Structural Evolution of Genetically Engineered Silklike Protein

Chapter 12 Structural Evolution of Genetically Engineered Silklike Protein Polymers 1

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J. Philip Anderson , Matthew Stephen-Hassard , and David C. Martin

Downloaded by MONASH UNIV on October 29, 2012 | http://pubs.acs.org Publication Date: December 3, 1993 | doi: 10.1021/bk-1994-0544.ch012

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Macromolecular Science and Engineering Center and Department of Materials Science and Engineering, College of Engineering, University of Michigan, Ann Arbor, MI 48109-2136 The effect of processing on the evolution of crystal structure and morphology in a silk-like protein polymer, SLPF, were examined by transmission electron microscopy. A schematic ternary phase diagram was developed for the SLPF, water and formic acid system. Polymer swelling by solvent vapor was monitored by wide angle x-ray scattering. The disruption of the SLPF crystalline structure occured in steps consistent with the Lotz and Keith crankshaft model of Silk I.

Via recombinant DNA technology, designed protein polymers can be produced to obtain specific physical, chemical and biological properties of interest (7). These materials incorporate functional sequences which are modeled after segments of existing natural proteins. The synthesis of well-defined macromolecules by molecular biological techniques has been used to produce a new class of materials which is rapidly expanding (2-4). For the optimal application and future design of these protein polymers it necessary to understand the relationship between processing, structure and function. SLPF (silk-like polymer with fibronectin cell attachment functionality) is one such protein polymer, commercially designed and produced by Protein Polymer Technologies, Inc. for general use as a stable promoter of cellular attachment to a variety of surfaces. The amino acid sequence of SLPF is shown in Figure 1 by single character code (5). SLPF can be used to coat artificial surfaces like polystyrene culture dishes to promote both the adhesion and spreading of cells (6-7). To optimize performance for a variety of applications in vitro and in vivo, it is of interest to determine the influence of crystal structure and morphology on the biological and physical properties of SLPF. The utility of SLPF depends on generating autoclavible and biocompatible crystalline polymorphs and morphologies which have the ability to present cell attachment sites in an accessible manner. The crystalline structure of natural silk has been explored by x-ray diffraction (8-10). In an attempt to gain more information on the fiber processing in vivo, the silk producing glands from Bombyx mori were air dried and on x-ray analysis they were found to have a noticeably different crystal structure from the fibrous silk normally extruded from the insect (77). The unextruded protein structure (Silk I) was found to be easily drawn into the extruded polymorph, Silk Π. Lotz and Keith proposed an orthorhombic unit cell for Silk I [space group 1^2^ (a = 0.472 nm, b = 1.44 nm, 0097-6156/94/0544-0137$06.00/0 © 1994 American Chemical Society In Silk Polymers; Kaplan, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


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SILK POLYMERS: MATERIALS SCIENCE AND BIOTECHNOLOGY

c = 0.96 nm)] from electron microscopy, selected area electron diffraction and x-ray powder diffraction experiments on single crystals of poly(glycine-alanine) (72). The Lotz and Keith model for Silk I is a compact sheet form with die polypeptide backbone in a crankshaft conformation. The molecules pack in the unit cell so that the (020) distance between hydrogen-bonded sheets is on the order of 0.74 nm (Figure 2). To provide a model consistent with the antiparallel character observed in extended forms similar to Silk I, die complete crankshaft model consists of sheets composed of the antiparallel chains, in a regular alternating pattern. As in the extended β pleated sheets, Silk I crankshaft sheets stack to fill space. The face of the resulting solid, perpendicular to the chains, is thought to be composed of regular hairpin turns, where the chains switch direction and proceed back through the crystal. Studies of the crystal structure and morphology of SLPF films are described in more detail elsewhere (73). Dry SLPF powder and thin films were found to be consistent with the crankshaft model for Silk I. The fundamental component of SLPF films are whisker-shaped crystallites which have a nearly monodisperse width (12 nm +/- 2 nm) and variable lengths. These whisker crystallites associate into larger aggregates which are termed sheaves. Here we describe how crystallinity and morphology of SLPF varies with processing conditions. This research is focused on the structural evolution of genetically engineered protein polymers during processing into films and fibers. Currently, the manner in which higher order structure develops from folding and cooperative association in macromolecular systems is a central theme of biophysics and materials science. Because of the fidelity of control which it is possible to exert with the genetic methods of polymer synthesis we expect these systems to provide access to a variety of unanswered questions concerning the nature of self-organization and phase stability of condensed macromolecules. Experimental SLPF (batch S4) was supplied by Protein Polymer Technologies, Inc. in powder form (ProNectin F). The theoretical sequence, predicted genetically by die synthetic nucleotide sequence which encodes SLPF, is shown in Figure 1 by the single character amino acid code (5) and corresponds to a molecular weight of 73 kDa. The segmented copolymer was produced by recombinantly engineered E. coli cells, then purified from other cellular components by a process involving the extraction of the lysate with 5M LiBr solution. The protein was precipitated using ammonium sulfate, washed, and dialyzed against deionized water. The insoluble SLPF suspension was then lyophilized to a dry powder. Transmission electron microscopy (TEM) and wide angle x-ray scattering (WAXS) were used in conjunction with solubility and swellabUity experiments to obtain basic information relevant to the processing of SLPF. All experiments were performed at room temperature. T E M . Sample preparation for TEM involved carbon coating freshly cleaved mica sheets to form a continuous amorphous film in a Denton evaporator. Formic acid (9597%) was purchased from Aldrich Chemical Co. and used as received. Solutions containing typically one mg of SLPF per ml of formic acid were atomized and sprayed onto the carbon film. The carbon coated mica, covered with solution droplets, was left in a laboratory hood. Upon solvent evaporation, which occurred in seconds, crystallized droplets of SLPF were left on the surface of the carbon film. This film was then floated off the mica onto deionized water. Submerged 400 mesh copper TEM grids were lifted with tweezers to capture SLPF droplet-covered carbon film. The samples were examined with a Phillips EM 420 operating at 120 kV. A variety of magnifications were used, typically 3 kX to 20 kX.

In Silk Polymers; Kaplan, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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{HEAD}- [(Silk-Like) Fibronectin Segment] -{TAIL} 9

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{(fMJDPVWjQRR^^ -[(GAGAGS) G AAVTGRGDSPAS AAGY] 9

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{(GAGAGS) G AG AMDPGRYQLS AGRYHYQLVWCQK} 2


Figure 1. Amino acid sequence of SLPF showing the design which consists of a head, crystallizable center, and tail. Here we use the single-letter amino acid code (5). G, A, and S code for glycine, alanine, and serine, the principal amino acids in crystalline regions of Bombyx mori silk.

[100] Projection

[001] Projection

(020)

planes:

h y d r o g e n - b o n d e d sheet p a c k i n g

Figure 2. Schematic diagram of the Lotz and Keith crankshaft model for Silk I shown in the [100] and [001] crystallographic projections. The crankshaft conformation of the chain is evident in the [100] projection. The molecules hydrogen-bond face to face, with the result that the spacing between the hydrogen bonded sheets (i.e., the (020) spacing) is on the order of 0.7 nm. In Silk Polymers; Kaplan, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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Variations in polymer concentration and water content of the solutions lead to a variety of SLPF morphologies and degrees of crystallinity. The nature and extent of SLPF crystallinity was estimated by observation and distinction of SLPF whisker crystallites and sheaf structures. Other morphological features of the films were observed as described in more detail below.


Solubility and Swelling. SLPF on 0.5 mm etched glass WAXS sample holders were exposed to a variety of atmospheres produced by a range of water and formic acid mixtures. Dry SLPF power was held above a 50 ml formic acid/water solution, in a closed petri dish. Solution composition was variedfrom0 to 100% formic acid. The duration of swelling by solvent vapor was 24 hours. Weight gain of the samples was monitored with a Mettler AE analytical balance, while structural changes were assayed by WAXS. The solubility of SLPF in mixtures of water and formic acid was examined by adding increasing amounts of polymer until precipitation occurred. WAXS. A Rigaku Geigerflex theta-theta x-ray system was used to perform WAXS on dry SLPF powder and solvent-swollen films. The instrument was equipped with a 1.5 kW Cu Κα tube which emits x-ray radiation at a wavelength of 0.154 nm, a graphite monochromator and a Nal scintillation type detector. Detailed WAXS runs were conducted at 0.5 degrees per minute with sampling at 0.05 degree intervals. The samples are held horizontally in the instrument with both the source and detector moving during the WAXS experiment, making it possible to analyze the microstructure during transitions from swollenfilmsand solutions to dry powder and vice versa. Results and Discussion Part of a ring droplet with an interior consisting of plane oriented whiskers and a periphery of radially oriented sheaves shown in Figure 3. The whisker crystallites create a patchwork structure in the droplet interior. Immediately beyond the sheaves at the edge of the droplet there was an outer ring of apparently amorphous material. The lack of contrast between the whiskers and their matrix supports the idea that uncrystallized material is surrounding these well crystallized whiskers. Inringdroplets like that shown in Figure 3, there is often less SLPF in the center then at the periphery. There is further evidence for semi-crystallinity in SLPF droplets. The photomicrograph in Figure 4 shows regions entirely devoid of SLPF, indicated by the lightest contrast. The slightly darker patches are believed to be amorphous SLPF. Crystallization consistent with dense whisker nuclei is found only in the thickest parts of the film. Shown in Figure 5 is a highly crystalline film in which the sheaf morphology is well developed. This film is reminiscent of the spherulitic texture commonly seen in thin films of crystallizable synthetic polymers (14) and previously observed in natural proteins (15). This data indicates that synthetic and natural proteins behave in some manners consistent with conventional semi-crystalline polymers. As more information becomes available, it is reasonable to expect additional areas where proteins can serve as model systems for understanding polymer morphology and organization or vice versa. In general, experiments have indicated that the morphology becomes less well defined as the amount of water content increases. At low water content, the whisker crystallites are large and distinct As the amount of water increases, the amorphous fraction of the film grows as the whiskers shorten and distort To reveal the relationship between processing pathway and structure we have developed a ternary phase diagram scheme to describe the relative amount of protein polymer (SLPF), solvating component (formic acid) and non-solvating component (water) during film orfiberformation, as shown in Figure 6. Because of the fact that SLPF is not folly soluble in water but is nearly so in formic acid, there is two-phase


Genetically Engineered Silklike Protein Polymers


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Figure 3. ΤΈΜ micrograph of an SLPF droplet with patchwork whisker crystalline interior and radially oriented sheaf periphery.


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Figure 4. TEM micrograph with droplets showing a variation in the extent of crystallinity with droplet thickness. There is evidence for amorphous material in the thinnest regions of the film.


Genetically Engineered SUklike Protein Polymers


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Figure 5. TEM micrograph showing spherulitic structure of SLPF similar to that commonly seen in thin films of semi-crystalline synthetic polymers.


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Protein

Structural Evolution of Genetically Engineered Silklike Protein

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