. Polym. Chem.) 1997, 38 (2), 73–74. (57) Grubb, D. T.; Ji, G. Int. J. Biol. Macromol. 1999, 24 (2-3), 203–10. (58) Shao, Z.; vollrath, F.; Sirichaisit, J.; Young, R. J. Polymer 1999, 40 (10), 2493–2500. (59) Work, R. W. J. Exp. Biol. 1985, 118, 379–404. (60) Liu, Y.; Shao, Z. Z.; Vollrath, F. Biomacromolecules 2008, 9 (7), 1782– 1786. (61) Savage, K. N.; Gosline, J. M. J. Exp. Biol. 2008, 211 (12), 1937– 1947. (62) Savage, K. N.; Gosline, J. M. J. Exp. Biol. 2008, 211 (12), 1948–1957.

BM100399X