Tryptophan lifetime

The phosphorescence lifetimes of various proteins at room temperature are given in Table 3.1. Some variability in the lifetimes reported from lab to lab is evident, possibly due to different enzyme preparation, removal of oxygen (see below), or other conditions. Nevertheless, when measured under the same conditions, it is apparent that the tryptophan lifetimes vary dramatically from protein to protein. Alkaline phosphatase exhibits the longest lifetime from a protein in solution with a lifetime of 1.5—1.7 s at 22°C, approaching the lifetime of 5.5 s at 77 K. The lifetime of free indole in solution is 15—30 /is at 22°C.(38 39) Therefore, in the absence of other quenching mechanisms, the lower limit for the phosphorescence lifetime of a fully exposed tryptophan moiety in a protein should be about 20 /is. 

Fucaloro and Forster (1985) found substantially different behavior for the hydration dependence of the tryptophan lifetime of chymotrypsino-gen A (Fig. 24). Below 0.15 h the lifetime was constant above 0.15 h the lifetime decreased from the dry protein value of about 3 nsec to the dilute solution value of near 2 nsec at a hydration level above 0.4. The change in lifetime near 0.15 h is sharp, as for a phase change. The per-colative phase transition of lysozyme is at this hydration level. 

However, diffusion of the reactive QM out of the enzyme active site is a major concern. For instance, a 2-acyloxy-5-nitrobenzylchloride does not modify any nucleophilic residue located within the enzyme active site but becomes attached to a tryptophan residue proximal to the active site of chymotrypsin or papain.23,24 The lack of inactivation could also be due to other factors the unmasked QM being poorly electrophilic, active site residues not being nucleophilic enough, or the covalent adduct being unstable. Cyclized acyloxybenzyl molecules of type a could well overcome the diffusion problem. They will retain both the electrophilic hydroxybenzyl species b, and then the tethered QM, in the active site throughout the lifetime of the acyl-enzyme (Scheme 11.1). This reasoning led us to synthesize functionalized... 

Fig. 1 N-formyltryptophanamide used for QM calculations on a tryptophan trimmed from a protein structure, also showing the two kinds of electron density shifts that control Trp fluorescence wavelength (red) and intensity/lifetime (green)...

Fig. 3 Transition energy for S0 — Sj (red) and S0 — CT(black) for a tryptophan during a 2 ns QM-MM trajectory of the human eye lens protein yD-crystallin showing typical fluctuations due to rapid changes in local electrostatic potentials at the atoms of the chromophore. This Trp has a low quantum yield because the CT state is near the Sj state much of the time. Heterogeneity in lifetime and wavelength are evident in both states because regions of 100 ps are seen having distinctly different average energies...

Malo GD, Pouwels LJ, Wang M, Weichsel A, Montfort WR, Rizzo MA, Piston DW, Wachter RM (2007) X-ray structure of Cerulean GFP a tryptophan-based chromophore useful for fluorescence lifetime imaging. Biochemistry 46 9865-9873... 

RNA. Both proteins contain no tryptophan S8 contains three tyrosines, and S15 contains two tyrosines. The tyrosine emission of these two proteins represents a case in which the quantum yield is higher in the native than the denatured protein. The average fluorescence lifetime, however, was little affected by denaturation no change was observed for S8, and the average lifetime decreased about 12% for S15. The collisional quenchers 1 and Cs + had essentially equivalent access to the tyrosines of both proteins, either in the native or denatured state, and comparison of the bimolecular quenching constants with that of free tyrosine suggested that the tyrosines were all well exposed. The mechanism for the reduction in quantum yield upon denaturation, apparently a static interaction, has not been elucidated. 

Dynamic In this case, the distribution of lifetimes is the result of the electronically excited tryptophan chromophore being perturbed by and colliding with the surrounding groups of atoms in the protein molecule and with solvent molecules. 

One would expect that lowering the temperature or increasing the viscosity of the solvent would increase the width of the lifetime distribution, since both factors may affect the rate of transitions between microstates. If this rate is high as compared with the mean value of the fluorescence lifetime, the distribution should be very narrow, as for tryptophan in solution. When the rate of transitions between microstates is low, a wide distribution would be expected. 

The elucidation of the intramolecular dynamics of tryptophan residues became possible due to anisotropy studies with nanosecond time resolution. Two approaches have been taken direct observation of the anisotropy kinetics on the nanosecond time scale using time-resolved(28) or frequency-domain fluorometry, and studies of steady-state anisotropy for xFvarying within wide ranges (lifetime-resolved anisotropy). The latter approach involves the application of collisional quenchers, oxygen(29,71) or acrylamide.(30) The shortening of xF by the quencher decreases the mean time available for rotations of aromatic groups prior to emission. 

In order to avoid complications caused by excitation energy transfer between tryptophan residues, most investigations have been performed with proteins containing one tryptophan residue per molecule. When studying protein solutions, there are difficulties in separating the effects of rotation of entire protein molecules and of the chromophores themselves relative to their environment in the protein matrix. It is usually assumed that intramolecular motions are more rapid and manifest themselves as short-lived components of anisotropy decay curves or in depolarization at short emission lifetimes. 

The most typical case is probably that in which tryptophan residues undergo rotations during the excited-state lifetime with a small angular amplitude (up to 30°).(30 69) Similar motions are observed in protein-dye complexes. We will consider below the qualitative models which have been put forward to describe such motions. 

These data may be explained in terms of the above mechanism of the long-wavelength shift of fluorescence spectra for red-edge excitation. The properties of the environment of the tryptophan residues in the proteins studied are such that during the lifetime of the excited state, structural relaxation of the surrounding dipoles fails to proceed. Studies of the dependence of the... 

J. R. Lakowicz and G. Weber, Nanosecond segmental mobilities of tryptophan residues in proteins observed by lifetime-resolved fluorescence anisotropies, Biophys. J. 32, 591-601 (1980). 

Several routes are possible to populate the triplet state. The triplet excited state can, in principle, be directly excited from the ground state, but a low extinction coefficient associated with the S0 to T, transition (reflected in the long lifetime) makes direct excitation an inefficient process for tryptophan. The triplet state can be thermally populated, but for tryptophan the large energy gap between the ground state and the triplet state makes this process unfavorable. Energy transfer from a higher state can also populate the... 

Tryptophan at 77 K in rigid solution has a phosphorescence quantum yield of 0.17(20) and a lifetime of 6 s. These values at 77 K are relatively invariant from protein to protein and do not vary significantly between buried and exposed tryptophans.(21,22) If one assumes that the intersystem crossing yield is a constant, a calculation of the quantum yield of indole phosphorescence can be roughly estimated from the lifetimes. The phosphorescence yield is related to lifetime by... 

At 77 K the position of the 0-0 band is generally blue shifted for exposed tryptophans and red shifted for buried tryptophans. Along with a shift in wavelength to the red, the phosphorescence lifetime decreases.(28) The single tryptophan of human serum albumin shows red-shifted phosphorescence and D - L triplet zero-field splitting, indicating that it is in a hydrophobic environment. 29 ... 

Table 3.1. Phosphorescence Lifetimes from Tryptophan-Containing Proteins at Ambient Temperatures under Anaerobiosis...

The range of six orders of magnitude for lifetimes of tryptophan phosphorescence in proteins at room temperature is larger than for fluorescence. The lower limit for fluorescence lifetime is about 0.5 ns, while the upper limit is 8 ns.(21> Typical values range from 3 to 5 ns. 

It has been observed that for some proteins the room temperature phosphorescence lifetimes are increased in D20. The phosphorescence lifetime of liver alcohol dehydrogenase is 300 ms in H2Oand 500 ms in D2O.<10) Phosphorescence lifetimes are often dramatically increased by exchanging hydrogen with deuterium. The reason for this is that decay rates are affected by overtones of the C-H or N-H stretch. In the case of tryptophan in... 

Room temperature phosphorescence can be observed from dried proteins. Sheep wool keratin(47) has a phosphorescence lifetime of 1.4 s. Six lyophilized proteins were shown to exhibit phosphorescence at room temperature.(48) The spectra were diffuse, and the lifetime was non-single-exponential, which the authors interpreted as due to inhomogeneous distribution of tryptophans. As the protein was hydrated, the phosphorescence lifetime decreased. This decrease occurred over the same range of hydration where the tryptophan fluorescence becomes depolarized. Hence, these results are consistent with the idea that rigidity of the site contributes to the lifetimes. 

For single-tryptophan proteins there is some correlation between blue-shifted fluorescence emission maximum and phosphorescence lifetime (Table 3.2). Another correlation is that three of the proteins which exhibit phosphorescence, azurin, protease (subtilisin Carlsberg), and ribonuclease Tlt are reported to show resolved fluorescence emission at 77 K. Both blue-shifted emission spectra and resolved spectra are characteristic of indole in a hydrocarbon-like matrix. 

Table 3.2. Correlation between Fluorescence Emission Maximum and Phosphorescence Lifetime in Single-Tryptophan Proteins 1...

Other groups within the protein may affect excited states. Disulfide bonds quench the excited states of tryptophan. For instance, at 77 K the phosphorescence lifetime of native lysozyme is low, 1.4s reduction of the disulfide bonds or denaturation gave the typical phosphorescence lifetime of 5.6 s.(49) Therefore, the absence of phosphorescence at room temperature from this protein is likely to be due to quenching of both the singlet and the triplet state. 

Other groups may cause shortening of the lifetime. The phosphorescence of parvalbumin is quenched by free tryptophan with a quenching rate constant of about 10s M i s l (D. Calhoun, unpublished results). A more extensive survey of proteins or model compounds with known distances between tryptophans is needed to study how adjacent tryptophans affect the lifetime. It should be noted that at low temperature the phosphorescence lifetime of poly-L-tryptophan is about the same as that of die monomer.(12) This does not necessarily mean that in a fluid solution tryptophan-tryptophan interaction could not take place. Thermal fluctuations in the polypeptide chain may transiently produce overlap in the n orbitals between neighboring tryptophans, thus resulting in quenching. 

It is clear that the wide range of protein phosphorescence lifetimes is due to various specific quenching mechanisms kq) or due to flexibility of the tryptophan site, thereby affecting km. It also follows that phosphorescence will be very sensitive to conformational fluctuations since subtle changes in distance or orientation relative to a specific quenching moiety within the protein will affect the lifetimes dramatically. The phosphorescence emission from protein tryptophan remains relatively unexplored in terms of investigation of dynamic structure-function relationships. 

Dorn anus el al.(74> proposed that the ratio of phosphorescence intensity to lifetime, P/t), of tryptophan phosphorescence as a function of temperature be used to distinguish heterogeneity in emission from multitryptophan proteins. Since different tryptophans within one protein show different temperature-... 

Bismuto et alF compared the phosphorescence from both tuna and sperm whale apomyoglobin. The emission occurs from a tryptophan in the A helix. The temperature dependence of lifetime and the position of the 0-0 vibrational band differ as a function of temperature for the two proteins. The authors interpreted their results to indicate that the microenvironment of the tryptophan in sperm whale apomyoglobin possesses a higher degree of internal flexibility than that in the tuna protein. 

Vanderkooi et al.(m) examined the phosphorescence from tryptophan in sarcoplasmic reticulum vesicles and the purified Ca transport ATPase at room temperature in deoxygenated solutions. The phosphorescence decay is multiexponential the lifetime of the long-lived component of phosphorescence is 22 ms. Addition of ATP or vanadate decreased the phosphorescence yield. The Ca2+-ATPase of the sarcoplasmic reticulum alternates between two conformations, called Ei and E2, during Ca2+ transport. The observations were interpreted to indicate that either the binding of vanadate or phosphate to the phosphorylation site of the ATPase or the induced shift in the conformation from the i to the E2 state produced the phosphorescence quenching. 

For horse liver alcohol dehydrogenase, denaturation by guanidine hydrochloride resulted in a decrease in phosphorescence lifetime parallel with loss of activity.(79) With urea as a denaturant, the decrease in phosphorescence lifetime appeared cooperative, and it is suggested that the denaturant loosened intramolecular interactions (such as hydrogen bonds), resulting in greater fluidity of the tryptophan environment.(80)... 

Berger and Vanderkooi(88) studied the depolarization of tryptophan from tobacco mosaic virus. The major subunit of the coat protein contains three tryptophans. The phosphorescence decay is non-single-exponential. At 22°C the lifetime of the long component decays with a time constant of 22 ms, and at 3°C the lifetime is 61 ms. The anisotropy decay is clearly not singleexponential and was consistent with the known geometry of the virus. 

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