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Structures of Differently Aggregated and Precipitated Forms of γB Crystallin: An FTIR Spectroscopic and EM Study

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Abstract:

The lens protein, γB-crystallin, precipitates during cataract formation. As a recombinant protein, in aqueous solution, γB aggregates and precipitates upon heating, cooling, exposure to ultraviolet light, or refolding from a denatured state. We have studied soluble γB crystallin, as well as each of the above aggregated forms, to determine whether γB's polypeptide chain is differently organized in each form. For this purpose, we used : (a) Fourier Transform Infra Red (FTIR) spectroscopy in the horizontal attenuated total reflectance (HATR) mode, to examine changes in secondary structural content, and (b) transmission electron microscopy (TEM) to examine gross morphological differences. The peak of the γB FTIR amide I band shifts from ~1633 cm-1 to ~1618 cm-1 in heat-, UV- and refolding-induced γB precipitates, indicating that narrow beta sheets with fewer strands and higher strand twist angles are becoming reorganized into wider, more planar sheets containing larger numbers of shorter strands, with smaller twist angles. In contrast, in cold-induced precipitates, a loss of anti-parallel beta sheet content is observed. This difference is partly explained by the differential effects of temperature on different non-covalent interactions stabilizing protein structures. The native beta sheet content of γB crystallin (~50%) is raised in heat- (~60%) and refolding-induced (~58%) precipitates, but lowered in cold- (~41%), and UV-induced (~44%) precipitates. Cold precipitates also display ~26% helical content. All four aggregates have distinctively different morphological characteristics; this appears to be in keeping with their distinctively different secondary structural contents.





Keywords: Gamma crystallin; UV-induced precipitation; cold precipitation; conformational changes; electron microscopy (EM); fourier transform infrared (FTIR) spectroscopy; protein secondary structure; thermal aggregation

Document Type: Research Article

DOI: https://doi.org/10.2174/092986610791760414

Publication date: 2010-09-01

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  • Protein & Peptide Letters publishes short papers in all important aspects of protein and peptide research, including structural studies, recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, drug design etc. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallisation, and preliminary structure determinations of biologically important proteins are acceptable. Purely theoretical papers are also acceptable provided they provide new insight into the principles of protein/peptide structure and function.
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