title: Role of the C-terminal extensions of alpha-crystallins. Swapping the C-terminal extension of alpha-crystallin to alphaB-crystallin results in enhanced chaperone activity.
abstract: Several small heat shock proteins contain a well conserved alpha-crystallin domain, flanked by an N-terminal domain and a C-terminal extension, both of which vary in length and sequence. The structural and functional role of the C-terminal extension of small heat shock proteins, particularly of alphaA- and alphaB-crystallins, is not well understood. We have swapped the C-terminal extensions between alphaA- and alphaB-crystallins and generated two novel chimeric proteins, alphaABc and alphaBAc. We have investigated the domain-swapped chimeras for structural and functional alterations. We have used thermal and non-thermal models of protein aggregation and found that the chimeric alphaB with the C-terminal extension of alphaA-crystallin, alphaBAc, exhibits dramatically enhanced chaperone-like activity. Interestingly, however, the chimeric alphaA with the C-terminal extension of alphaB-crystallin, alphaABc, has almost lost its activity. Pyrene solubilization and bis-1-anilino-8-naphthalenesulfonate binding studies show that alphaBAc exhibits more solvent-exposed hydrophobic pockets than alphaA, alphaB, or alphaABc. Significant tertiary structural changes are revealed by tryptophan fluorescence and near-UV CD studies upon swapping the C-terminal extensions. The far-UV CD spectrum of alphaBAc differs from that of alphaB-crystallin whereas that of alphaABc overlaps with that of alphaA-crystallin. Gel filtration chromatography shows alteration in the size of the proteins upon swapping the C-terminal extensions. Our study demonstrates that the unstructured C-terminal extensions play a crucial role in the structure and chaperone activity, in addition to generally believed electrostatic "solubilizer" function.
authors.affiliation: Centre for Cellular and Molecular Biology, Hyderabad, Andhrapradesh 500 007, India.
authors.forename: Saloni Yatin
authors.initials: SY
authors.lastname: Pasta
authors.forename: Bakthisaran
authors.initials: B
authors.lastname: Raman
authors.forename: Tangirala
authors.initials: T
authors.lastname: Ramakrishna
authors.forename: Ch Mohan
authors.initials: ChM
authors.lastname: Rao
mesh_terms: D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D001483:Base Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D017931:DNA Primers; D005456:Fluorescent Dyes / Q000737:chemistry; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D038201:alpha-Crystallins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000817:Anilino Naphthalenesulfonates; D017931:DNA Primers; D005456:Fluorescent Dyes; D018832:Molecular Chaperones; D038201:alpha-Crystallins; C027132:1-anilino-8-naphthalenesulfonate