Effect of phosphorylation on alpha B-crystallin: differences in stability, subunit exchange and chaperone activity of homo and mixed oligomers of alpha B-crystallin and its phosphorylation-mimicking mutant.
Phosphorylation appears to be one of the modulators of chaperone functions of small heat shock proteins. However, the role of phosphorylation is not completely understood. We have investigated the structural and functional consequences of a phosphorylation-mimicking mutation in alpha B-crystallin, a small heat shock protein with chaperone activity. We have used a phosphorylation-mimicking mutant, 3D alpha B-crystallin, in which all the three phosphorylatable serine residues are replaced with aspartic acid. 3D alpha B-Crystallin showed enhanced chaperone-like activity towards DTT-induced aggregation of insulin, heat-induced aggregation of citrate synthase and SDS-induced amyloid fibril formation of alpha-synuclein. Fluorescence and circular dichroism spectroscopic studies showed that 3D alpha B-crystallin exhibits lower stability towards urea-induced denaturation compared to alpha B-crystallin. Subunit exchange studies using fluorescence resonance energy transfer showed that 3D alpha B-crystallin exhibits an observable increase in subunit exchange compared to alpha B-crystallin. Since only part of alpha B-crystallin is phosphorylated in vivo, our subunit exchange studies indicate that formation of mixed oligomers between the unphosphorylated and phosphorylated subunits are likely to play a role in vivo. Our study shows that mixed-oligomer formation modulates the chaperone-like activity. We propose that the degree of phosphorylation of the alpha B-crystallin oligomers and temperature are key modulators to achieve a wide range of chaperone capabilities of the small heat shock protein, alpha -crystallin.
Centre for Cellular and Molecular Biology, Hyderabad 500 007, India.
Md Faiz
MF
Ahmad
Bakthisaran
B
Raman
Tangirala
T
Ramakrishna
Ch Mohan
ChM
Rao
D019943:Amino Acid Substitution; D000682:Amyloid / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D002950:Citrate (si)-Synthase / Q000378:metabolism; D019281:Dimerization; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D050888:Heat-Shock Proteins, Small / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D007328:Insulin / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism*; D010766:Phosphorylation; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013501:Surface-Active Agents / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology; D038203:alpha-Crystallin B Chain / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051844:alpha-Synuclein / Q000378:metabolism
D000682:Amyloid; D050888:Heat-Shock Proteins, Small; D007328:Insulin; D018832:Molecular Chaperones; D021122:Protein Subunits; D011994:Recombinant Proteins; D013501:Surface-Active Agents; D038203:alpha-Crystallin B Chain; D051844:alpha-Synuclein; D001224:Aspartic Acid; D012967:Sodium Dodecyl Sulfate; D014508:Urea; D002950:Citrate (si)-Synthase; D004229:Dithiothreitol