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Protein Science (2004), 13:1288-1294. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Proteins can convert to {beta}-sheet in single crystals

Run Zheng1, Xiaojing Zheng1, Jian Dong and Paul R. Carey

Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106

(RECEIVED December 5, 2003; FINAL REVISION February 3, 2004; ACCEPTED February 10, 2004)



Abstract

Raman microscopy was used to follow conformational changes in single protein crystals. Crystals of native insulin and of the 5S and 12S subunits of the enzyme transcarboxylase showed a mixture of Raman marker bands signifying {alpha}-helix, {beta}-sheet and nonordered secondary structure. However, by reducing the S–S bonds in the insulin crystal, or by lowering the pH for the 5S crystal, or by soaking substrates into the 12S crystal, the secondary structure in each crystal became predominantly {beta}-sheet. The {beta}-form crystals could be dissolved only with difficulty and yielded high–molecular weight protein aggregates, indicating that the {beta}-sheet formation involves intermolecular contacts. Although their morphology appeared unchanged, the crystals no longer diffracted X-rays. Using crystals that had not been exposed to laser light, the dye thioflavin T formed highly fluorescent complexes with the "{beta}-transformed" crystals but not with the native crystals.

Keywords: Raman microscopy; protein secondary structure; "{beta}-transformed" crystals; thioflavin

Abbreviations: TC: transcarboxylase • 5S: a subunit of TC that carboxylates pyruvate • 12S: a subunit of TC that transfers carboxylate from methylmalonyl-CoA to biotin • Ni-NTA: a nickel-charged agarose resin

Reprint requests to: Paul R. Carey, Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106; e-mail: prc5{at}cwru.edu; fax: (216) 368-3419.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03550404.

Supplemental material: see www.protein.science.org

1 These authors contributed equally to this article.


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