Heat-induced conformational change of human lens recombinant alphaA- and alphaB-crystallins.
OBJECTIVE
To determine which component of lens alpha-crystallin is responsible for heat-induced transition, conformational change and high molecular weight (HMW) aggregation.


METHODS
Recombinant alphaA- and alphaB-crystallins were used. Temperature dependent changes were probed by Trp fluorescence and circular dichroism (CD) measurements. HMW aggregates were induced by heating at 62 degrees C for 1-2 h and then cooling to room temperature. The nature of HMW aggregation was studied with fluorescent probes, 4,4'-dianilino-1, 1'-binaphthalene-5,5'-disulfonic acid (bis-ANS) and thioflavin T (ThT).


RESULTS
CD and Trp fluorescence revealed that alphaB-crystallin was more susceptible than alphaA-crystallin to heat-induced conformational change and aggregation. At temperatures greater than 70 degrees C, alphaB-crystallin precipitated but alphaA-crystallin remained soluble. Both bis-ANS and ThT probes displayed increased fluorescence intensity with HMW aggregation, but the increase for bis-ANS was greater with alphaB-crystallin than with alphaA-crystallin, while the reverse was true for ThT.


CONCLUSIONS
These results indicate that alphaB-crystallin is more susceptible than alphaA-crystallin to heat-induced conformational change and aggregation and are consistent with the notion that alphaA- and alphaB-crystallins have different biochemical and biophysical properties in spite of their high degree of homology.
Liang
J J
JJ
Center for Ophthalmic Research, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA. jliang@rics.bwh.harvard.edu
Sun
T X
TX
Akhtar
N J
NJ
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D052160:Benzothiazoles; D002942:Circular Dichroism; D003459:Crystallins / Q000737:chemistry* / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry; D006358:Hot Temperature; D006801:Humans; D007908:Lens, Crystalline / Q000737:chemistry*; D008970:Molecular Weight; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013844:Thiazoles / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D052160:Benzothiazoles; D003459:Crystallins; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D013844:Thiazoles; C009462:thioflavin T; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D014364:Tryptophan