Biomacromolecules 2004, 5, 758-767
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Structural Conformation of Spidroin in Solution: A Synchrotron Radiation Circular Dichroism Study Cedric Dicko,*,†,§ David Knight,† John M. Kenney,‡,§ and Fritz Vollrath†,# Department of Zoology, Oxford University, OX1 3PS United Kingdom, Department of Physics, East Carolina University, North Carolina 27858, Institute for Storage Ring Facilities, University of Aarhus, 8000 Aarhus C., Denmark, and Department of Zoology, University of Aarhus, 8000 Aarhus C., Denmark Received September 24, 2003
Spider silk is made and spun in a complex process that tightly controls the conversion from soluble protein to insoluble fiber. The mechanical properties of the silk fiber are modulated to suit the needs of the spider by various factors in the animal’s spinning process. In the major ampullate (MA) gland, the silk proteins are secreted and stored in the lumen of the ampulla. A particular structural fold and functional activity is determined by the spidroins’ amino acid sequences as well as the gland’s environment. The transition from this liquid stage to the solid fiber is thought to involve the conversion of a predominantly unordered structure to a structure rich in beta-sheet as well as the extraction of water. Circular dichroism provides a quick and versatile method for examining the secondary structure of silk solutions and studying the effects of various conditions. Here we present the relatively novel technique of synchrotron radiation based circular dichroism as a tool for investigating biomolecular structures. Specifically we analyze, in a series of example studies on structural transitions induced in liquid silk, the type of information accessible from this technique and any artifacts that might arise in studying self-assembling systems. Introduction The extremely tough and virtually insoluble filaments of spider dragline silk are formed in a complex process from a liquid crystalline spinning dope.1 Our understanding of this process is largely derived from studies of gross morphology, in molecular anisotropy.2-8 The conformation, stability and behavior of spider dragline silk protein (spidroin) before and during fiber processing, remains poorly understood. Circular dichroism (CD)9-11 is an ideal technique for studying secondary structural transitions in proteins (such as the spidroins) in solution.12 CD spectroscopy measures the differential absorption between left-handed and right-handed circularly polarized light as a function of wavelength, typically in the visible and ultraviolet (UV). It is uniquely sensitive to sample chirality or overall asymmetry. CD applications can be largely grouped into quantifying the secondary structural content, as well as into monitoring conformational changes. Synchrotron Radiation Circular Dichroism (SRCD) The study of biological materials in the ultraviolet and visible part of the photon energy spectrum is useful as this region corresponds to peptide bond-dependent electronic transitions in biomolecules, hence providing information on * To whom correspondence should be addressed. Tel: + 44 1865 271216. Fax: + 44 1865 281253. E-mail:
[email protected]. † Oxford University. ‡ East Carolina University. § Storage Ring Facilities, University of Aarhus. # Department of Zoology, University of Aarhus.
Figure 1. Qualitative comparison of flux in CD (dashed line) and SRCD (full line). The flux functions are estimates based on the flux at 180 nm in both instruments.
their secondary structures.11,13-18 Conventional CD spectrometers19,20 with xenon sources generally produce a strong signal in the visible and ultraviolet region (>240 nm). However, at shorter wavelengths, the photon fluxes tend to decrease rapidly (Figure 1) due to the strong absorption of the optics and the sample. Even so, well-tuned instruments routinely reach 190 nm and, with careful work, even 175 nm. The reduced performance of the conventional CD machines below 240 nm is, in part, remedied by time-consuming multiple-scan averaging and thorough N2 purging. Nevertheless, since the 1960s, CD has been one of the most valuable techniques for investigating protein structures in solution. Since the early 1980s, synchrotron radiation has been used as a light source for CD spectrometers21,22 although until recently with limited application to biological systems. During the past few years, developments in instrumentation have resulted in SRCD
10.1021/bm034373e CCC: $27.50 © 2004 American Chemical Society Published on Web 03/30/2004
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Structural Conformation of Spidroin in Solution
spectrometers that are suitable for examining protein conformation and folding under a wide range of experimental conditions. The extreme brightness and wide spectral range (Figure 1) of SRCD spectrometers allow data to be collected with high spatial resolution and low noise background. More importantly, they allow data collection under near in vivo conditions, i.e., in buffer solutions and at protein concentrations more in keeping with cellular environments than those used for other structure determination techniques. All these parameters make SRCD a valuable tool for studying concentrated, light-scattering spidroin solutions. However, the high photon flux raises the issue of sample degradation and integrity. Although a recent study23 demonstrated that SRCD does not seem to damage protein samples, we feel that more work in this direction is needed. In conclusion, the great contribution of SRCD structural investigation lies in its ability to record spectra down to 130 nm as well as extending the scope of experimental conditions and thus greatly extending the range of conventional CD measurements. All in all, SRCD thus provides a great potential for more detailed structural fold recognition24 as we shall demonstrate below. Spectral Features and Information Content of SRCD and CD Data In proteins and peptides, CD signals arise from a number of chromophores in specific areas of the visible and ultraviolet regions. Electronic transitions of interest span a large range of wavelengths including the near UV (250300 nm), the far UV (190-250 nm), and the vacuum UV (VUV) (
