Biomacromolecules 2005, 6, 1405-1413
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Effects of Electrospinning and Solution Casting Protocols on the Secondary Structure of a Genetically Engineered Dragline Spider Silk Analogue Investigated via Fourier Transform Raman Spectroscopy Jean S. Stephens,*,†,‡ Stephen R. Fahnestock,§ Robin S. Farmer,† Kristi L. Kiick,† D. Bruce Chase,§ and John F. Rabolt*,† Department of Materials Science and Engineering and Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19716, and Dupont Central Research and Development, Wilmington, Delaware 19780 Received November 5, 2004; Revised Manuscript Received February 23, 2005
Micrometer and submicrometer diameter fibers of recombinant dragline spider silk analogues, synthesized via protein engineering strategies, have been electrospun from 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and compared with cast films via Raman spectroscopy in order to assess changes in protein conformation that may result from the electrospinning process. Although the solvent casting process was shown to result in predominantly β-sheet conformation similar to that observed in the bulk, the electrospinning process causes a major change in conformation from β-sheet to R-helix. A possible mechanism involving electric field-induced stabilization of R-helical segments in HFIP solution during the electrospinning process is discussed. Introduction Dragline spider silk is one of the strongest protein materials known, yielding properties that are comparable by weight to high-strength steel.1 The basis for this lies in part in the amino acid sequence of the silk fibroin (silk protein), which imparts block copolymer-like behavior to the protein: hard segments form a crystalline structure that is embedded in an elastic matrix.2 In dragline spider silk, the hard segment is a highly crystalline β-sheet formed by an alanine- (A-) rich portion of the sequence while the elastic region is composed of a glycine- (G-) rich region of the sequence.3 The chain conformation of the elastic region is not clearly defined but probably consists of disordered, R-helical, and β-sheet conformations. Recent studies have shown the presence of R-helical conformation in the elastic region with some portions being incorporated into β-sheet structures.4 Figure 1 illustrates the proposed dragline fibroin superstructure (hard/soft segment organization) and also shows the amino acid sequences for consensus repeats of the dragline spider silk fibroins, spidroins 1 and 2. Dragline spider silk has been studied for decades and has been proposed for use in a wide variety of applications, such as biomedical materials (i.e., tissue engineering, sutures, wound dressings) and protective apparel (body armor).4,5 The major drawback for the use of dragline spider silk in commodity applications is that, unlike silkworms, which can * Corresponding authors: e-mail (J.S.S.)
[email protected]; (J.F.R.)
[email protected]. † Delaware Biotechnology Institute. ‡ Present address: 100 Bureau Dr., Stop 8543, Polymers Division, NIST, Gaithersburg, MD 02899. § Dupont Central Research and Development.
Figure 1. Dragline spider silk fibroin amino acid sequences and schematic of conformational structure. Adapted from ref 3.
produce large quantities of silk, spiders are not as readily domesticated.5 Over the past decade and longer, an increasing number of investigators have endeavored to overcome this difficulty via the cloning and expression of both natural and synthetic spider dragline silk genes in a variety of expression hosts.6-14 These investigations have resulted in the production of a variety of silk-like proteins of academic and commercial interest, with sequences that capture many of the properties of natural silk and/or that permit manipulation of the assembly of the hard segments. Of most direct relevance to the studies reported here, Fahnestock and co-workers at Dupont have created analogues of dragline silk of the golden orb weaver (Nephila claVipes).15,16 Artificial genes were designed to encode amino acid sequences that mimic the N. claVipes spidroin 1 (DP-1) and spidroin 2 (DP-2) and were then incorporated into bacteria and yeast expression hosts for production of dragline silk analogue proteins that have the amino acid sequences listed in Figure 2. X-ray diffraction (XRD) characterization of the solution spun fibers of spidroin 1 silk analogue (DP-1) in HFIP and subjected to postspin draw have a larger Bragg spacing than the natural silk (7.5 Å as compared to 5.3 Å, respectively).3 This may be a result
10.1021/bm049296h CCC: $30.25 © 2005 American Chemical Society Published on Web 04/16/2005
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Biomacromolecules, Vol. 6, No. 3, 2005
Figure 2. Silk analogue amino acid sequence.
of part of the soft segment crystallizing in conjunction with the hard segment and therefore resulting in an expanded β-sheet region. The increase in Bragg spacing could alternatively indicate the induction of an alternate crystalline polymorph, that of Silk I, where one amino acid adopts nearly an R-helical conformation and another adopts nearly a β-sheet conformation, creating a crankshaft conformation.17 The increased Bragg spacing of the DP-1 silk analogue fibers, as spun from HFIP solution, may therefore suggest that the presence of alternative crystalline structures contributes to the observed decreased strength and increased tensile modulus of fibers of the recombinant protein as compared to the natural silk protein. These previous results illustrate the known importance of chain conformation and processing methods in controlling the properties of polypeptide-based materials. The continued development of polypeptide-based materials for applications in both biomedical and nanotechnology arenas has increased the importance of developing a detailed understanding of the impact of processing parameters on the resulting structures of these materials. It has been known for decades that the secondary structure of various polypeptides (e.g., homopolypeptides and block-co-polypeptides) can be altered under conditions of high stress, changes in pH, and as a result of heating or cooling,18,19 conditions that are likely to occur during the processing of proteins for application purposes. However, polymer conformational changes that result during fiber formation via electrospinning have not been particularly widely studied, and investigations to elucidate potential electrospinning-induced conformational changes are needed. The electrospinning process is a fiber formation method that can readily produce continuous nanometer-diameter fibers, with an average fiber diameter of 100-500 nm. Electrospinning utilizes electrostatic forces to generate the fibers from polymer solutions or melts.20 The electrospinning apparatus generally consists of a syringe/needle or pipet, which is filled with a polymer solution or melt, and a highvoltage, low-current power supply. A charge is applied to the solution through an electrode attached to the high-voltage power supply (0-30 kV). At a critical voltage, referred to as the starting voltage, the charge overcomes the surface tension of the solution, resulting in the formation of an electrically charged jet (electrospinning jet).21-23 The jet is accelerated to a counterelectrode that is a set distance away, where the fibers are collected as a nonwoven fibrous membrane. The counterelectrode is generally grounded but can be held at a charge opposite to that of the solution.
Stephens et al.
Electrospinning requires only a small amount of polymer (
