Conformation of gamma-crystallins of the calf lens: Effects of temperature and denaturing agents

Abstract

The secondary and tertiary conformations of calf γ-crystallin were monitored by circular dichroism (CD) measurements in the near and far ultraviolet regions. The secondary structures of the various fractions of γ-crystallin do not possess any α-helix conformation as judged by the far ultraviolet CD spectra. Each of the various γ-crystallin fractions exhibited a unique and complex near ultraviolet CD spectrum, reflecting the contributions of tryptophanyl, tyrosyl and phenylalanyl residues to the optical activity. The low molecular weight β-crystallin fraction, β s, possesses its own characteristic secondary and tertiary structural parameters. β s-crystallin contains about 10% of α-helical structure and its CD wavelength profile in the near ultraviolet is different from that observed for the various γ-crystallin fractions. One per cent sodium dodecyl sulfate and 5 m-guanidine hydrochloride were very effective in the disruption of the secondary and tertiary structure of γ-crystallin as judged from the changes observed in the CD spectra. On the other hand, 8 m-urea caused only minor perturbation in the CD wavelength profile, suggesting that γ-crystallin did not denature at high urea concentration. The conformation of γ-crystallin as a function of temperature was monitored by CD measurements between −20°C and +60°C at pH values of 5·5 and 8·5 and at various protein concentrations ranging from 1 to 40 mg/ml. No significant spectroscopic changes were observed within this temperature range. The independence of the CD spectra from temperature suggests that the aromatic residues of γ-crystallin are in a rigid environment and do not possess appreciable conformational motility. Previous work Zigman and Lerman 1965 has suggested that γ-crystallin (which was classified as a “cryoprotein”) when cooled, changes its conformation in such a way as to cause precipitation. While conformational changes as a function of temperature could be detected by optical measurements in other cryoproteins, we have failed to detect such changes in γ-crystallins. The fact that γ-crystallin precipitates at relatively high concentration in a very narrow pH range as the temperature is lowered, can simply be explained as isoelectric precipitation.