Subunit exchange of small heat shock proteins. Analysis of oligomer formation of alphaA-crystallin and Hsp27 by fluorescence resonance energy transfer and site-directed truncations.
alphaA-Crystallin, a member of the small heat shock protein (sHsp) family, is a large multimeric protein composed of 30-40 identical subunits. Its quaternary structure is highly dynamic, with subunits capable of freely and rapidly exchanging between oligomers. We report here the development of a fluorescence resonance energy transfer method for measuring structural compatibility between alphaA-crystallin and other proteins. We found that Hsp27 and alphaB-crystallin readily exchanged with fluorescence-labeled alphaA-crystallin, but not with other proteins structurally unrelated to sHsps. Truncation of 19 residues from the N terminus or 10 residues from the C terminus of alphaA-crystallin did not significantly change its subunit organization or exchange rate constant. In contrast, removal of the first 56 or more residues converts alphaA-crystallin into a predominantly small multimeric form consisting of three or four subunits, with a concomitant loss of exchange activity. These findings suggest residues 20-56 are essential for the formation of large oligomers and the exchange of subunits. Similar results were obtained with truncated Hsp27 lacking the first 87 residues. We further showed that the exchange rate is independent of alphaA-crystallin concentration, suggesting subunit dissociation may be the rate-limiting step in the exchange reaction. Our findings reveal a quarternary structure of alphaA-crystallin, consisting of small multimers of alphaA-crystallin subunits in a dynamic equilibrium with the oligomeric complex.
Bova
M P
MP
Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, California 90095, USA.
McHaourab
H S
HS
Han
Y
Y
Fung
B K
BK
D003459:Crystallins / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes; D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry*; D007700:Kinetics; D046911:Macromolecular Substances; D018832:Molecular Chaperones; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000378:metabolism; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence
D003459:Crystallins; D005456:Fluorescent Dyes; D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D011994:Recombinant Proteins