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Chapter 41,450

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

Mandal, K.; Chakrabarti, B.

Biochemistry 27(12): 4564-4571

1988


ISSN/ISBN: 0006-2960
PMID: 3166999
DOI: 10.1021/bi00412a051
Accession: 041449850

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.

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