Proteins decay kinetics

Broos J, Maddalena F, Hesp BH (2004) In vivo synthesized proteins with monoexponential fluorescence decay kinetics. J Am Chem Soc 126 22-23... 

In addition, the quenching of the fluorescence of fluorophore groups in protein molecules by neighboring groups(35) and its temperature dependence, t36) energy transfer of electronic excitation and its dependence on excitation wavelength,(1) the type of emission decay kinetics,(1,2) and changes... 

In this section, the information on structure and dynamics of proteins which may be obtained from direct observations of fluorescence decay will be considered. This type of information is afforded by methods which permit fluorescence decay kinetics to be followed with picosecond and nanosecond resolution. 

There are several conformational arrangements with different interactions of the fluorophores with surrounding groups of atoms. Such interactions may affect differently nonradiative deexcitation processes in the excited state, and the decay times for these conformational states will differ. If each of these states is characterized by singleexponential decay kinetics, then the number of constants f, will correspond to the number of aromatic groups in the protein that are in conformationally different states. 

Proteins having one chromophore per molecule are the simplest and most convenient in studies of fluorescence decay kinetics as well as in other spectroscopic studies of proteins. These were historically the first proteins for which the tryptophan fluorescence decay was analyzed. It was natural to expect that, for these proteins at least, the decay curves would be singleexponential. However, a more complex time dependence of the emission was observed. To describe the experimental data for almost all of the proteins studied, it was necessary to use a set of two or more exponents.(2) The decay is single-exponential only in the case of apoazurin.(41) Several authors(41,42) explained the biexponentiality of the decay by the existence of two protein conformers in equilibrium. Such an explanation is difficult to accept without additional analysis, since there are many other mechanisms leading to nonexponential decay and in view of the fact that deconvolution into exponential components is no more than a formal procedure for treatment of nonexponential curves. 

One can expect that the analysis of continuous distributions of electronic excited-state lifetimes will not only provide a higher level of description of fluorescence decay kinetics in proteins but also will allow the physical mechanisms determining the interactions of fluorophores with their environment in protein molecules to be elucidated. Two physical causes for such distributions of lifetimes may be considered ... 

In the majority of cases, fluorescent labels and probes, when studied in different liquid solvents, display single-exponential fluorescence decay kinetics. However, when they are bound to proteins, their emission exhibits more complicated, nonexponential character. Thus, two decay components were observed for the complex of 8-anilinonaphthalene-l-sulfonate (1,8-ANS) with phosphorylase(49) as well as for 5-diethylamino-l-naphthalenesulfonic acid (DNS)-labeled dehydrogenases.(50) Three decay components were determined for complexes of 1,8-ANS with low-density lipoproteins.1 51 1 On the basis of only the data on the kinetics of the fluorescence decay, the origin of these multiple decay components (whether they are associated with structural heterogeneity in the ground state or arise due to dynamic processes in the excited state) is difficult to ascertain. 

A. Grinvald and J. Z. Steinberg, Fast relaxation processes in a protein revealed by the decay kinetics of tryptophan fluorescence, Biochemistry 13, 5170-5177 (1974). 

Selected entries from Methods in Enzymology [vol, page(s)] . Applications, 246, 335 [immunoassay, 246, 343-344 nucleic acids, 246, 344-345 photoreceptors, 246, 341-343 protein conformation, 246, 339-340 protein-membrane interactions, 246, 340-341 two-dimensional imaging, 246, 345] energy level diagram, 246, 336 excited state decay kinetics, 246, 337-338 in-... 

The Photoactive Yellow Protein (PYP) is the blue-light photoreceptor that presumably mediates negative phototaxis of the purple bacterium Halorhodospira halophila [1]. Its chromophore is the deprotonated trans-p-coumaric acid covalently linked, via a thioester bond, to the unique cystein residue of the protein. Like for rhodopsins, the trans to cis isomerization of the chromophore was shown to be the first overall step of the PYP photocycle, but the reaction path that leads to the formation of the cis isomer is not clear yet (for review see [2]). From time-resolved spectroscopy measurements on native PYP in solution, it came out that the excited-state deactivation involves a series of fast events on the subpicosecond and picosecond timescales correlated to the chromophore reconfiguration [3-7]. On the other hand, chromophore H-bonding to the nearest amino acids was shown to play a key role in the trans excited state decay kinetics [3,8]. In an attempt to evaluate further the role of the mesoscopic environment in the photophysics of PYP, we made a comparative study of the native and denatured PYP. The excited-state relaxation path and kinetics were monitored by subpicosecond time-resolved absorption and gain spectroscopy. 

In other media like micelles, cyclodextrin, binary solvent mixtures, and proteins (47-55), lifetime distributions are routinely used to model the decay kinetics. In all of these cases the distribution is a result of the (intrinsic or extrinsic) fluorescent probe distributing simultaneously in an ensemble of different local environments. For example, in the case of the cyclodextrin work from our laboratory (53-55), the observed lifetime distribution is a result of an ensemble of 1 1 inclusion complexes forming and coexisting. These complexes are such that the fluorescent probe is located simultaneously in an array of environments (polarities, etc.) in, near, and within the cyclodextrin cavity, which manifest themselves in a distribution of excited-state lifetimes (53-55). In the present study our experimental results argue for a unimodal lifetime distribution for PRODAN in pure CF3H. The question then becomes, how can a lifetime distribution be manifest in a pure solvent ... 

Even in proteins containing a sin e tryptophan residue, multiexponential decay kinetics have been observed suggesting mobility of the protein structure during emission . However, recent time-resolved fluorescence studies of the tryptophan zwitterion and tryptophan peptides indicate that this fluorc hore does not decay... 

Here we will review the work of Sauer, Mathis, Acker and van Best in their original attempt to characterize the iron-sulfur center FeS-X, an electron carrier with a more negative redox potential and positioned ahead of FeS-A and FeS-B in the electron-transfer chain, and thus an intermediary electron donor to FeS-A and FeS-B. When the oxidized FeS-A/B proteins serve as secondary acceptors, the photochemically reduced forms have been found to re-reduce the photooxidized primary donor P700. In parallel experiments, Sauer etal. characterized FeS-X indirectly by determining the decay kinetics of photooxidized PTOO" in its recombination with reduced FeS-X" by kinetic spectrophotometric measurements. In the process, some insight was also gained indirectly regarding the nature of FeS-A and FeS-B. 

Fig. 9 (C) shows a plot of data points obtained in a manner similar to that illustrated in panel (B) but at other temperatures ranging from 13 to 225 K and for times ranging from 25 s to 10 m, as tabulated in Fig. 9 (D). In Fig. 9 (C), the percentages ofP700 lost ( ) and reduced FeS-A" lost (o) agree well with the expected values derived from the decay kinetics monitored by optical spectroscopy cf. Fig. 5). Thus, the close match between the dark decay of both species supports the notion that reduced iron-sulfur protein FeS-A is the component that is recombining with P700. ...

In the previous chapter we presented an overview of protein fluorescence. We described the spectral properties of the aromatic amino acids and how these properties depend on protein structure. We now extend this discussion to include time-resolved measurements of intrinsic protein flu( scence. Prior to 1983, most measurements of time-resolved fluorescence were performed using TCSPC. The instruments employed for these measurements typically used a flashlamp etcitation source and a standard dynode-chain-type PMT. Such instruments provided instrument response functions with a half-width near 2 ns, which is comparable to thedecay time of most proteins. The limited repetition rate of the flashlamps, near 20 kHz. resulted in data of modest statistical accuracy, unless the acquisition times were excessively long. Given the complexity of protein intensity and anisotropy decays, and the inherent difficulty of resolving multiexponential processes. ii was difficult to obUun definitive information on the decay kinetics of proteins. 

Representative fluorescence decays from whole cells in the presence of dithionite are shown in Fig. 2. The decay curve shown in the top panel is from cells luutx)iing a plasmid that encodes wild type capsukaus reaction center and antenna proteins. The bulk of the decay occurs with a time constant of40-45 ps, followed by a much lower amount of longer-lived delayed fluorescence with a multiexponential decay. The delayed fluorescence includes the 10 ns lifetime ofP Bj e". The mutants examined thus far show one of two phenotypes with regard to their fluorescence decay curves. The decay kinetics from one group of mutants are similar to the parent strain (Fig. 2, middle panel). The other mutants tested all had emission decays that were very similar to each other but different from the decay of either the parent strain or the mutants described above. An example of one of these decays is shown in the bottom panel of Fig. 2. 

From our experiments it can be concluded that the state of hydration sets a conformational state in the protein which controls the decay kinetics of the relatively stable M-412 intermediate in the protein, whereas the earlier shortlived intermediates (the K- andL-compo-nents) are not affected to any critical degree. Thus, the early events are obviously occurring within a microenvironment of the protein which is isolated from interaction with the surface of the membrane protein structure. 

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