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Biophysical Journal 86:404-410 (2004)
© 2004 The Biophysical Society

Protein Fiber Linear Dichroism for Structure Determination and Kinetics in a Low-Volume, Low-Wavelength Couette Flow Cell

Timothy R. Dafforn *, Jacindra Rajendra {dagger}, David J. Halsall {ddagger}, Louise C. Serpell § ¶ and Alison Rodger {dagger}

* Department of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom; {dagger} Department of Chemistry, University of Warwick, Warwick CV4 7AL, United Kingdom; {ddagger} Department of Clinical Biochemistry and § Department of Haematology, University of Cambridge, Cambridge Institute of Medical Research, Cambridge CB2 2XY, United Kingdom; and Neurobiology Division, Medical Research Council Centre, Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom

Correspondence: Address reprint requests to Timothy R. Dafforn, Department of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. E-mail: tim.dafforn{at}hotmail.com.

High-resolution structure determination of soluble globular proteins relies heavily on x-ray crystallography techniques. Such an approach is often ineffective for investigations into the structure of fibrous proteins as these proteins generally do not crystallize. Thus investigations into fibrous protein structure have relied on less direct methods such as x-ray fiber diffraction and circular dichroism. Ultraviolet linear dichroism has the potential to provide additional information on the structure of such biomolecular systems. However, existing systems are not optimized for the requirements of fibrous proteins. We have designed and built a low-volume (200 µL), low-wavelength (down to 180 nm), low-pathlength (100 µm), high-alignment flow-alignment system (couette) to perform ultraviolet linear dichroism studies on the fibers formed by a range of biomolecules. The apparatus has been tested using a number of proteins for which longer wavelength linear dichroism spectra had already been measured. The new couette cell has also been used to obtain data on two medically important protein fibers, the all-ß-sheet amyloid fibers of the Alzheimer's derived protein Aß and the long-chain assemblies of {alpha}1-antitrypsin polymers.




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