Silk protein structures

R613 F. Hagn and H. Kessler, Spider Silk Proteins. Structure and Function of Spider Silk Proteins by Nuclear Magnetic Resonance Spectroscopy , GIT Lahor-Fachzeitschrift, 2011, 55, 838. 

Liivak, O., Blye, A., Shah, N., Jehnski, L.W., 1998. A microfabricated wet-spinning apparatus to spin fibers of silk proteins. Structure—property correlations. Macromolecules 31 (9), 2947-2951. 

The secondary structure of a protein is the shape adopted by the polypeptide chain—in particular, how it coils or forms sheets. The order of the amino acids in the chain controls the secondary structure, because their intermolecular forces hold the chains together. The most common secondary structure in animal proteins is the a helix, a helical conformation of a polypeptide chain held in place by hydrogen bonds between residues (Fig. 19.19). One alternative secondary structure is the P sheet, which is characteristic of the protein that we know as silk. In silk, protein... 

Hayash C.I., Shipley N., and Lewis R., Hypotheses that correlate the sequence, structure, and mechanical properties of spider silk proteins, Int. J. Biol. Macromol., 24, 271, 1999. 

Parkhe AD, Seeley SK, Gardner K (1997) Structural studies of spider silk proteins in the fiber. J Mol Recognit 10 1-6... 

Takeuchi, A., Ohtsuki, C., Miyazaki, T., Kamitakahara, M., Ogata, S., Yamazaki, M., Furutani, Y., Kinoshita, H. and Tanihara, M. (2005) Heterogeneous nudeation of apatite on protein Structural effect of silk seridn. Journal of the Royal Society Interface, 2, 373-378. 

Silk proteins (spidroins in spiders and fibroins in Lepidoptera insects) are assembled into well-defined nanofibrillar architectures (Craig and Riekel, 2002 Eby et al., 1999 Inoue et al., 2000b, 2001 Li et al., 1994 Putthanarat et al, 2000 Vollrath et al., 1996). Spidroins and fibroins are largely constructed from two chemically distinct repetitive motifs or blocks (Table I), an insoluble crystalline block and a soluble less-crystalline block (Craig, 2003 Fedic et al., 2002 Hayashi and Lewis, 2000 Hayashi et al., 1999). The crystalline blocks are composed of short side-chained amino acids in highly repetitive sequences that give rise to /1-sheet structures. 

To understand the role of silk protein design and -structure assembly, it is important to consider the exact sequence of events in the aggregation... 

Fig. 3. Solubility of silk proteins in solution as a function of time. Low solubility corresponds to protein aggregation. The fast and slow aggregations are observed in vitro (Dicko et al., 2004a), whereas the stable helical conformation (storage structure) is observed in vivo (Dicko et al., 2004b,d). This illustrates the inherent instability of silk protein in solution and shows the /(-sheet polymorph structure as the most stable form. In other words, the spiders actively control and modulate the unavoidable silk protein aggregation prior to fiber formation.

A possible mechanism for such tight control is illustrated in Fig. 5. Clearly, increasing the protein concentration has a dramatic impact on the secondary structures of silk proteins in solution. The low concentration silk protein solution at 1% w/v is dominated by disordered structures or equally possible a polyproline II type structure (Sreerama and Woody, 2003). 

Fig. 4. Time-induced conformational change of spider silk protein (spidroin) in solution. Solutions of silk proteins at 1% w/v in distilled water were monitored using circular dichroism. The graph shows a change in secondary structure with time. The silk proteins underwent a kinetically driven transition from a partially unfolded structure to a -sheet-rich structure (from Dicko et al., 2004c). ( ) after 0 days, (O) after 1 day, and (A) after 2 days. The conformational change appeared faster at 20°C compared to 5°C, suggesting a hydrophobically driven mechanism. (Copyright 2004 American Chemical Society.)...

Fig. 5. The effect of protein-protein interactions on Nephila edulis major ampullate circular dichroism spectra in solution. A change in secondary structure with increasing concentration is observed. At low concentration (minimal protein-protein interactions) silk proteins appear partially unfolded in solution. At higher concentration (higher protein-protein interactions) silk proteins refold into a helix-like structure, most likely a molten-like globule (from Dicko et al., 2004c). This final molten structure would facilitate local chain rearrangement while preserving the global structure for protein storage and transport. (Copyright 2004 American Chemical Society.)...

Fig. 6. Structural stability of major ampullate silk protein in constrained Nephila edulis. The graph shows a time series of circular dichroism spectra of major ampullate (MA) protein at 1% w/v in distilled water. The spiders prior to dissection were prevented from spinning, but fed and watered for at least 2 weeks. With time, the secondary structure of silk protein is becoming more and more disordered. The arrow indicates increasing time (days). Note that the amino acid composition of the silk protein was similar to that of a native N. edulis spider. Interestingly, silk protein extracted from the constrained spider did not respond to denaturing conditions (detergents, alcohols, pH, and salts Dicko et al, 2004a, 2005).

In addition to traditional X-ray techniques to study silk (Bram etal., 1997 Lotz and Cesari, 1979 Riekel et al., 1999a Warwicker, 1960), other structural tools have helped unravel various aspects of silk protein conformation. These include solid-state NMR (Asakura et al., 1983, 1988, 1994 Beek et al., 2000, 2002) studies of native and regenerated silk together with and studies of isotopically edited silks, which have dramatically improved the model of structure distribution within silk fibers (Beek et al., 2000, 2002). 

In summary, the physiological control of silk protein conversion shows an ingenious balance of activating and inhibiting mechanisms that are dependent on composition and sequence arrangement (Krejchi et al., 1994). Denaturing effects observed in silks appear to be identical to those found in amyloid-forming proteins, and they principally alter the competitive outcome of the hydration of nonpolar and polar residues (Anfinsen, 1973 Dill, 1990 Dobson and Karplus, 1999 Kauzmann, 1959). The key differences to amyloids may lie in the hierarchical level of the structures (Muthukumar et al., 1997) involved in the assembly of silks compared to amyloids. 

Inoue et al. (2003) found that silk proteins will form rodlike structures and that those structure will assemble into comblike or fabric-like superstructure. The scale differences between the rods (nanometers) and the superstructure (micrometers) would suggest that the rod formation is governed by amyloid fibril formation and that the supramolecular arrangement is governed by the properties of the rod (Oroudjev et al., 2002 Putthanarat et al., 2000), namely surface interaction and hydration. Three levels of association could be considered (i) within the proteins internal /1-strands will organize to form intra /1-sheet structures, (ii) /1-sheets from neighboring molecules will associate to form fibril subunits, and (iii) the fibril subunits will further associate to form larger fibrils or rods. 

Dicko, C., Knight, D., Kenney, J. M., and Vollrath, F. (2004b). Secondary structures and conformational changes in flagelliform, cylindrical, major, and minor ampullate silk proteins. Temperature and concentration effects. Biomacromolecules 5, 2105-2115. 

Some protein structures limit the kinds of amino acids that can occur in the J3 sheet. When two or more /3 sheets are layered close together within a protein, the R groups of the amino acid residues on the touching surfaces must be relatively small. J3-Keratins such as silk fibroin and the fibroin of spider webs have a very high content of Gly and Ala residues, the two amino acids with the smallest R groups. Indeed, in silk fibroin Gly and Ala alternate over large parts of the sequence. 

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