Heat and concentration effects on the small heat shock protein, alpha-crystallin

alpha-Crystallin, a major protein of the mammalian lens, plays a vital role in maintaining the structural stability and transparency of the lens. It p erforms this function through chaperone-like activity; it has recently been reported that heating alpha-crystallin enhances this ability, The present studies, using both time-resolved and steady-state fluorescence methods, we re carried out to compare the conformational changes that result from heati ng with those that result from increasing protein concentration (up to 70 m g/mL), The relative fluorescence quantum yield from tryptophan (Trp) presen t in alpha-crystallin increases and then decreases with a concomitant shift of the emission maximum to longer wavelengths when either heating times or protein concentrations are increased. The time profile of fluorescence dec ay was resolved into three components with lifetimes of ca 0.5, 3 and 7 ns and emission maxima of ca 340, 342 and 350 nm, respectively. With longer he ating time or increasing concentrations the contribution from the longer-li ved component increases at the expense of the shorter-lived species. These data indicate that with heating or at higher concentrations the internal Tr p residues move to the surface of the protein giving a more hydrophobic ext erior and possibly explain the reported increased chaperone activity upon h eating. As a result of the concentration studies, alpha-crystallin may be m ore efficient in its chaperone activity in vivo than has been determined by in vitro experiments.