Spectral Relaxation in Proteins

In contrast to the exten ve use of labeled membranes, diere are relatively few studies of time-dependent spectral relaxation around tryptophan residues in proteins. The possibility of time-dependent shifts was suggested by the detection of a negative preexponential factor for one pro-tein and by the increase of mean lifetime with emission wavelength. More recently, there have been measurements of TRES of tryptophan in solvents and in proteins diat provide good evidence that tryptophan residues can display time-dependent spectral shifts. Time-dependent spectral shifts of tryptophan can be readily observed in solvents at low temperature. Figwe 7.16 shows the example of NATA in isobityl alcohol at 

Spectra relaxation of indole has also been observed using the FD method. In this case the frequency responses were dependent on the observation wavelength. The phase angles were larger and the modulations waller 

TRES have also been obsoved for proteins. The ngle-trypto[dian protein sUq ylococcal nudease was examined in glycerol/water at -40 These conditions were 

Because of the rapid rales of spectral relaxation, it is more lUfficult to measure protein TRES in aqueous solution at rocnn temperature. However, a few measurement have been pofcxmed. Adrenocorticotropin (ACTH) is a anall pqitide hormone in which the single tryptophan 

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Lakowicz JR. On spectral relaxation in proteins. Photochem. Photobiol. 2000 72 421 37. 

The fluorescence behaviour of a fluorophore is also influenced by the solvent, especially the solvent polarity [308]. Moreover, when a molecule is excited the solvent molecules around it rearrange. Consequently, energy is transferred to the solvent, with the result that the emission spectrum is red-shifted. Solvent (or spectral) relaxation in water happens on the time scale of a few ps. However, the relaxation times in viscous solvents and in dye-protein constructs can be of the same order as the fluorescence lifetime. The measurement of the solvent relaxation can therefore be used to obtain information about the local environment of fluorescent molecules [485]. 

Observation of reorientational dynamics of dipolar groups surrounding the fluorophore in response to changes in the dipole moment of the fluorophore occurring upon electronic excitation. Such dynamics result in the appearance of spectral shifts with time,(1 ) in changes of fluorescence lifetime across the fluorescence spectrum,(7,32) and in a decrease in the observable effects of selective red-edge excitation.(1,24 33 34) The studies of these processes yield a very important parameter which characterizes dynamics in proteins— the reorientational dipolar relaxation time, xR. 

As shown above, the intrinsic fluorescence spectra of proteins as well as coenzyme groups and probes shift within very wide ranges depending on their environment. Since the main contribution to spectral shifts is from relaxational properties of the environment, the analysis of relaxation is the necessary first step in establishing correlations of protein structure with fluorescence spectra. Furthermore, the study of relaxation dynamics is a very important approach to the analysis of the fluctuation rates of the electrostatic field in proteins, which is of importance for the understanding of biocatalytic processes and charge transport. Here we will discuss briefly the most illustrative results obtained by the methods of molecular relaxation spectroscopy. 

The fluorescent probe 2,6-TNS and other similar aminonaphthalene derivatives (1,8-ANS, DNS) were considered to be indicators of the polarity of protein molecules, and they were assumed to be bound only to hydrophobic sites on the protein surface. The detection of considerable spectral shifts with red-edge excitation has shown that the reason for the observed short-wavelength location of the spectra of these probes when complexed to proteins is not the hydrophobicity of their environment (or, at least, not only this) but the absence of dipole-relaxational equilibrium on the nanosecond time scale. Therefore, liquid solvents with different polarities cannot be considered to simulate the environment of fluorescent probes in proteins. 

In this chapter results of the picosecond laser photolysis and transient spectral studies on the photoinduced electron transfer between tryptophan or tyrosine and flavins and the relaxation of the produced ion pair state in some flavoproteins are discussed. Moreover, the dynamics of quenching of tryptophan fluorescence in proteins is discussed on the basis of the equations derived by the present authors talcing into account the internal rotation of excited tryptophan which is undergoing the charge transfer interaction with a nearby quencher or energy transfer to an acceptor in proteins. The results of such studies could also help to understand primary processes of the biological photosynthetic reactions and photoreceptors, since both the photoinduced electron transfer and energy transfer phenomena between chromophores of proteins play essential roles in these systems. 

The effects of solvent polarity are best imderstood by specific examples. To model the fluorescence emission of proteins we examine spectra for iV-acetyl-L-tryptophanamide (NATA). This molecule is analogous to tryptophan in proteins. It is a neutral molecule, and its emission is more homogeneous than that of tryptophan itself. In solvents of increasing polarity the emission spectra shift towards longer wavelengths (Fig. 5). The emission maxima of NATA in dioxane, ethanol and water are 333, 344 and 357, respectively. These solvents are non-viscous, so the emission is dominantly from the relaxed state (Fig. 4). The spectral shifts can be used to calculate the change in dipole moment which occurs upon excitation [6]. More typically, the emission spectrum for a sample is compared with that foimd for the same fluorophore in various solvents, and the environment judged as polar or non-polar. While this approach is qualitative, it is simple and reliable, and does not involve the use of theoretical models or complex calculations. 

The measurement of one-bond coupling constants from the distance between the multiplet components in HSQC spectrum becomes unreliable for large proteins due to the differential relaxation that can severely broaden one of the components, and even make it undetectable. Luy and Marino proposed to overcome this problem by introduction of J-modulation to the sharp TROSY component. The JE-TROSY experiment starts with a spin-echo J-evolution period, which is followed by a traditional TROSY detection sequence. As the result the resonances are independently modulated by the single-bond coupling, which leads to the displacement of the cross-peak in the independent spectral dimension after the 3D Fourier transformation. This displacement is measured either relative to zero frequency if real FT is used in the J-dimension, or as the splitting between the pseudo multiplet components if the complex FT is applied. The spectral width in the J-dimension is normally set to twice the value of the... 

Why is spectral relaxation rarely observed in proteins One partial answo is because diis ect is uaudly not considered and tbe heterogeneity of protein decays is interpr ed in terms cmifornuitioial stales. Also, die time-dependent shifts are small and somewhat difficult to observe. Finally, spectral relnXStion probably occurs mostly on a subnanosecond timescale, so that diis process is mostly complete prior to emission. 

Renger, T., Marcus, R.A. On the relation of protein dynamics and exciton relaxation in pigment-protein complexes an estimation of the spectral density and a theory for the calculation of optical spectra. J. Chem. Phys. 116, 9997-10019 (2002)... 

Subpicosecond and picosecond motions are related to localized vibrations. According to [23], it appears that the main contribution to absorption in the spectral interval from 1 to 200 cm is caused by the hydrogen bond kinetics of the protein structural elements and of the bound water rather than by the excitation of the protein structure. Although this kind of motion primarily includes the solvent, it probably provides a viscous damping for the fast conformational fluctuations, and thus can play a certain role in the relaxation process. As noted by the authors, the most important time scales are the nanosecond and the microsecond ones. Corresponding motions determine the internal mobility in proteins, as well as in an enzyme action. Otherwise, Carreri and Gratton are sure that the motions in the millisecond-second time scale are not important for the determination of the catalytic properties of an enzyme. The authors discussed mainly the conformational fluctuations which take place near the conformationally equilibrium state of a protein globule. 

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