Picosecond fluorescence decay in photolyzed lens protein alpha-crystallin.
ABSTRACT 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.The Journal of Physical Chemistry 04/2002; 98(37). DOI:10.1021/j100088a030 · 2.78 Impact Factor
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ABSTRACT: α-Crystallin, a major protein of the mammalian lens, plays a vital role in maintaining the structural stability and transparency of the lens. It performs this function through chaperone-like activity; it has recently been reported that heating α-crystallin enhances this ability. The present studies, using both time-resolved and steady-state fluorescence methods, were carried out to compare the conformational changes that result from heating with those that result from increasing protein concentration (up to 70 mg/mL). The relative fluorescence quantum yield from tryptophan (Trp) present in α-crystallin increases and then decreases with a concomitant shift of the emission maximum to longer wavelengths when either heating times or protein concentrations are increased. The time profile of fluorescence decay was resolved into three components with lifetimes of ca 0.5, 3 and 7 ns and emission maxima of ca 340, 342 and 350 nm, respectively. With longer heating time or increasing concentrations the contribution from the longer-lived component increases at the expense of the shorter-lived species. These data indicate that with heating or at higher concentrations the internal Trp residues move to the surface of the protein giving a more hydrophobic exterior and possibly explain the reported increased chaperone activity upon heating. As a result of the concentration studies, α-crystallin may be more efficient in its chaperone activity in vivo than has been determined by in vitro experiments.Photochemistry and Photobiology 01/2000; 71(4). DOI:10.1562/0031-8655(2000)0710470HACEOT2.0.CO2 · 2.68 Impact Factor
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ABSTRACT: Human seminal plasma prostatic inhibin (HSPI) is one of the first seminal plasma proteins, which has been isolated, purified, and characterized. HSPI contains two tryptophans at positions 32 and 92 along its 94 amino acid primary sequence. Among the three fluorescence quenchers acrylamide (neutral), potassium iodide (anionic), and cesium chloride (cationic), potassium iodide is found to quench the tryptophan fluorescence more compared to the other two. The fluorescence decay of HSPI is biexponential with lifetimes 5.86 and 2.44 ns. The Stern-Volmer quenching curves in the case of fluorescence intensity and average lifetime are identical, indicating that the quenching mechanism is purely dynamic. The decay associated spectra of the two lifetimes show that the two tryptophans are solvent-exposed. The fluorescence quenching data is in favor of associating the two Lifetimes to separate tryptophans. The fluorescence anisotropy decay of HSPI is single exponential with a correlation time of 9.2 ns, which is due to the rotation of entire protein. Absence of any fast component in the anisotropy decay indicates that the two tryptophans are in motionally restricted, rigid environments. In NMR studies, the cross-peak patterns observed in 2D-COSY and 2D-NOESY spectra of HSPI gave unambiguous evidence that each of the two tryptophans is sterically constrained and exists in a single rotamer population.The Journal of Physical Chemistry B 07/1998; 102(28). DOI:10.1021/jp9733983 · 3.38 Impact Factor