Structure and stability of gamma-crystallins: tryptophan, tyrosine, and cysteine accessibility.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
Mandal
K
K
Eye Research Institute, Retina Foundation, Boston, Massachusetts 02114.
Chakrabarti
B
B
D000818:Animals; D002417:Cattle; D003459:Crystallins; D003545:Cysteine; D013050:Spectrometry, Fluorescence; D014364:Tryptophan; D014443:Tyrosine
D003459:Crystallins; D014443:Tyrosine; D014364:Tryptophan; D003545:Cysteine