title: Structures of differently aggregated and precipitated forms of gamma B crystallin: an FTIR spectroscopic and EM study.
abstract: The lens protein, gamma B-crystallin, precipitates during cataract formation. As a recombinant protein, in aqueous solution, gamma B aggregates and precipitates upon heating, cooling, exposure to ultraviolet light, or refolding from a denatured state. We have studied soluble gamma B crystallin, as well as each of the above aggregated forms, to determine whether gamma B'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 gamma B FTIR amide I band shifts from approximately 1633 cm(-1) to approximately 1618 cm(-1) in heat-, UV- and refolding-induced gamma B 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 gamma B crystallin (approximately 50%) is raised in heat- (approximately 60%) and refolding-induced (approximately 58%) precipitates, but lowered in cold- (approximately 41%), and UV-induced (approximately 44%) precipitates. Cold precipitates also display approximately 26% helical content. All four aggregates have distinctively different morphological characteristics; this appears to be in keeping with their distinctively different secondary structural contents.
authors.affiliation: Department of Protein Science & Engineering, Institute of Microbial Technology (IMTECH), Chandigarh 160036, India.
authors.forename: Uzma
authors.initials: U
authors.lastname: Fatima
authors.forename: Swati
authors.initials: S
authors.lastname: Sharma
authors.forename: Purnananda
authors.initials: P
authors.lastname: Guptasarma
mesh_terms: D046529:Microscopy, Electron, Transmission / Q000379:methods*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D038222:gamma-Crystallins / Q000737:chemistry* / Q000648:ultrastructure*
chemical_list: D038222:gamma-Crystallins