Properties of Synthetic Spider Silk Fibers Based on Argiope aurantia

May 6, 2008 - Spiders have evolved a complex system of silk producing glands. Each of the glands produces silk with strength and elasticity tailored t...
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Biomacromolecules 2008, 9, 1506–1510

Articles Properties of Synthetic Spider Silk Fibers Based on Argiope aurantia MaSp2 Amanda E. Brooks,*,† Shane M. Stricker,† Sangeeta B. Joshi,‡ Timothy J. Kamerzell,‡ C. Russell Middaugh,‡ and Randolph V. Lewis† Department of Molecular Biology, University of Wyoming, 1000 East University Avenue, Laramie, Wyoming 82070, and Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66045 Received October 9, 2007; Revised Manuscript Received March 11, 2008

Spiders have evolved a complex system of silk producing glands. Each of the glands produces silk with strength and elasticity tailored to its biological purpose. Sequence analysis of the major ampullate silk reveals four highly conserved concatenated blocks of amino acids: (GA)n, An, GPGXX, and GGX. While the GPGXX motif, which has been hypothesized to be responsible for the extensibility of the fiber, displays natural variation in its precise sequence arrangement and content, correlating these differences with particular fiber properties has been difficult. Three genetic constructs based on the Argiope aurantia sequence were engineered to progressively increase the number of GPGXX repeats in a head-to-tail assembly prior to interruption by another motif. Circular dichroism and Fourier transform infrared spectroscopy of synthetic spider silk spin dopes show secondary structures that correspond to an increase in the repeat number of GPGXX regions and an increase in the extensibility of synthetically spun recombinant fibers. Producing a designer high-performance fiber with a specific combination of strength and extensibility for medical, military, and commercial applications has continued to elude material scientists. Orb-weaving spiders, however, can produce an array of up to six high-performance silk fibers and a specialized glue.1 One of the six solid fibers, major ampullate dragline silk, is a molecular composite of two main proteins: MaSp1 and MaSp2.2,3 Each of the two main proteins polymerizes differently and makes a discrete contribution to the extreme toughness and moderate extensibility of the major ampullate fiber.4 The basis for such a contribution is not merely due to the overall amino acid composition of the proteins but, more importantly, lies in the molecular arrangement of the repetitive amino acid sequences of MaSp1 and MaSp2. The amino acids (such as alanine (A), glycine (G), and proline (P)) are organized in a series of concatenated motifs to give spider silk a balance of strength and extensibility5–7 tailored to its use. Each of four general motifs, polyalanine (An), polyglycine/alanine ((GA)n), GPGXX (X ) G, Q, Y, A, S), and GGX (X ) L, Y, S, A), appears to be associated with both structural and functional aspects of the fiber.5 Thus, manipulating the iterated amino acid block structure and multicomponent protein arrangement of major ampullate silk provides a method with which to investigate the underlying molecular architecture responsible for both strength and extensibility.6 Understanding the relationship between spider silk protein sequences and the function of the solid fiber is a multifaceted problem. First, the precise nature of the interaction and association of MaSp1 and MaSp2, both in the liquid crystalline * To whom correspondence should be addressed. Phone: (307) 766-6380. Fax: (307) 766-5098. E-mail: [email protected]. † University of Wyoming. ‡ University of Kansas.

state in the gland as well as in the fiber, is currently unknown; however, the presence of both proteins in the major ampullate fiber is well documented.2,3 Second, there are three levels of variability that affect a native spider silk fiber: (1) interspecies, which is predicated to be the greatest variable, (2) intraspecies or variability among individuals of the same species, and (3) individual or variation among fibers from the same individual.8 This variability can also manifest itself in multiple ways: (1) the specific amino acid sequence of the repetitive units, (2) the ratio of MaSp1 to MaSp2, and (3) the mechanical properties of the fiber.8–10 Historically, the basis for the prevailing correlations between amino acid sequence and the mechanical properties of the solid silk fibers have been established by studies of primarily Nephila claVipes silk fibers. However, Argiope aurantia silk fibers have slightly different repeat units (Table 1) as well as slightly altered mechanical behavior.9–11 Unfortunately, interspecific differences in the mechanical properties of dragline silk are obscured by complex and intrinsic fiber variability.8 Production of a synthetic version of spider silk holds the potential to diminish the variation seen in natural fibers, thus allowing the development of spider silk-based biomaterials. A key to comprehending how distinct functions can arise from a limited set of proteins lies in the molecular arrangement of the amino acid motifs and their subsequent impact on protein structure. By varying these arrangements, insights into the structure/function relationship of silk proteins should help to establish predictable patterns of protein behavior that can in turn be used to produce designer biomaterials. To explore the effect of altering the amino acid motif architecture on the mechanical properties of a solid silk fiber, a series of engineered Argiope aurantia MaSp2 based fibers were produced with increasing amounts of the Argiope aurantia specific GPGXX

10.1021/bm701124p CCC: $40.75  2008 American Chemical Society Published on Web 05/06/2008

Properties of Synthetic MaSp2 Silk Proteins

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Table 1a monomer unit

repeat times

DNA size (bp)

protein size (kD)

1E(GGYGPGAGQQGPGSQGPGSGGQQGPGGQ)1GPYGPSA8 2E(GGYGPGAGQQGPGSQGPGSGGQQGPGGQ)2GPYGPSA8 3E(GGYGPGAGQQGPGSQGPGSGGQQGPGGQ)3GPYGPSA8

16 12 8

2043 2547 2367

63 71 67

a Each monomer unit was doubled to give approximately 2 kb of DNA and to produce synthetic MaSp2 proteins. The exact sizes of the DNA constructs as well as the proteins are shown in the table. Note that protein molecular weights are shown without the presence of the N-terminal and C-terminal 6 histidine tag.

motif (Table 1). Each fiber was then evaluated for amino acid content, secondary structure, and altered mechanical properties.

Materials and Methods All chemicals used were obtained from Sigma Aldrich (St. Louis, MO) and all solutions were made with Milli-Q water unless otherwise stated. Cloning. Three different constructs were made based on the Argiope aurantia specific MaSp2 GPGXX motif amino acid sequence (GGYGPGAGQQGPGSQGPGSGGQQGPGGQ). The number of concatenated motifs was increased sequentially (i.e. 1E ) 1, 2E ) 2, 3E ) 3) prior to interruption by a linker (GPYGPS) and a polyalanine motif (A8) to produce the series of constructs. Two synthetic genes were synthesized by Midland Reagents (Supporting Information) to generate monomer units (Table 1) using a compatible nonregenerable cloning strategy,12 which permitted for the incorporation of both N-terminal and C-terminal histidine tags. All restriction enzymes used for the cloning strategy were purchased from New England Biolabs (Beverly, MA). Protein Expression and Purification. Each of the final three constructs: 1E × 16, 2E × 12, and 3E × 8 were expressed from pET30a in BL21DE3 (Novagen, Madison, WI) E. coli in a 10 L fermentorscale culture at 37 °C. The culture was permitted to reach an OD600 of approximately 1, upon which IPTG (isopropyl-beta-D-thiogalactopyranoside; Biosynth AG, Switzerland) was added to a 1 mM final concentration. After induction, the culture was allowed to produce the synthetic silk protein for 2.5 h, at which time the cells were harvested by centrifugation at 5500 rpm for 20 min. Cell pellets were resuspended in a 3:1 weight-to-volume ratio of binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris (Fisher Chemicals, Fair Lawn, NJ), pH 7.9) and frozen at -80 °C until purification. To purify the silk-like protein of interest, the terminal histidine tags of the protein were captured using nickel affinity chromatography (Ni-NTA resin (Novagen)). The manufacturer’s protocol was utilized with the following modifications. The cells were lysed with 0.5% lysozyme, 0.01% DNase, and 2% deoxycholate acid sodium salt monohydrate (MP Biomedicals, Aurora, OH). Additionally, to ensure complete lysis, the resuspended cells were also sonicated (Misonix Sonicator 3000) for approximately five minutes. The soluble and insoluble fractions were separated by centrifugation (9500 rpm for approximately 10 min). Protein in the soluble fraction was allowed to bind to the Nickel affinity resin for approximately 30 min on an orbital shaker (S500, VWR, West Chester, PA). To compete with nonspecific resin interactions, a 30 mM imidazole wash (30 mM imidazole (Fisher Chemicals), 0.5 M NaCl, 20 mM Tris, pH 7.9) followed by a 60 mM imidazole wash (60 mM imidazole, 0.5 M NaCl, 20 mM Tris, pH 7.9) were used. The strip fraction (100 mM EDTA, 0.2 M Tris, 0.5 M NaCl) relied on the chelating affects of EDTA (Fisher Chemicals) to elute the altered MaSp2. This fraction was dialyzed against Milli-Q water using a stirred cell with a 30 K membrane (Millipore Amicon, Billerica, MA). SDS-PAGE and Western Blot Analysis. All SDS PAGE gels were run in the recommended buffer using precast 4-20% gradient gels (iGELS, Life Therapeutics, Clarkston, GA) at 100 V for approximately 1 h. Coomassie staining with Bio-Safe Coomassie (Bio-Rad, Hercules, CA) allowed the proteins to be visualized. A dual color protein standard (Bio-Rad, Hercules, CA) was included on all gels. A standard Western blot protocol was followed. Briefly, the proteins were transferred onto a nitrocellulose membrane (Pierce, Rockford, IL). The efficiency of

the transfer was assessed using Ponceau (0.5% Ponceau S (Mallinckrodt, Paris, KY), 0.1% acetic acid (EMD, San Diego, CA)) staining. The blot was then blocked using 5% dry milk TBS Tween (TBST, 0.24% Tris, 0.8% NaCl, 0.05% Tween 20 (Calbiochem, San Diego, CA)) solution after which it was probed with a primary antihistidine antibody (1:5000 dilution in TBST) (Novagen) and a secondary HRP conjugated antimouse antibody (1:5000 dilution) (Promega, Madison, WI). All washes were done with TBST. Following reaction with ECL (enhanced chemiluminescence, Amersham, Picataway, NJ) according to the manufacturer’s protocol, the blot was exposed to film (Super RX FujiFilm, Japan) for up to 10 min and developed according to standard protocols. Marks were made on the blot prior to exposure to film. These marks were subsequently used to align the nitrocellulose to the X-ray film. The blot was scanned with the nitrocellulose behind it to visualize the ladder, which cannot normally be visualized on the X-ray film exposure. Amino Acid Analysis. A small sample of each protein was submitted to Dr. Michael Hinman in our laboratory at the University of Wyoming for amino acid analysis. Following complete acid hydrolysis with 6 M HCl at 155 °C, the proteins were fluorescently labeled using a Waters AccQ Fluor Reagent Kit (Millipore, Milford, MA) by the manufacturer’s instructions and then analyzed by HPLC (L-6200 Hitachi Fluorometer). Fiber Spinning and Mechanical Testing. Following SDS-PAGE and Western blot analyses, the proteins were lyophilized to dryness (Labconco Centrivap). Spin dopes were made using 1,1,1,3,3,3hexafluro-2-propanol (HFIP; TCI America, Portland, OR) to dissolve the lyophilized protein and make a 10-12% solution. The solution was extruded from a 1 mL Hamilton syringe (Hamilton, Reno, NV) through 0.005 in. Peek tubing (VWR) at a syringe plunger speed of 2 mm/min into an isopropanol coagulation bath. The Peek tubing was positioned so that the tip just broke the surface tension of the isopropanol coagulation bath. The fiber was then mounted in either 5.5 or 2.5 cm lengths on a mechanical testing card.13 Each fiber was viewed under a Nikon Eclipse E200 microscope at 40× magnification and measured at five different places along the fiber (Image J 1.32) then averaged to determine the diameter of each fiber. Subsequently, every sample was tested on an MTS Synergie 100 (MTS corporation, Eden Prairie, MN) using a custom-built 10 g load cell (Transducer Techniques, Temecula, CA) in a simple tension test at a rate of 2 mm/min. The data was plotted in Excel (Mac V11.2) and a polynomial regression curve was fitted to reflect the highest possible R2 with the lowest degree polynomial. Oneway ANOVA was used to compare fibers from the different constructs to look for significant differences in stress, strain, and Young’s modulus. Circular Dichroism Studies. CD studies were performed with a Jasco-810 spectrophotometer equipped with a Peltier temperature controller to determine the secondary structure of silk protein constructs. Protein stock solutions were prepared at 1 mg/mL in hexafluoroisopropanol (HFIP) and were diluted 5-fold in PBS for the CD studies (final concentration of 20% HFIP). The concentration of the protein constructs was measured by UV absorption spectroscopy (A280 nm, E1cm0.1% ) 0.646, 0.645, and 0.612 for 1EX16, 2EX12, and 3EX8, respectively). Far UV spectra of proteins (0.2 mg/mL) were collected at 25 °C in the range of 195-260 nm using a 0.1 cm path length cuvette sealed with a Teflon stopper. A scanning rate of 15 °C/hr and a scanning speed of 20 nm/min with a 1 nm resolution were employed. Each protein was scanned in triplicate and averaged by the computer software.

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Figure 1. (A) Biosafe Coomassie stained SDS PAGE (polyacrylamide gel electrophoresis). A 4-20% gradient gel was run. Each construct is represented by two lanes. The first is a prepurification sample; whereas, the second sample is after nickel affinity purification (1E×16, 2E×12, 3E×8 (left to right.)). (B) Western blot of each of the three constructed proteins (right to left) BSA was included on the far left as a negative control. The blot was probed with an R-His antibody and exposed to film for 10 min.

Figure 2. Polynomial regression curves18–20 for 1E×16 (9 fibers tested, gray), 2E×12 (11 fibers tested, black), 3E×8 (7 fibers tested, light gray). The overall ratio of polyalanine is decreased as the number of GPGXX motifs increases.

The CD signals were converted to molar ellipticity using the Jasco Spectra Manager software.

Results Each of the sequences described above (Table 1) was cloned and verified by sequencing, and the protein was subsequently produced in BL21-DE3 E. coli (Figure 1). Multiple positive clones were obtained for each sequence. Although proteins were produced from each positive clone, only those clones that exhibited the highest synthetic protein yield were used for all subsequent analysis and experiments. Following purification, dialysis, and lyophilization, the identity of each protein was initially confirmed by amino acid analysis (Table S1), and the effectiveness of the engineering was evaluated by assessing the mechanical properties of the synthetic fibers (Figure 2, Figure S1, Table 2). Native spider silk proteins are maintained as an aqueous solution at very high concentrations in secretory glands (20-30%); however, after the natural MaSp1 and Masp2 proteins are spun into a solid fiber, they display very limited solubility. Similarly, the synthetic silk proteins manifest very limited solubility after lyophilization. Therefore, dissolving the proteins in HFIP made synthetic spin dopes and fibers were spun as described in Materials and Methods. Notably, post spin

draw techniques were not used in this study because we were interested in the properties of “as-spun” fibers. Multiple fibers from each construct were tested (9 1E×16 fibers, 11 2E×12 fibers, and 7 3E×8 fibers). Fiber diameters varied between 60 and 101 µm, having an average diameter of 83.85 µ (( 18.6 µ) for 1E×16, between 36 and 105 µm, having an average diameter of 74.1 µ (( 33.9 µ) for 2E×12, and between 45 and 142 µm, having an average diameter of 72.6 µ (( 34.2) for 3E×8. Comparing the stress/strain curves from each of the resulting fibers shows that the mechanical properties are altered by modifications in the amino acid motif composition of the synthetic MaSp2 fibers (Figure 2, Table 2). However, one-way ANOVA only reveals significant differences between the strain capacity for 1E×16 or 2E×12 fibers and those form 3E×8. Variation is well documented to be an inherent property of natural fibers;8 however, the source of synthetic fiber variation (as reflected by the R2 values of the polynomial regressions) is unclear. Additionally, synthetic fibers in general lack the yield point that characterizes native fibers,10 which may reflect dramatic differences in fiber processing (i.e., spinneret design, microenvironment, and the fundamental difference between synthetic fiber extrusion versus natural fiber drawing). Polynomial regression curves in Figure 2 were separated into their component data series and the data from each fiber plotted individually. Analysis of data sets from each tested fiber revealed that, analogous to native silk fibers,10 the individual data sets segregated into three distinct clusters based on mechanical behavior. Subsequent polynomial regression curves of these clusters show better fit, as shown by the R2 values (Figure S1). Notably, the 1E×16, which show a yield point (stress ) 4.6 × 107, strain ) 2.6), fibers segregated almost equally into the high strength and high extensibility clusters, while the 2E×12 fibers segregated into 40% high extensibility, 40% high strength, and 20% moderate strength and extensibility. The 3E×8 fibers had a distribution of 50% high extensibility, 25% high strength, and 25% moderate strength and extensibility. Circular dichroism spectra of synthetic silk protein solutions show that as the number of “GPGXX” units increase there is a corresponding increase in type II β-turns (Figure 3), as reflected by an increase in the characteristic type II β-turn minima near 230 nm. Such an increase is consistent with an increase in elasticity (Figure 2). Organic solvents such as HFIP are known to promote formation of R-helical secondary structure.16,17 Because use of HFIP for synthetic protein spin dope was necessitated by the low solubility of the synthetic silk proteins,

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Table 2a stress (Pascal) 1E×16 2E×12 3E×8 a

6.60 × 10 ( 5.1 × 10 4.95 × 107 ( 7.8 × 106 1.93 × 106 ( 2.4 × 106 6

6

strain (%)

Young’s modulus (Pascal)

1.5 ( 0.3 3.6 ( 2.6 19 ( 2.2

4.47 × 106 ( 3.7 × 106 4.40 × 107 ( 3.1 × 107 3.68 × 104 ( 4.1 × 104

Summary of the average and standard deviation for the stress, strain, and Young’s modulus for 1E×16, 2E×12, and 3E×8.

Figure 3. Average CD spectra for three recombinant silk proteins in 10 mM PBS: 1E×16 (green), 2E×12 (purple), 3E×8 (red). As the number of “GPGXX” specific motifs is increased prior to interruption by the linker and polyalanine domains, the percentage of type II β-turn secondary structure increases, as evidenced by the decrease (in intensity) of the minima between 225 and 230 (nm).

it is thus important to evaluate the structure of the protein in HFIP. Therefore, the structure of the proteins was also evaluated in HFIP using FTIR (Table S2). Generally, the FTIR spectra show an increase in β- turns as the number of concatenated MaSp2 motifs is increased from 1 to 3, although a significant amount of R-helix is also detected (Table S2).

Discussion There are currently over 37000 known species of spiders;21 however, current studies that (1) characterize spider silk proteins and fibers and (2) establish correlations between the primary structure of spider silk proteins and the mechanical function of the fiber have been limited mostly to Nephila claVipes silk fibers. Argiope aurantia major ampullate silk fibers, which exhibit slightly different mechanical properties,10,11 have a MaSp2 repeat unit with distinct sequence features.9 One unique component of the Argiope aurantia GPGXX motif is exactly one highly conserved GGY motif that preludes the main GPGXX motif in each repeat unit (Table 1). This is not surprising because one of the general characteristics of an elastomeric protein is the presence of a nonelastic domain (GGY and An) that could potentially form intermolecular cross-links necessary for fiber strength.22,23 This characteristic of elastomeric proteins motivated us to explore the impact of engineering an Argiope aurantia MaSp2 based sequence, which increased the number of iterated “GPGXX” motifs (concurrently decreasing the molar percent of alanine, i.e. 20% for 1E×16, 14% for 2E×12, 11% for 3E×8) on the strength of the fiber. Although the amount of alanine shows a consistent decline, which is expected to correspond with decreased fiber strength, it was unknown whether a decrease in the molar percent of alanine would result in a corresponding linear reduction in strength. With three elastic repeats, the fiber shows only a third of the

strength of a fiber with one or two repeats (qualitatively this difference is greater); concurrently, it shows approximately 8× more strain to break (Figure 2). There are no statistically significant differences between one elastic repeat and two elastic repeat according to one-way ANOVA because of the large standard deviations; however, despite evidence of a qualitative difference when considering a visual comparison of the stress/ strain curves produced by these two different fibers, the differences between fibers produced with one elastic repeat versus two elastic repeats are not dramatic and could indicate a thresh hold of glycine proline content that must be reached to see a dramatic difference in the fiber.15 Notably, natural Argiope aurantia major ampullate fibers have an average strength of approximately 2 orders of magnitude greater and an average strain to break approximately 2× greater than the best synthetic fibers. Interestingly, 1E×16 clones had a lower yield (Figure 1). This large discrepancy could be explained by a combination of (1) the presence of both MaSp1 and MaSp2 proteins in the natural fibers, (2) the larger size of native spider silk proteins, (3) the greater effectiveness of the natural silk spinning apparatus, and (4) the natural structure of the silk protein in solution and the subsequent conversion to a natural solid phase structure.23,24 Due to the large variation between fibers, quantitative comparisons are estimates at this stage, but they clearly suggest that the relationship between the molar percent alanine and the fiber strength is not linear. It should be noted that natural spider silk fibers also show a large amount of variation.8 Variation can best be modeled by considering the best fit polynomial regression analyses, which also allows distinct clusters of fibers to be identified from both synthetic (Figure S1) and natural fibers.10 Although all fibers in this study were spun in the same manner, the lack of a post spin draw process and slightly

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changing ambient conditions could have introduced a element of variability; thus, much of the variation in synthetic fibers should be eliminated after the histidine tags are cleaved off the synthetic silk proteins and as the spinning process is further refined by adding steps such as postspin draw, which are known to decrease the diameter and variability (Costas Karatzas, Nexia Biotechnology, personal communication). Despite variation in the diameters and mechanical properties of the fibers, comparisons of secondary structure abundance do show an increase in type II β-turn structure as the number of iterated GPGXX motifs increases. In addition, intermolecular β-sheets start to appear when the GPGXX module is doubled prior to interruption by an intervening motif (Table S2, Figure 3). This is somewhat counterintuitive because the numbers of polyalanine motifs, which are known to form β-sheet structure, have decreased, while the number of the GPGXX modules has concurrently increased. However, recent evidence has suggested that glycine and proline in combination promotes a stabilized yet self-assembling elastomeric structure.25 Thus, the conformation and solubility of natural and synthetic silk proteins are partially a result of the increased percent of glycine and proline.14,15 Consequently, this increase in β-structure may be due to conformational changes that produced increased flexibility and subsequently enhanced turn content. These tendencies in secondary structure suggest that future work is needed to address not only the change in secondary structure abundance but, more importantly, should consider the precise structural organization of the synthetic fibers using nuclear magnetic resonance (NMR). The results presented here confirm that engineering the amino acid sequence can alter the mechanical properties of a synthetic silk fiber. This study also reveals that increases in extensibility (as a result of engineering) are not linear and raises some interesting questions regarding the precise relationship between amino acid sequence and mechanical properties. Consequently, the mechanical properties of major ampullate silk (dragline) cannot be solely attributed to divergent silk protein sequences. There must also be an element of fiber production that contributes to the material properties and, hence, the inter- and intraspecies variation. Such possibilities will continue to be investigated during solution-phase studies to consider the pH and composition of the spin dope. Acknowledgment. This study was supported with grants from the NIH, NSF, and Department of Defense.

Brooks et al.

Supporting Information Available. Additional methods and data. This material is available free of charge via the Internet at http://pubs.acs.org.

References and Notes (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25)

Vollrath, F. Sci. Am. 1992, 70. Xu, M.; Lewis, R. V. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 7120. Hinman, M. B.; Lewis, R. V. J. Biol. Chem. 1992, 267 (27), 19320. Sponner, A.; Unger, E.; Grosse, F.; Weisshart, K. Nat. Mater. 2005, 4, 772. Hayashi, C. Y.; Lewis, R. V. Science 2000, 287, 1477. Hayashi, C. Y.; Shipley, N. H.; Lewis, R. V. Int. J. of Biol. Macromol. 1999, 24, 271. Gatsey, J.; Hayashi, C.; Motruik, D.; Woods, J.; Lewis, R. V. Science 2001, 291, 2603. Madsen, B.; Shao, Z. Z.; Vollrath, F. Int. J. Biol. Macromol. 1999, 24 (2-3), 301. Brooks, A. E.; Steinkraus, H. B.; Nelson, S. R.; Lewis, R. V. Biomacromolecules 2005, 6 (6), 3095. Brooks, A. E.; Brothers, T. J.; Creager, M. S.; Lewis, R. V. J. Appl. Biomater. Biomech. 2007, 5 (3), 158. Lewis, R. V.; Hinman, M.; Kothakota, S.; Fournier, M. J. Protein Expression Purif. 1996, 7 (4), 400. Brooks, A. E.; Creager, M. S.; Lewis, R. V. Biomed. Sci. Instrum. 2005, 41, 1. Stanger, H. E.; Gellman, S. H. J. Am. Chem. Soc. 1998, 120, 4236. Raucher, S.; Baud, S.; Miao, M.; Keeley, F. W.; Pomes, R. Structure 2006, 14 (11), 1667. Hirota, N.; Mizuno, K.; Goto, Y. Protein Sci. 1997, 6, 416. Roccatano, D.; Fioroni, M.; Zacharias, M.; Colombo, G. Protein Sci. 2005, 10, 2582. 1E×16: y ) 26855x6-672028x5 + 6E+06x4-3E+07x3 + 5E+07x27E+06x + 2E+06. 2E×12: y ) y ) 1E+06x6-3E+07x5 + 1E+08x4-1E+08x3 + 8E+07x2-3E+06x + 2E+06. 3E×8: y ) 2.6893x6-139.6x5 + 2310.2x4-9686.2x3-83388x2 + 765805x + 555601. Coddington, J. National Geographic. http://news.nationalgeographic. com/news/2004/06/0624_040624_tvspider.html (accessed June, 2004). Suzuki, Y.; Matsuoka, T.; Iimura, Y.; Fujiwara, H Insect Biochem. Mol. Biol. 2002, 32 (6), 599. Asakura, T.; Suita, K.; Kameda, T.; Afonin, S.; Ulrich, A. S. Magn. Reson. Chem. 2004, 42 (2), 258. Lefevre, T. Biomacromolecules 2007, 8 (8), 2342. Dicko, C.; Knight, D.; Kenney, J. M.; Vollrath, F. Biomacromolecules. 2004, 5 (3), 758. Trevino, S. R.; Schaefer, S.; Schooltz, J. M.; Pace, C. N. J. Mol. Biol. 2007, 373 (1), 211.

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