Tryptophan Phosphorescence Lifetime Research Articles

Direct excitation of tryptophan phosphorescence. A new method for triplet states investigation

We studied room temperature phosphorescence of tryptophan (TRP) embedded in poly (vinyl alcohol) films. With UV (285 nm) excitation, the phosphorescence spectrum of tryptophan appears at about 460 nm. We also observed the TRP phosphorescence with blue light excitation at 410 nm, well outside of the S0 →S1 absorption. This excitation reaches the triplet state of tryptophan directly without the involvement of the singlet excited state. The phosphorescence lifetime of tryptophan is in the sub-millisecond range. The long-wavelength direct excitation to the triplet state results in high phosphorescence anisotropy which can be useful in macromolecule dynamics study via time-resolved phosphorescence.

Curcumin Prevents Aggregation in α-Synuclein by Increasing Reconfiguration Rate

α-Synuclein is a protein that is intrinsically disordered in vitro and prone to aggregation, particularly at high temperatures. In this work, we examined the ability of curcumin, a compound found in turmeric, to prevent aggregation of the protein. We found strong binding of curcumin to α-synuclein in the hydrophobic non-amyloid-β component region and complete inhibition of oligomers or fibrils. We also found that the reconfiguration rate within the unfolded protein was significantly increased at high temperatures. We conclude that α-synuclein is prone to aggregation because its reconfiguration rate is slow enough to expose hydrophobic residues on the same time scale that bimolecular association occurs. Curcumin rescues the protein from aggregation by increasing the reconfiguration rate into a faster regime.

Room Temperature Phosphorescence from Tryptophan and Halogenated Tryptophan Analogs in Amorphous Sucrose

Abstract. Tryptophan phosphorescence lifetime and quantum yield are sensitive to the local environment. The phosphorescence from tryptophan analogs, however, has not been studied. We report here data on the room temperature phosphorescence of tryptophan, 4‐, 5‐ and 6‐fluoro‐DL‐tryptophan (4‐F‐trp, 5‐F‐trp and 6‐F‐trp) and 5‐bromo‐DL‐tryptophan (5‐Br‐trp) embedded in glassy powders of freeze‐dried sucrose. In aqueous solution, the absorption of the analogs was either blue‐shifted (4‐F‐trp), red‐shifted (5‐F‐trp and 5‐Br‐trp) or not shifted (6‐F‐trp) with respect to tryptophan. The phosphorescence emission spectra of all analogs were red‐shifted compared to trp (442 nm) with maxima at 446 nm (5‐F‐trp), 451 mn (6‐F‐trp), 452 nm (5‐Br‐trp) and 469 nm (4‐F‐trp). The 5‐F‐trp and 6‐F‐trp analogs had emission intensities similar to tryptophan (relative quantum yields of 0.68 and 0.91, respectively, compared to tryptophan), while the intensities of the 4‐F and 5‐Br analogs were lower (relative quantum yields of 0.039 and 0.022, respectively). All analogs exhibited complex decay behavior requiring several exponentials for an adequate fit; the average lifetimes were all lower than that of trp (1039 ms). The average lifetimes of the fluorinated analogs (5‐F, 721 ms; 6‐F, 482 ms and 4‐F, 35 ms) scaled approximately with the relative quantum yields while that of 5‐Br (0.53 ms) was significantly lower. Analysis of the individual lifetimes suggested that the fluorinated analogs differ in their sensitivity to environmental interactions, with 5‐F‐ and 6‐F‐trp quenched 1.5‐2‐fold and 4‐F‐trp about 23‐fold more efficiently than tryptophan. The red‐shifted 5‐F‐trypto‐phan analog, which has been incorporated into proteins, may provide an alternative phosphorescence probe for selective phosphorescence detection of a specific protein in a complex mixture.

In vitro renaturation of bovine beta-lactoglobulin A leads to a biologically active but incompletely refolded state.

When bovine beta-lactoglobulin (beta-LG) was refolded after extensive denaturation in 4.8 M guanidine hydrochloride (GuHCl), the functional activity of the protein, retinol binding, as measured by the enhancement of this ligand's fluorescence, was completely recovered. In contrast, the room-temperature tryptophan phosphorescence lifetime of the refolded protein, a local measure of the residue environment, was approximately 10 ms, significantly shorter than the phosphorescence lifetime of the untreated native protein (approximately 20 ms). The lability of the freshly refolded protein, as monitored by following the time course of its unfolding when incubated in 2.5 M GuHCl through the change in fluorescence intensity at 385 nm, was also determined and found to be increased significantly relative to untreated native protein. In contrast to the long term postactivation conformational changes detected previously in Escherichia coli alkaline phosphatase (Subramaniam V, Bergenhem NCH, Gafni A, Steel DG, 1995, Biochemistry 34:1133-1136), we found no changes in either the lability or phosphorescence decays of beta-LG during a period of 24 h. Our results are in agreement with the report by Hattori et al. (1993, J Biol Chem 268:22414-22419), using conformation-specific monoclonal antibodies to recognize native-like structure, that long-term changes occur in the protein conformation, compared with the native structure, on refolding.

Time-resolved tryptophan phosphorescence spectroscopy: a sensitive probe of protein folding and structure

Room-temperature tryptophan phosphorescence from proteins is a sensitive probe of the local structural environment of the luminescent amino acid. Tryptophan phosphorescence lifetimes can vary over 3-4 orders of magnitude depending on the structural rigidity of the emitter environment and the proximity of quenching interactions, and this sensitivity can be used to characterize the local structural environment of the chromophore. Tryptophan phosphorescence lifetime can be easily monitored as a function of time, and thus is capable of producing effectively real-time information on dynamic processes in proteins. We discuss the origins and characteristics of tryptophan phosphorescence, and its application to monitoring folding processes in proteins and to studying protein conformation and flexibility. We also present the extension of phosphorescence techniques to study, for the first time, triplet emission at room temperature from tryptophan residues engineered into specific positions as reporters of protein structure.

Phosphorescence lifetime of tryptophan in proteins.

This investigation enquires into the factors that are responsible for the wide range of room-temperature Trp phosphorescence lifetimes (tau) in proteins. By exploiting the enhanced sensitivity and time resolution of phosphorescence measurements, experiments were conducted to evaluate the triplet quenching potential of each amino acid side chain. From the magnitude of the Stern-Volmer rate constant it is concluded that, among the amino acids, quenching reactions at 20 degrees C are quite effective with His, Tyr, Trp, cysteine, and cystine, with rate enhancements of 20 and 50 times when the side chains of Tyr and His, respectively, are in the ionized form. The distance dependence of the quenching interaction, estimated from the quenching of internal Trp residues in proteins separated from the amino acid in solution by a protein spacer of various thickness, emphasizes the very short-range nature of the process. The importance of these side chains, and to some extent that of the peptide linkage, as intrinsic quenchers of Trp phosphorescence in proteins was also confirmed with short synthetic peptides prepared appositely with only one type of these residues. Finally, very short (microseconds) phosphorescence lifetimes of Trp residues in proteins were shown to be invariably associated with the presence of Tyr or Cys in the immediate neighborhood of the chromophore. From a survey of the amino acids that are nearest neighbors to Trp in proteins and the corresponding value of tau it was established that, in the absence of His, Tyr, Trp, and Cys, tau is > or = 1 ms and appears to reflect mainly the local fluidity of the protein structure. Otherwise, tau can be much shorter, and for bulky His, Tyr, and Trp side chains it seems to depend dramatically on the mutual chromophore-quencher orientation. In these cases the triplet decay kinetics is shown to be a complex function of temperature, pH, and flexibility of the protein site.

Direct Kinetic Evidence for Triplet State Energy Transfer from Escherichia coli Alkaline Phosphatase Tryptophan 109 to Bound Terbium

The addition of excess Tb3+ to metal-depleted Escherichia coli alkaline phosphatase results in enhanced luminescence from enzyme-bound terbium, which increases with sample deoxygenation and exhibits a tryptophan-like excitation spectrum. Following pulsed excitation at 280 nm, the time-resolved terbium emission shows a negative prefactor associated with a submillisecond rise time, which is independent of the concentration of dissolved oxygen. The absence of a build-up phase and similarity in lifetime in the decay kinetics of directly excited (488 nm) terbium allows for the assignment of the submillisecond component in the 280 nm excited sample to bound terbium. The results of the steady state and time-resolved experiments suggest that the time evolution of alkaline phosphatase-bound terbium emission is determined by energy transfer (kET approximately 360 and 120 s-1) from the triplet state of tryptophan to terbium followed by terbium decay. This model is based on the observations that 1) the tryptophan phosphorescence lifetime (previously assigned to Trp109) corresponds to the longer component of the terbium emission and 2) the long-lived emission is enhanced, as is the Trp109 phosphorescence, by deoxygenation. An energy transfer mechanism involving the Trp109 triplet state is shown to be inconsistent with a dipole-dipole process and is best understood as a through-space electron exchange over a donor-acceptor distance of 9-10 A.

Phosphorescence reveals a continued slow annealing of the protein core following reactivation of Escherichia coli alkaline phosphatase.

When Escherichia coli alkaline phosphatase (AP) is refolded in vitro after extensive denaturation in 6.2 M guanidine hydrochloride, the enzymatic activity reaches its asymptotic value in 1 h at 24 degrees C. In contrast, the structural rigidity of the hydrophobic core of the protein, monitored by the recovery of the tryptophan phosphorescence lifetime, returns to its characteristic native-like value over several days. Moreover, the protein lability, measured by the rate of inactivation in 4.5 M guanidine hydrochloride, also changes on a time scale much longer than the recovery of activity. These results clearly demonstrate that while the return of enzymatic activity, the traditional measure of the attainment of the native state, indicates that AP has refolded to its final, active conformation, the phosphorescence data indicate otherwise. In the context of the rugged energy landscape model [Frauenfelder, H., et al. (1991) Science 254, 1598-1603], the slow annealing of the hydrophobic core is consistent with the presence of high-energy barriers that separate fully active intermediates along the folding pathway. The data suggest that the core of the protein undergoes continued structural rearrangements affecting the rigidity of the protein environment surrounding the emitting tryptophan and the protein lability long after the return of enzyme activity.

Glycerol effects on protein flexibility: a tryptophan phosphorescence study

In exploring the dynamic properties of protein structure, numerous studies have focussed on the dependence of structural fluctuations on solvent viscosity, but the emerging picture is still not well defined. Exploiting the sensitivity of the phosphorescence lifetime of tryptophan to the viscosity of its environment we have used the delayed emission as an intrinsic probe of protein flexibility and investigated the effects of glycerol as a viscogenic cosolvent. The phosphorescence lifetime of alcohol dehydrogenase, alkaline phosphatase, apoazurin and RNase T1, as a function of glycerol concentration was studied at various temperatures. Flexibility data, which refer to rather rigid sites of the globular structures, point out that, for some concentration ranges glycerol, effects on the rate of structural fluctuations of alcohol dehydrogenase and RNase T1 do not obey Kramers' a power law on solvent viscosity and emphasize that cosolvent-induced structural changes can be important, even for inner cores of the macromolecule. When the data is analyzed in terms of Kramers' model, for the temperature range 0-30 degrees C one derives frictional coefficients that are relatively large (0.6-0.7) for RNase T1, where the probe is in a flexible region near the surface of the macromolecule and much smaller, less than 0.2, for the rigid sites of the other proteins. For the latter sites the frictional coefficient rises sharply between 40 and 60 degrees C, and its value correlates weakly with molecular parameters such as the depth of burial or the rigidity of a particular site. For RNase T1, coupling to solvent viscosity increases at subzero temperatures, with the coefficient becoming as large as 1 at -20 degrees C. Temperature effects were interpreted by proposing that solvent damping of internal protein motions is particularly effective for low frequency, large amplitude, structural fluctuations yielding highly flexible conformers of the macromolecule.

PHOSPHORESCENCE LIFETIME STUDIES OF INTERACTIONS BETWEEN SERUM ALBUMINS AND SODIUM DODECYL SULFATE

Binding of sodium dodecyl sulfate (SDS) to bovine serum albumin (BSA) and human serum albumin (HSA) in aqueous solutions at room temperature induces significant changes in the phosphorescence lifetime of tryptophan (Trp) residues. A steep rise of the phosphorescence lifetime from 1.9 ms to 10.0 ms for BSA and from 1.9 ms to 5.5 ms for HSA is observed when the total SDS concentration increases from 0.0 mM to 0.22 mM at 1 mg/mL protein concentration. As the total SDS concentration is further increased to 2.2 mM, a slower increase in the phosphorescence lifetime is observed, from 10.0 ms to 19.5 ms for BSA and from 5.5 ms to 7.2 ms for HSA. It appears that the phosphorescence lifetime modifications are mainly due to an increase of protein matrix rigidity around Trp residues. The observed differences (between HSA and BSA) allow us to distinguish the contribution of the two Trp residues to the BSA phosphorescence.

Nucleutide- and temperature- induced changes in myosin subfragment-1 structure

The effects of nucleotide binding and temperature on the internal structural dynamics of myosin subfragment 1 (S1) were monitored by intrinsic tryptophan phosphorescence lifetime and fluorescence anisotropy measurements. Changes in the global conformation of S1 were monitored by measuring its rate of rotational diffusion using transient electric birefringence techniques. At 5°C, the binding of MgADP, MgADP,P and MgADP,V (vanadate) progressively reduce the rotational freedom of S1 tryptophans, producing what appear to be increasingly more rigidified S1-nucleotide structures. The changes in the luminescence properties of the tryptophans suggest that at least one is located at the interface of two S1 subdomains. Increasing the temperature from 0 to 25°C increases the apparent internal mobility of S1 tryptophans in all cases and, in addition, a reversible temperature-dependent transition centered near 15°C was observed for S1,S1-MgADP and S1-MgADP,P, but not for S1-MgADP,V. The rotational diffusion constants of S1 and S1-MgADP were measured at temperatures between 0 and 25°C. After adjusting for the temperature and viscosity of the solvent, the data indicate that the thermally induced transition at 15°C comprises local conformational changes, but no global conformational change. Structural features of S1-MgADP,P, which may relate to its role in force generation while bound to actin, are presented.

Tryptophan phosphorescence as a monitor of protein flexibility

Abstract Recent development on the triplet-state spectroscopy of tryptophan are reviewed. The application of tryptophan phosphorescence lifetime In proteins is discussed with regards to the potentialities it offers in probing the dynamical structure of these macromolecules and as a sensitive monitor of ligand-induced conformational changes.

Quenching of room temperature protein phosphorescence by added small molecules.

A number of molecular agents that can efficiently quench the room temperature phosphorescence of tryptophan were identified, and their ability to quench the phosphorescence lifetime of tryptophan in nine proteins was examined. For all quenchers, the quenching efficiency generally follows the same sequence, namely, N-acetyltryptophanamide (NATA) greater than parvalbumin approximately lactoglobulin approximately ribonuclease T1 greater than liver alcohol dehydrogenase greater than aldolase greater than Pronase approximately edestin greater than azurin greater than alkaline phosphatase. Quenching rate constants for O2 and CO are relatively insensitive to protein differences, while H2S and CS2 are somewhat more sensitive. These small molecule agents appear to act by penetrating into the proteins. However, penetration to truly buried tryptophans is less favorable than previously suggested; in five proteins studied, quenching efficiency by O2 is 20-1000 times lower than for NATA, and up to 10(5) lower for H2S and CS2. Larger and more polar quenchers--including organic thiols, conjugated ketones and amides, and anionic species--were also studied. The efficiency of these quenchers does not correlate with quencher size or polarity, the quenching reaction has low energy of activation, and quenching rates are insensitive to solvent viscosity. These results indicate that the larger quenchers do not approach the buried tryptophans by penetrating into the proteins, even on the long phosphorescence time scale, and are also inconsistent with a mechanism in which quencher encounter with the tryptophan occurs in free solution, as in a protein-opening reaction. The results obtained suggest that the quenching process involves a long-range radiationless transfer.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorescence quenching of l(-)-tryptophan in dimethyl sulfoxide at 77 K by trivalent lanthanide ions

Quenching of the corrected phosphorescence emission of tryptophan in dimethyl sulfoxide by trivalent lanthanide ions Ln 3+ can be interpreted in terms of a static model leading to triplet quenching constants k Q T ranging from 1.3 × 10 1 to 9.1 × 10 1 M −1. The values of k Q T increase with increasing spectral overlap and J values of the ground state of the appropriate lanthanide ions. The phosphorescence lifetime of tryptophan is virtually independent of [Ln 3+] and has a value of 6.15 ± 0.25 s, in agreement with the static model of triplet quenching.

Nature of phototransformation of phytochrome As probed by intrinsic tryptophan residues.

The phototransformation of the photomorphogenic photoreceptor phytochrome was probed by the intrinsic luminescence of the tryptophan (Trp) residues. The red light absorbing form of phytochrome (Pr) showed a decreased tryptophan phosphorescence intensity, compared to that of the far-red light absorbing form of phytochrome (Pfr), and a delayed fluorescence from the chromophore upon excitation of the tryptophan residues with 290-nm light. The tryptophan phosphorescence in both Pr and Pfr showed decreased lifetimes (0.29 and 1.84 s, respectively) compared to that of the free tryptophan (6.00 s). In addition, the decay kinetics of the delayed fluorescence in Pr showed a short-lifetime component (0.24 s), which is similar to the tryptophan phosphorescence lifetime value. This is due to an efficient triplet-singlet (3Trp-1Pr) energy transfer in the Pr from. The increases in the tryptophan phosphorescence quantum yield and lifetime in the Pfr form have been interpreted on the basis of chromophore reorientation on the protein surface as a result of the Pr lead to Pfr phototransformation. The Stern-Volmer plot of the quenching data further confirms preferential exposure of the tryptophan residues in the Pfr form (46% "exposed' tryptophan residues in the Pr form as compared to 72% in the Pfr form). These results provide strong support for the hydrophobic model of Pfr [Hahn, T. R., & Song, P. S. (1981) Biochemistry 20, 2602-2609).

Phosphorescence of alkaline phosphatase of E. coli in vitro and in situ

Escherichia coli K-12, which is rich in alkaline phosphatase, exhibits phosphorescence characteristic of tryptophan at room temperature. E. coli mutants which do not have alkaline phosphatase do not show long-lived phosphorescence. The phosphorescence spectrum and lifetime of E. coli K-12 was similar to that of purified alkaline phosphatase from E. coli. These results indicate that the long-lived tryptophan phosphorescence in E. coli is likely to be derived from alkaline phosphatase in situ. The temperature dependence of tryptophan phosphorescence life-time of purified alkaline phosphatase and E. coli K-12 differ; this may imply that alkaline phosphatase in E. coli may be associated with the cell envelope and is therefore protected against structural changes in the protein which result in increased phosphorescence decay rates.