Characterization of lens alpha-crystallin tryptophan microenvironments by room temperature phosphorescence spectroscopy.
Room temperature phosphorescence techniques were used to study the structural and dynamic features of the tryptophan residues in bovine alpha-crystallin. Upon excitation at 290 nm, the characteristic signature of tryptophan phosphorescence was observed with an emission maximum at 442 +/- 2 nm. The phosphorescence intensity decay was biphasic with lifetimes of 5.4 ms (71%) and 42 ms (29%). Phosphorescence quenching measurements strongly suggest that each component corresponds to one class of tryptophans with the more buried residues having the longer emission lifetime. Three small-molecule quenchers were surveyed, and in order of increasing quenching efficiency: iodide less than nitrite less than acrylamide. A heavy-atom effect was observed in iodide solutions, and an upper limit of 5% was placed on the quantum yield of triplet formation in iodide-free solutions, while the phosphorescence quantum yield was estimated to be approximately 3.2 x 10(-4). The temperature dependence of the phosphorescence lifetime was measured between 5 and 40 degrees C. Arrhenius plots exhibited discontinuities at 26 and 29 degrees C for the short- and long-lived components, respectively, corresponding to abrupt transitions in segmental flexibility. Denaturation studies revealed conformational transitions between 1 and 2 M guanidine hydrochloride, and 4 and 6 M urea. Long-lived phosphorescence lifetimes of 3 and 7 ms were measured in 6 M guanidine hydrochloride and 8 M urea, respectively, suggesting that some structural features are preserved even at very high concentrations of denaturant. Our studies demonstrate the sensitivity of room temperature phosphorescence spectroscopy to the structure of alpha-crystallin, and the applicability of this technique for monitoring conformational changes in lens crystallin proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Berger
J W
JW
Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia 19104.
Vanderkooi
J M
JM
D020106:Acrylamide; D000178:Acrylamides; D003459:Crystallins; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007454:Iodides; D008163:Luminescent Measurements; D009573:Nitrites; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D013057:Spectrum Analysis / Q000379:methods; D013696:Temperature; D013816:Thermodynamics; D014364:Tryptophan; D014508:Urea / Q000494:pharmacology
D000178:Acrylamides; D003459:Crystallins; D006146:Guanidines; D007454:Iodides; D009573:Nitrites; D020106:Acrylamide; D014364:Tryptophan; D014508:Urea; D019791:Guanidine