Thermodynamic and kinetic characterization of calf lens gammaF-crystallin

Gamma-crystallin is reported to be conformationally stable because of its internal structural symmetry, and gammaF (gammaIVa) is the most stable among the various gamma-crystallin gene products. However, there is no detailed report on its thermodynamic and kinetic stability. In the present study, detailed unfolding of gammaF-crystallin was investigated by equilibrium and kinetics methods with fluorescence and far-UV CD spectroscopic measurements. The GdnHCl-induced unfolding curves probed by Trp emission maximum and intensity showed a sharp single-step transition. Upon widening the unfolding transition with the use of urea in 1.5 M GdnHCl, a more proper fit for thermodynamic analysis was obtained. GammaF-Crystallin underwent a straightforward two-state process (N <==> U) without showing any measurable amount of intermediate. The conformational stability, as measured by deltaG(D)H2O (approximately 9 kcal/mol), indicates that gammaF-crystallin is a very stable protein. The high activation energy deltaG++H2O (approximately 24 kcal/mol), calculated from unfolding kinetics monitored by far-UV CD at 218 nm, also indicates that the native and unfolded states are separated by a high activation energy barrier.