Effects Of Nucleotides On The Structure Of Alpha-Crystallin, Using Fluorescence Spectroscopy

Alpha-crystallin, a major protein found in the eye lens has been shown to be a member of the small heat-shock proteins and a molecular chaperone. Most molecular chaperones require for their function the binding of a nucleotide, such as ATP. However, no nucleotide(s) have yet been conclusively identified to bind α-crystallin. Therefore, we have investigated the possible nucleotide binding to α-crystallin by determining the intrinsic fluorescence of the protein while in the presence of varying concentrations of several different nucleotides. Screening of the nucleotides was performed at 25°C as well as 37°C. Of the nucleotides screened, most showed no significant changes of the proteins’ fluorescence. However, fluorescence spectroscopy does seem to indicate that the nucleotides GTP and GDP interact with α-crystallin. Titration of α-crystallin with increasing amounts of GTP showed a proportional quenching of the fluorescence intensity with no significant shift of the maxima wavelength. Binding by GTP was further supported by the use of the fluorescent analog TNP-GTP whose fluorescence intensity and wavelength maxima were significantly changed upon binding to the protein.