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 γBs 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, conformational changes, thermal aggregation, cold precipitation, UV-induced precipitation, protein secondary structure, fourier transform infrared (FTIR) spectroscopy, electron microscopy (EM)
Protein & Peptide Letters
Title: Structures of Differently Aggregated and Precipitated Forms of γB Crystallin: An FTIR Spectroscopic and EM Study
Volume: 17 Issue: 9
Author(s): Uzma Fatima, Swati Sharma and Purnananda Guptasarma
Affiliation:
Keywords: Gamma crystallin, conformational changes, thermal aggregation, cold precipitation, UV-induced precipitation, protein secondary structure, fourier transform infrared (FTIR) spectroscopy, electron microscopy (EM)
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 γBs 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.
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Fatima Uzma, Sharma Swati and Guptasarma Purnananda, Structures of Differently Aggregated and Precipitated Forms of γB Crystallin: An FTIR Spectroscopic and EM Study, Protein & Peptide Letters 2010; 17 (9) . https://dx.doi.org/10.2174/092986610791760414
DOI https://dx.doi.org/10.2174/092986610791760414 |
Print ISSN 0929-8665 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5305 |
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