Picosecond fluorescence decay in photolyzed lens protein α-crystallin
Institute for Molecular Science, Okazaki, Japan. Biochemistry
(Impact Factor: 3.02).
06/1993; 32(18):4787-92. DOI: 10.1021/bi00069a013
Photolysis of calf lens protein alpha-crystallin in aqueous solutions has been monitored by observing changes in fluorescence decay following UV irradiation at 308 nm. The fluorescence decay was biexponential in dark controls and in photolyzed solutions. The recovered lifetime components in pH 7.4 phosphate buffer at 23 degrees C were 3.5 and 0.5 ns before irradiation and 2.7 and 0.5 ns after irradiation. As the UV dose increased, the relative weighting coefficient of the 2.7-ns decay component decreased, and that of the 0.5-ns component increased, resulting in an overall lifetime shortening. Similar results were obtained in 5 M guanidine hydrochloride solutions where lifetime components of 2.7 and 0.5 ns were observed. These observations were in contrast to the behavior of tryptophan monomer solutions which did not show any change in fluorescence decay kinetics upon UV photolysis but only a reduced fluorescence intensity. Steady-state fluorescence spectra and fluorescence quantum yields were also measured at 23 degrees C for unirradiated bovine alpha-crystallin and gave phi F = 0.11 +/- 0.01 in pH 7.4 buffer and phi F = 0.10 +/- 0.01 in 5 M guanidine hydrochloride solutions. The combined steady-state and fluorescence decay data were consistent with assignment of the long-lived fluorescence decay component in alpha-crystallin to emission from Trp-9, which is known to photolyze relatively rapidly. The short decay component was assigned to Trp-60, which photolyzed much more slowly. We thus provide an example of using steady-state photochemical data to assign fluorescence decay components in a multi-tryptophan protein.
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ABSTRACT: Time-resolved fluorescence intensity and anisotropy decay measurements have been carried out on barstar, the inhibitor protein of the bacterial ribonuclease, barnase. The intrinsic fluorescence of the three tryptophans in this protein have been used to characterize the molten globule-like conformation at pH 3 (A form) and the native conformation at p H 7 (N state). The fluorescence intensity decay could be fitted to a sum of two exponentials with lifetimes of 4.1 and 1.5 ns at pH 7 (N state) and three exponentials with lifetimes of 4.9, 1.5, and 0.2 ns at pH 3 (A form). The emergence of the 0.2-ns component was pH dependent with a pKof -4.5. Fluorescence quenching by iodide has shown that the tryptophan (Trp) residues are solvent inaccessible at p H 7 and partially exposed at pH 3 (A form). Quenching by acrylamide has suggested that the 1.5-ns decay component arises from one of the three Trp residues and the 4.1-11s component arises from the remaining two Trp residues. Of the latter, one is buried and the other is highly accessible to acrylamide. Decay of fluorescence anisotropy has shown that the Trp residues are rigidly held and do not have any segmental mobility at pH 7. The A form is characterized by a high level of aggregation and a high degree of internal motion. The aggregated A form could be relevant in the folding pathway of barstar when the possibility of interaction of molten globular form with chaperone proteins is recognized. Comparison of the dynamic behavior of the Cys - Ala mutant with that of the wild type has shown the proximity of SH group(s) to Trp residues.
Available from: Patricia J T A Groenen
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ABSTRACT: α-Crystallin is a high-molecular-mass protein that for many decades was thought to be one of the rare real organ-specific proteins. This protein exists as an aggregate of about 800 kDa, but its composition is simple. Only two closely related subunits termed αA- and αB-crystallin, with molecular masses of approximately 20 kDa, form the building blocks of the aggregate.
The idea of organ-specificity had to be abandoned when it was discovered that α-crystallin occurs in a great variety of nonlenticular tissues, notably heart, kidney, striated muscle and several tumors. Moreover αB-crystallin is a major component of ubiquinated inclusion bodies in human degenerative diseases.
An earlier excitement arose when it was found that αB-crystallin, due to its very similar structural and functional properties, belongs to the heat-shock protein family. Eventually the chaperone nature of α-crystallin could be demonstrated unequivocally.
All these unexpected findings make α-crystallin a subject of great interest far beyond the lens research field.
A survey of structural data about α-crystallin is presented here. Since α-crystallin has resisted crystallization, only theoretical models of its three-dimensional structure are available. Due to its long life in the eye lens, α-crystallin is one of the best studied proteins with respect to post-translational modifications, including age-induced alterations. Because of its similarities with the small heat-shock proteins, the findings about α-crystallin are illuminative for the latter proteins as well.
This review deals with: structural aspects, post-translational modifications (including deamidation, racemization, phosphorylation, acetylation, glycation, age-dependent truncation), the occurrence outside of the eye lens, the heat-shock relation and the chaperone activity of α-crystallin.
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ABSTRACT: The major eye-lens protein alpha-crystallin is known to possess a remarkable sequence homology to the low molecular weight heat-shock proteins and has been shown to protect several proteins against thermally induced aggregation. In this work we demonstrate that the rapid aggregation of rabbit muscle phosphoglycerate kinase during incubation at 52 degrees C is completely inhibited in presence of 1/3 moles alpha-crystallin monomer per mole enzyme. Upon irradiation by UV light, tryptophan fluorescence intensity of alpha-crystallin declines, reflecting the destruction of these residues. A remarkable correlation is revealed between the reduction in alpha-crystallin fluorescence during UV-irradiation and the loss of its ability to protect phosphoglycerate kinase against aggregation. Since a loss of tryptophan fluorescence in intact eye lenses in vivo has been demonstrated to occur upon exposure to UV light, as well as during aging, it is proposed that the enhanced rate of lens opacification and cataract formation, as well as the increased levels of damaged lens proteins, which accumulate under these conditions, are the result of the gradual loss of the chaperone-protein efficacy of alpha-crystallin.
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