Journal
PROTEIN SCIENCE
Volume 13, Issue 5, Pages 1288-1294Publisher
WILEY
DOI: 10.1110/ps.03550404
Keywords
Raman microscopy; protein secondary structure; beta-transformed crystals; thioflavin
Categories
Funding
- NIDDK NIH HHS [R01 DK053053, DK53053] Funding Source: Medline
- NIGMS NIH HHS [GM54072, R01 GM054072, R56 GM054072] Funding Source: Medline
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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.
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