Subunit exchange demonstrates a differential chaperone activity of calf alpha-crystallin toward beta LOW- and individual gamma-crystallins.
The chaperone activity of native alpha-crystallins toward beta(LOW)- and various gamma-crystallins at the onset of their denaturation, 60 and 66 degrees C, respectively, was studied at high and low crystallin concentrations using small angle x-ray scattering (SAXS) and fluorescence energy transfer (FRET). The crystallins were from calf lenses except for one recombinant human gamma S. SAXS data demonstrated an irreversible doubling in molecular weight and a corresponding increase in size of alpha-crystallins at temperatures above 60 degrees C. Further increase is observed at 66 degrees C. More subtle conformational changes accompanied the increase in size as shown by changes in environments around tryptophan and cysteine residues. These alpha-crystallin temperature-induced modifications were found necessary to allow for the association with beta(LOW)- and gamma-crystallins to occur. FRET experiments using IAEDANS (iodoacetylaminoethylaminonaphthalene sulfonic acid)- and IAF (iodoacetamidofluorescein)-labeled subunits showed that the heat-modified alpha-crystallins retained their ability to exchange subunits and that, at 37 degrees C, the rate of exchange was increased depending upon the temperature of incubation, 60 or 66 degrees C. Association with beta(LOW)- (60 degrees C) or various gamma-crystallins (66 degrees C) resulted at 37 degrees C in decreased subunit exchange in proportion to bound ligands. Therefore, beta(LOW)- and gamma-crystallins were compared for their capacity to associate with alpha-crystallins and inhibit subunit exchange. Quite unexpectedly for a highly conserved protein family, differences were observed between the individual gamma-crystallin family members. The strongest effect was observed for gamma S, followed by h gamma Srec, gamma E, gamma A-F, gamma D, gamma B. Moreover, fluorescence properties of alpha-crystallins in the presence of bound beta(LOW)-and gamma-crystallins indicated that the formation of beta(LOW)/alpha- or gamma/alpha-crystallin complexes involved various binding sites. The changes in subunit exchange associated with the chaperone properties of alpha-crystallins toward the other lens crystallins demonstrate the dynamic character of the heat-activated alpha-crystallin structure.
Putilina
Tatiana
T
Laboratoire de Minéralogie-Cristallographie, CNRS and P6-P7 Universities, Case 115, 4 Place Jussieu, F75252 Paris Cedex 05, France.
Skouri-Panet
Fériel
F
Prat
Karine
K
Lubsen
Nicolette H
NH
Tardieu
Annette
A
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002417:Cattle; D002850:Chromatography, Gel; D005452:Fluoresceins / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D007908:Lens, Crystalline / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D009282:Naphthalenesulfonates / Q000494:pharmacology; D011485:Protein Binding; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D014965:X-Rays; D038201:alpha-Crystallins / Q000737:chemistry* / Q000378:metabolism; D038204:beta-Crystallins / Q000737:chemistry* / Q000378:metabolism; D038222:gamma-Crystallins / Q000737:chemistry* / Q000378:metabolism
D005452:Fluoresceins; D018832:Molecular Chaperones; D009282:Naphthalenesulfonates; D013439:Sulfhydryl Reagents; D038201:alpha-Crystallins; D038204:beta-Crystallins; D038222:gamma-Crystallins; C015296:5-iodoacetamidofluorescein; C006421:1,5-I-AEDANS