Decreased heat stability and increased chaperone requirement of modified human betaB1-crystallins.
OBJECTIVE
To determine how deamidation and partial loss of the N- and C-terminal extensions alter the heat stability of betaB1-crystallin.


METHODS
Human lens betaB1, a deamidated betaB1, Q204E, and alphaA-crystallins were expressed. Truncated betaB1 was generated by proteolytic removal of part of its terminal extensions. The aggregation and precipitation of these proteins due to heating was monitored by circular dichroism and light scattering. The effect of heat on the stability of both monomers and oligomers was investigated. The flexibility of the extensions in wild type and deamidated betaB1 was assessed by 1H NMR spectroscopy.


RESULTS
With heat, deamidated betaB1 precipitated more readily than wild type betaB1. Similar effects were obtained for either monomers or oligomers. Flexibility of the N-terminal extension in deamidated betaB1 was significantly reduced compared to the wild type protein. Truncation of the extensions further increased the rate of heat-induced precipitation of deamidated betaB1. The presence of the molecular chaperone, alphaA-crystallin, prevented precipitation of modified betaB1s. More alphaA was needed to chaperone the truncated and deamidated betaB1 than deamidated betaB1 or truncated betaB1.


CONCLUSIONS
Deamidation and truncation of betaB1 led to destabilization of the protein and decreased stability to heat. Decreased stability of lens crystallins may contribute to their insolubilization and cataract formation.
D002942:Circular Dichroism; D003641:Deamination; D006358:Hot Temperature; D006801:Humans; D008027:Light; D009682:Magnetic Resonance Spectroscopy; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D012542:Scattering, Radiation; D038202:alpha-Crystallin A Chain / Q000378:metabolism; D038206:beta-Crystallin B Chain / Q000737:chemistry* / Q000378:metabolism
D018832:Molecular Chaperones; D038202:alpha-Crystallin A Chain; D038206:beta-Crystallin B Chain