Structure and stability of .gamma.-crystallins: tryptophan, tyrosine, and cysteine accessibility

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Abstract
The solute perturbation techniques of fluorescence of tryptophan (Trp) and dye-labeled thiol groups of cysteine as well as phosphorescence of tyrosine (Tyr) were utilized to obtain information on the relative solvent exposure and accessibility of these residues in .gamma.-crystallins. Both acrylamide and iodide quenchers were used to evaluate the quenching parameters in terms of accessibility and charge characteristics of the proteins. Stern-Volmer plots reveal the presence of more than one class of Trp residues in .gamma.-III and .gamma.-IV, and these residues in .gamma.-II are least accessible compared to the other two. Both steady-state and lifetime quenching studies of the dye-labeled fluorescence indicate that distinct differences also exist among these crystallins in cysteine (Cys) accessibilities. All three proteins, .gamma.-II, .gamma.-III, and .gamma.-IV, show two distinct lifetime components of the dye-labeled Cys residues. Both components of .gamma.-II undergo dynamic quenching, whereas only the major component of the other two crystallins is affected by the quenchers. Addition of acrylamide causes a decrease in Tyr phosphorescence of .gamma.-III and .gamma.-IV, but no change in the emission of .gamma.-II. The decrease is attributed to the formation of a nonemittive ground-state complex between the acrylamide and Tyr of the proteins; the association constant, Ka, calculated from the emission data, has been considered as a measure of Tyr accessibility. Ka values indicate that Tyr residues in .gamma.-III are most exposed and accessible compared to those in the other two proteins. Results of quenching by iodide ion reveal significant differences in the surface charge of the proteins. This study demonstrates that despite the high degree of sequence homology and similarity in the secondary structure of these proteins, differences exist in the arrangements and microenvironments of Trp, Tyr, and Cys residues, causing significant variation in tertiary structure and charge characteristics. These specific molecular features may be primarily responsible for their remarkable denaturation and cryoprecipitation behavior and photoinduced aggregation.