Synthetic and Recombinant Domains from a Midge's Giant Silk Protein

Chapter 8 Synthetic and Recombinant Domains from a Midge's Giant Silk Protein Role for Disulfide Bonds Steven T. Case and Stanley V. Smith

Downloaded by CORNELL UNIV on July 26, 2016 | http://pubs.acs.org Publication Date: December 3, 1993 | doi: 10.1021/bk-1994-0544.ch008

Department of Biochemistry, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216-4505

Silk proteins from the midge (Chironomus tentans, Diptera) are unique, in comparison to silkworm, spider and caddis fly silks, in their containing appreciable quantities of cysteine. At least six midge silk proteins are composed of tandem repeats containing cysteines in highly conserved positions. Repeats in the spI family of proteins can be divided into subrepeat (SR) and constant (C) regions, the latter of which contains four invariant cysteines. A combination of gel electrophoresis, sulfhydryl assays, and amino acid analyses were used to demonstrate that a synthetic C region peptide and a recombinant C+SR protein can fold and form two intramolecular disulfide bonds. These results suggest an additional role for cysteines in the pathway of midge silk protein assembly. Cysteines in proteins serve several important functions: stabilization of folding through formation of intramolecular disulfide bonds, covalent coupling of protein subunits by formation of intermolecular disulfide bonds, modification of enzyme activity by thiol-disulfide exchange and coordination of metals (1-4). The importance of these reactive and potentially interactive sites is often highlighted by their evolutionary conservation in homologous proteins. For example, subunit chains are joined by invariant cysteines in vertebrate immunoglobulins, Β and Τ lymphocyte antigen receptors and major histocompatibility proteins (1). The presence of cysteine makes silk from the midge, Chironomus tentans, unique among silks from aquatic and non-aquatic insects (such as caddis flies and silkworms, respectively) and spiders. Six of the midge's ten major silk proteins are composed of tandem repeats that include a conserved pattern of two or more cysteines (5), implying an important role for this residue. The spl family of midge silk proteins consists of four 1000 to 1500 kDa proteins composed of 130 to 150 tandem copies of a "core repeat" (6-8). Core repeats are divisible into a variable subrepeat (SR) region and a constant (C) region.

0097-6156/94/0544-0091$06.00/0 © 1994 American Chemical Society Kaplan et al.; Silk Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


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SILK POLYMERS: MATERIALS SCIENCE AND BIOTECHNOLOGY

SR regions are 30 to 60 residues long with shorter repeats containing the motif, basic residue-Pro-acidic residue. The C region contains 30 residues of which four are invariant cysteines. It has been proposed that the formation of midge silk fibers involves alignment of core repeats on adjacent segments of spl molecules (5). Intermodular electrostatic interactions could occur between parallel SR regions; intermolecular disulfide bonds could occur between parallel C regions. Formation of these disulfide bonds would contribute to silk stability and insolubility. In order to study the structure and molecular interaction of spl domains, C and SR peptides representative of each domain were constructed. In addition, a gene encoding a recombinant protein with C and SR domains fused as a monomelic core repeat was synthesized. This paper reports that both the synthetic C peptide (synC) and recombinant £ And SR (rC AS) protein can form intramolecular disulfide bonds. These results suggest an additional role for cysteines in the pathway of silk formation in the midge. Materials and Methods Peptide Synthesis. The synthesis and purification of the 30-residue C domain peptide from spla has been described (9). It is referred to here as synC and has the following amino acid sequence (single-letter code): RCGEAMRKAEAEKCARRNGRFNAEKCRCEE. Recombinant Protein. rCAS represents a monomelic 82-residue core repeat from spla. Its sequence, MRKAEAEKCARRNGRFNAEKCRCEEAGKPERNEEPEKGEKPRPEKPEKGEKPRPEKPEKGEKPRPEKPEKGEKPKPEKPEKGEKPRPER CGEAM, is encoded in a synthetic gene optimized for bacterial codon usage and expression using a T7 RNA polymerase-based system. The construction and insertion of this gene into an expression vector has already been outlined (10). rCAS was purified to apparent homogeneity by preparative isoelectric focusing and dialysis. Reduction and Reformation of Protein Disulfides. Both synC and rCAS were denatured and disulfide bonds reduced in 6 M urea, 10 mM dithiothreitol, 10 mM Tris-HCl, pH 8, and 1 mM EDTA, followed by chromatography over a column of Sephadex G-25 equilibrated in 10 mM HC1. To study refolding, samples were diluted into 1 mM EDTA, 100 mM Tris-HCl at various pH values and incubated overnight at room temperature at peptide and protein concentrations of 250/xM. Protein thiols were measured as described by Ellman (11). Gel Electrophoresis. rCAS was examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as described (12). synC was examined by SDS-PAGE optimized for small proteins (13). rCAS, synC and molecular weight markers were visualized by staining with Coomassie blue. Amino Acid Analysis. Denatured synC and rCAS were alkylated with and without prior reduction with dithiothreitol, as described above. Samples were subjected to gas phase hydrolysis in 6 Ν HC1 at 150°C for 70 minutes. Amino acids were analyzed under non-reducing conditions on a Hewlett Packard AminoQuant.

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

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Results Disulfides in synC. During the course of biophysical experiments aimed at learning the structure of synC (9), aliquots were examined by SDS-PAGE. When a sample of "native" synC (stored in H 0 for several months at 4°C) was treated with reducing agents, its electrophoretic mobility appeared to slightly decrease (Figure 1A). In addition, a minor, slower migrating band disappeared that may have been a dimer. Thiol assays indicated that there was less than 1 mole of protein sulfhydryl per mole of "native" synC; however, after denaturation and reduction, synC did contain measurable sulfhydryls. Amino acid analysis of "native" synC under non-reducing conditions revealed qualitatively the presence of cystine (two cysteines joined by a disulfide bond). Carboxymethyl-cysteine was only detectable in alkylated samples that were previously reduced. To see if disulfide bonds could reform, samples of reduced synC were incubated under various conditions (variations in ionic strength, presence of oxidizing and reducing agents) and disulfide bond formation monitored by the decrease of measurable sulfhydryls. Figure 2 illustrates the pH-dependent loss of sulfhydryl groups which is maximum at neutral to alkaline pH, the pH range where thiol groups are most reactive. The loss of detectable sulfhydryls and the predominant appearance of monomers by SDS-PAGE (Figure IB) suggested that synC was refolding and forming two intramolecular disulfide bonds. The presence of disulfides was confirmed qualitatively by the refolding-dependent detection of cystine.


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Disulfides in rCAS. To determine whether an adjacent SR domain (which lacks cysteine) would alter the ability of a C domain to form intramolecular disulfide bonds, we examined rCAS. The electrophoretic mobility of purified rCAS also decreased upon reduction (Figure 3). Results of an extensive biochemical characterization of "native" versus reduced and alkylated rCAS (including amino acid sequencing, sulfhydryl measurements, amino acid analyses and laser desorption time-of-flight mass spectrometry) were consistent with the following conclusion: purified rCAS contains two intramolecular disulfide bonds which can be reduced quantitatively to four sulfhydryl-containing cysteines. To ascertain whether these intramolecular disulfides can reform, parallel samples of synC and rCAS were incubated overnight at pH 8. Sulfhydryls were measured before and after the incubation and aliquots examined by SDS-PAGE. Under these conditions, the sulfhydryl content of synC and rCAS declined by more than 90%. SDS-PAGE indicated that the majority of both substrates refolded as monomelic molecules, although minor quantities of putative dimers and trimers were visible (Figure 4). These results suggest that synC and rCAS both have the intrinsic ability to fold and form intramolecular disulfide bonds. Discussion Since synC and rCAS can form intramolecular disulfide bonds, it would appear that, in spite of the presence of the SR region, the C domain of rCAS can fold so that pairs of cysteines are sufficiently close for disulfide bond formation.




Figure 1. SDS-PAGE of synC. (A) 5 μg samples of "native" (N) and reduced (R) synC. Before electrophoresis, samples were boiled in SDS sample buffer (12) lacking 0-mercaptoethanol. (B) S /*g aliquots of samples of reduced synC that were incubated overnight at room temperature in 10 mM HC1 or at the indicated pH at a peptide concentration of 250/xM. Samples were run on a 16% polyacrylamide gel, as described (13). The molecular weight markers (M) on each gel are: ovalbumin, 43 kDa; carbonic anhydrase, 29 kDa; β-lactoglobulin, 18 kDa; lysozyme, 14 kDa; bovine pancreatic trypsin inhibitor, 6 kDa; insulin, 2.3 and 3.4 kDa.



8. CASE & SMITH


Figure 2. The relative sulfhydryl content of synC (determined as moles SH/moles of peptide) that was reduced and incubated overnight in 10 mM H Q or at the indicated pH. Sulfhydryls were measured as described by Ellman (11).

Figure 3. Differences in the electrophoretic mobility of purified "native" (N) and reduced (R) rCAS on a 15% polyacrylamide gel (12). Protein molecular weight markers (M) are the same as in Figure 1.


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Figure 4. The electrophoretic mobility of synC (lanes 1 and 6) and rCAS (lanes 2 and 5) after reduction and refolding at peptide and protein concentrations of 250μΜ at pH 8, respectively. The sizes of some low (lane 3) and high (lane 4) molecular weight markers are indicated to the right. In addition to several proteins already in the low molecular weight mixture (Figure 1), high molecular weight markers include; myosin, 200 kDa; phosphorylase B, 97 kDa; and bovine serum albumin, 68 kDa.

What remains to be determined is whether or not the disulfide bonds in either "native" or refolded synC and rCAS are homogeneous (formed between unique pairs of cysteines) or heterogeneous (formed among all pairwise combinations of cysteines). We are currently sequencing tryptic fragments of rCAS to map disulfide-bonded cysteine pairs. Whether cysteine pairs are homo- or heterogeneous, one point remains: the C domain, with or without an adjacent SR, can fold and form intramolecular disulfide bonds. By extrapolating these results to tandem repeats in spls, one may postulate a role for the invariant cysteines in the pathway of spl assembly. Perhaps, concurrent with their synthesis, spl molecules are folded and stabilized by intramolecular disulfide bonds, one repeat at a time. This may maximize the solubility of these huge proteins by preventing aggregation prior to their secretion. In fact, the redox state of the endoplasmic reticulum does favor disulfide bond formation (14). If reduction of spl intramolecular disulfide bonds occurred after their secretion into the glandular lumen, where the protein concentration is higher than it is within the cells, their unfolding would occur in a highly concentrated protein solution favoring intermolecular interactions. Thus, if a change in redox state were encountered somewhere along the pathway of assembly and extrusion of the silk fiber, intermolecular disulfide bonds could be formed, contributing to the stability and insolubility of the ensuing silk fiber. At present, there is neither a site nor candidate molecule for accomplishing this task.


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Acknowledgments We thank Mike Wallace for performing the amino acid analyses. This work was supported by U.S. Army Research Office grant DAAL03-91-G-0239. Literature Cited


1. 2. 3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 13. 14.

Honjo, T.; Habu, S. Annu. Rev. Biochem. 1985, 54, 803-830. Ziegler, D. M. Annu. Rev. Biochem. 1985, 54, 305-329. Creighton, T. E.; Goldenber, D. P. J. Mol. Biol. 1984, 179, 497-526. Evans, R. M.; Hollenberg, S. M. Cell 1988, 52, 1-3. Case, S. T. ; Wieslander, L. In Structure, Cellular Synthesis and Assembly of Biopolymers; Case, S. T., Ed.; Results and Problems in Cell Differentiation; Springer-Verlag: Berlin, Heidelberg, 1992, Vol. 19; pp. 187-226. Wieslander, L.; Paulsson, G. Proc. Natl. Acad. Sci. USA 1992, 89, 45784582. Paulsson, G.; Bernholm, K.; Wieslander, L. J. Mol. Evol. 1992, 35, 205-216. Paulsson, G.; Höög, C.; Bernholm, K.; Wieslander, L. J. Mol. Biol. 1992, 225, 349-361. Wellman, S. E.; Hamodrakas, S. J.; Kamitsos, Ε. I.; Case, S. T. Biochim. Biophys Acta 1992, 1121, 279-285. Smith, S. V.; Case, S. T. Mat. Res. Soc. Symp. Proc. 1993, 292, (in press). Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70-77. Laemmli, U. K. Nature 1970, 227, 680-685. Schagger, H.; von Jagow, G. Anal. Biochem. 1987, 166, 368-379. Hwang, C.; Sinskey, A. J.; Lodish, H. F. Science 1992, 257, 1496-1502.

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Synthetic and Recombinant Domains from a Midge's Giant Silk Protein

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