Conformational change of human lens insoluble alpha-crystallin.
Human lens alpha-crystallin becomes progressively insoluble with age and is the major crystallin component in the water-insoluble (WI) fraction. The mechanism that causes the originally water-soluble (WS) alpha-crystallin to become insoluble is unknown. A conformational change by chemical modification may be the cause, but the nature of insolubility renders it impossible to study protein conformation in the WI fraction by most spectroscopic measurements. In the present study, alpha-crystallin in the WI fraction was extracted by urea and reconstituted to a folded protein by dialysis. The refolded urea-soluble (US) alpha-crystallin was compared with WS alpha-crystallin. The US alpha-crystallin has a greater amount of polymeric species, but fewer degraded subunits than the WS alpha-crystallin as shown by SDS-PAGE and Western blot. Circular dichroism (CD) measurements indicate that they have the same secondary structure but a different tertiary structure, possibly a partial unfolding in the US alpha-crystallin. This is supported by fluorescence measurements: Trp residues are more exposed and protein has a more-hydrophobic surface in the US than in the WS alpha-crystallin. Blue fluorescence further indicates that the US alpha-crystallin has a greater amount of pigment than the WS alpha-crystallin. Together, these results indicate that the US alpha-crystallin is a chemically and conformationally modified protein.
Sun
T X
TX
Center for Ophthalmic Research, Brigham and Women's Hospital, and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Akhtar
N
N
Liang
J J
JJ
D000368:Aged; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003459:Crystallins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008875:Middle Aged; D011487:Protein Conformation; D017510:Protein Folding; D012995:Solubility; D013050:Spectrometry, Fluorescence
D003459:Crystallins