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)
Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia 19104.
J W
JW
Berger
J M
JM
Vanderkooi
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