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Heat-induced conformational change of human lens recombinant alphaA- and alphaB-crystallins
J J Liang1, T X Sun, N J Akhtar
1Center for Ophthalmic Research, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA. jliang@rics.bwh.harvard.edu
Purpose:
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.