Taking into account the conformational dynamics of macromolecules when analyzing luminescent probe signals

A mathematical model is proposed that takes into account the specific features of electronic excitation energy transfer between the molecular probes sorbed on a polymeric chain in a liquid solution. The kinetics of the process is described from the standpoint of the stochastic changes in macromolecular conformations. The results of numerical solution of the problem are described, as well as the analytical expressions obtained in the context of perturbation theory for a low transfer rate and/or fast conformational dynamics of the macrochain. A channel of nonlinear (with respect to excitation) deactivation resulting from binary annihilation of closely spaced excited centers is considered. Expressions for the efficient rate of mutual quenching and delayed annihilation fluorescence of the probe are obtained. The time dependences of characteristic luminescent signals and the parametric curves of relative fluorescence quantum yield are given.     

Picosecond processes in chromatophores at various excitation intensities

The aim of this paper is to review and discuss the results obtained by fluorescence and absorption spectroscopy of bacterial chromatophores excited with picosecond pulses of varying power and intensity. It was inferred that spectral and kinetic characteristics depend essentially on the intensity, the repetition rate of the picosecond excitation pulses as well as on the optical density of the samples used. Taking the different experimental conditions properly into account, most of the discrepancies between the fluorescence and absorption measurements can be solved. At high pulse repetition rate (>10⁶ Hz), even at moderate excitation intensities (10¹⁰–10¹¹ photons/cm² per pulse), relatively long-lived triplet states start accumulating in the system. These are efficient (as compared to the reaction centers) quenchers of mobile singlet excitations due to singlet-triplet annihilation. The singlet-triplet annihilation rate constant in Rhodospirillum rubrum was determined to be equal to 10^-9 cm³ s^-1. At fluences >10¹² photons/cm² per pulse singlet-singlet annihilation must be taken into account. Furthermore, in the case of high pulse repetition rates, triplet-triplet annihilation must be considered as well. From an analysis of experimental data it was inferred that the singlet-singlet annihilation process is probably migration-limited. If this is the case, one has to conclude that the rate of excitation decay in light-harvesting antenna at low pumping intensities is limited by the efficiency of excitation trapping by the reaction center. The influence of annihilation processes on spectral changes is also discussed as is the potential of a local heating caused by annihilation processes. The manifestation of spectral inhomogeneity of light-harvesting antenna in picosecond fluorescence and absorption kinetics is analyzed.Abbreviations LHA light-harvesting antenna - RC reaction center     

Theory of exciton annihilation in complexes of a finite number of molecular sites

A theory of the kinematics of singlet exciton annihilation in complexes of a finite number of molecular sites is developed. The theory is based on a specific scheme suggested earlier by Gülen, Wittmershaus, and Knox [Biophys J. 49:469-477 (1986)]. It is adequate to address the excitation kinetics and dynamics in such systems, especially under high excitation intensities. A Pauli master equation is formulated and is solved to give explicit expressions for observables such as quantum yield and fluorescence intensity. The excitation intensity dependence of the observables is taken into account by introducing Poisson statistics. Details relevant to its application to the annihilation of excitons in photosynthetic systems and its connection to earlier theories are presented.     

Theory of Picosecond-Laser-Induced Fluorescence from Highly Excited Complexes with Small Numbers of Chromophores

The problem of singlet excitation kinetics and dynamics, especially at high excitation intensities, among a small number of chromophores of a given system has been addressed. A specific scheme for the kinetics is suggested and applied to CPII, a small chlorophyll (Chl)a/b antenna complex the fluorescence lifetime of which has been reported to be independent of excitation intensity over a wide intensity range of picosecond pulses. We have modeled the kinetics from the point of view that Chla molecules in CPII are Förster coupled so that a second excitation received by the group of Chla's either creates a state with two localized excitons or raises the first one to a doubly excited state. The data on CPII can be understood on the basis of a kinetic model that does not exclude exciton annihilation during the excitation pulse. The implied annihilation rate is consistent with our theoretical estimates of that rate obtained by applying excitation transfer theory to pairs of molecules both initially excited.     

Nonlinear annihilation of excitations in photosynthetic systems.

The theory of the singlet-singlet annihilation in quasi-homogeneous photosynthetic antenna systems is developed further. In the new model, the following important contributions are taken into account: 1) the finite excitation pulse duration, 2) the occupation of higher excited states during the annihilation, 3) excitation correlation effects, and 4) the effect of local heating. The main emphasis is concentrated on the analysis of pump-probe kinetic measurements demonstrating the first two above possible contributions. The difference with the results obtained from low-intensity fluorescence kinetic measurements is highlighted. The experimental data with picosecond time resolution obtained for the photosynthetic bacterium Rhodospirillum rubrum at room temperature are discussed on the basis of this theory.     

The temperature-induced conformational transition of immobilized chymotrypsinogen

A method has been suggested for obtaining kinetic thermodynamic data on conformational transitions of insoluble proteins by fluorescence measurements. The method was used for treatment of the temperature-induced conformational transition of chymotrypsinogen bonded to the hydroxyethyl methacrylate Spheron matrix. The bonding to Spheron causes destabilization of the native conformation of chymotrypsinogen. Two types of transition of immobilized chymotrypsinogen have been found which are controlled by first-order kinetics with different rate constants. The entropy and enthalpy changes were smaller than for free chymotrypsinogen in solution. The data obtained are interpreted as an effect of the physical interaction of the protein in the activated and denatured states with the polymeric matrix.     

Excited state annihilation in the photosynthetic unit

The kinetics of the in vivo fluorescence decays and fluorescence yields, as a function of excitation intensity, have been analysed with a model using excited state annihilation and time-dependent quenching processes. Triplet states, formed in the singlet-singlet annihilation processes, account for additional quenching of singlet states and the persistence of annihilation at longer times than the fluorescence lifetime. Together these processes give a satisfactory account of existing experimental data of the intensity dependence of fluorescence in vivo.     

Macromolecular conformation in solution. Study of carbonic anhydrase by the positron annihilation technique.

The structural features of carbonic anhydrase (carbonate hydro-lyase; EC 4.2.1.1) in aqueous solution were probed by the positron annihilation technique. The data obtained under varying conditions of temperature, pH, and enzyme concentration were interpreted in terms of the free volume model. The change of enzymic activity with temperature is accompanied by a change in free volume of the protein. Upon thermal denaturation an irreversible change in free volume of the molecule occurred. At low temperatures the protein-water interactions were investigated. These results are discussed in terms of current concepts of structure-function relationships in proteins. This study shows the sensitivity of the positron annihilation method toward the structure of proteins related to their overall conformation and to the nature of bound water.     

Role of diffusion potential on the flow of angiotensin II,bradykinin and related molecules through cellophane membranes

The diffusion of electrically charged peptides (angiotensin II, bradykinin and [Suc¹]angiotensin II) across tight cellophane membranes, obtained by different degrees of acetylation, shows a kinetic behaviour which was interpreted in the literature as indicative of the existence of different molecular conformations presenting slow interconversion velocities and different permeabilities across the membrane. A diffusion potential (Δψ) was found to be present across the membrane along diffusion experiments performed in low ionic strength. Upon annihilation of δψ by chemical voltage clamping (by equally increasing the ionic strength on both bathing solutions) the diffusion rate was decreased and the flow followed first order kinetics, indicating a major role of Δψ in the process. As the ionic strength increase could also affect molecular conformation, the role of Δψ on the diffusion of those molecules was tested by fitting flux and Δψ experimental results by an integrated form of Nernst-Planck flux equation. It is concluded that the deviation from first order diffusion kinetics, observed in low ionic strength, is solely due to the diffusion potential, and not to the existence of more than one molecular conformation in aqueous solution. This study was extended to amino acids and other related charged molecules.     

A step toward the prediction of the fluorescence lifetimes of tryptophan residues in proteins based on structural and spectral data

A method is presented that allows the calculation of the lifetimes of tryptophan residues on the basis of spectral and structural data. It is applied to two different proteins. The calcium binding protein from the sarcoplasm of the muscles of the sand worm Nereis diversicolor (NSCP) changes its conformation upon binding of Ca2+ or Mg2+. NSCP contains three tryptophan residues at position 4, 57, and 170, respectively. The fluorescence lifetimes of W57 are investigated in a mutant in which W4 and W170 have been replaced. The time resolved fluorescence properties of W57 are linked to its different microconformations, which were determined by a molecular dynamics simulation map. Together with the determination of the radiative rate constant from the wavelength of maximum intensity of the decay associated spectra, it was possible to determine an exponential relation between the nonradiative rate constant and the distance between the indole CE3 atom and the carbonyl carbon of the peptide bond reflecting a mechanism of electron transfer as the main determinant of the value for the nonradiative rate constant. This result allows the calculation of the fluorescence lifetimes from the protein structure and the spectra. This method was further tested for the tryptophan of Ha-ras p21 (W32) and for W43 of the Tet repressor, which resulted in acceptable values for the predicted lifetimes.     

Singlet-singlet annihilation kinetics in aggregates and trimers of LHCII

Singlet-singlet annihilation experiments have been performed on trimeric and aggregated light-harvesting complex II (LHCII) using picosecond spectroscopy to study spatial equilibration times in LHCII preparations, complementing the large amount of data on spectral equilibration available in literature. The annihilation kinetics for trimers can well be described by a statistical approach, and an annihilation rate of (24 ps)(-1) is obtained. In contrast, the annihilation kinetics for aggregates can well be described by a kinetic approach over many hundreds of picoseconds, and it is shown that there is no clear distinction between inter- and intratrimer transfer of excitation energy. With this approach, an annihilation rate of (16 ps)(-1) is obtained after normalization of the annihilation rate per trimer. It is shown that the spatial equilibration in trimeric LHCII between chlorophyll a molecules occurs on a time scale that is an order of magnitude longer than in Photosystem I-core, after correcting for the different number of chlorophyll a molecules in both systems. The slow transfer in LHCII is possibly an important factor in determining excitation trapping in Photosystem II, because it contributes significantly to the overall trapping time.     

Distribution of distances in thiopeptides by fluorescence energy transfer and frequency-domain fluorometry

Frequency-domain fluorescence spectroscopy was employed to examine the decays of tryptophan in Boc-Trp-Met-Asp-Phe-NH2 (donor) and (Formula: see text) (donor-acceptor pair). The efficiency of energy transfer in the thiopeptide amounted to 60%. The measured dispersion of fluorescence decay times was used to recover the donor-acceptor distance distribution. The parameters of the Gaussian distance distribution obtained for this peptide (r, the mean distance (9 A); hw, the halfwidth (25 A)) indicate the lack of a distinct favorable conformation.     

Fluorescence lifetime heterogeneity in aggregates of LHCII revealed by time-resolved microscopy.

Two-photon excitation, time-resolved fluorescence microscopy was used to investigate the fluorescence quenching mechanisms in aggregates of light-harvesting chlorophyll a/b pigment protein complexes of photosystem II from green plants (LHCII). Time-gated microscopy images show the presence of large heterogeneity in fluorescence lifetimes not only for different LHCII aggregates, but also within a single aggregate. Thus, the fluorescence decay traces obtained from macroscopic measurements reflect an average over a large distribution of local fluorescence kinetics. This opens the possibility to resolve spatially different structural/functional units in chloroplasts and other heterogeneous photosynthetic systems in vivo, and gives the opportunity to investigate individually the excited states dynamics of each unit. We show that the lifetime distribution is sensitive to the concentration of quenchers contained in the system. Triplets, which are generated at high pulse repetition rates of excitation (>1 MHz), preferentially quench domains with initially shorter fluorescence lifetimes. This proves our previous prediction from singlet-singlet annihilation investigations (Barzda, V., V. Gulbinas, R. Kananavicius, V. Cervinskas, H. van Amerongen, R. van Grondelle, and L. Valkunas. 2001. Biophys. J. 80:2409-2421) that shorter fluorescence lifetimes originate from larger domains in LHCII aggregates. We found that singlet-singlet annihilation has a strong effect in time-resolved fluorescence microscopy of connective systems and has to be taken into consideration. Despite that, clear differences in fluorescence decays can be detected that can also qualitatively be understood.     

两种不同红藻的R－藻红蛋白激发强度相关的皮秒（10~（－12）秒）荧光动力学研究

本文研究了分别从红藻多管藻（Ｐｏｌｙｓｉｐｈｏｎｉａｕｒｃｅｏｌａｔｅ）和条斑紫菜（Ｐｏｒｐｈｙｒａｙｅｚｏｅｎｓｉｓ）中提取的两种不同光谱类型的Ｒ－藻红蛋白Ｒ－ｐｈｙｃｏｅｒｙｔｈｒｉｎ激发强度相关的皮秒（１０－１２秒）荧光衰减动力学过程。结果发现：随激发光强增大,单重态－单重态激子湮灭发生（其衰减过程约为６０～８０皮秒）,并引起荧光量子产率下降。这两种Ｂ－藻红蛋白在相同光强激发下,表现出不同的单重态一单重态激子湮灭过程,主要因它们处于激发态的发色团数目不同所致。     

Conformation, length, and speed measurements of electrodynamically stretched DNA in nanochannels

A method is presented to rapidly and precisely measure the conformation, length, speed, and fluorescence intensity of single DNA molecules constrained by a nanochannel. DNA molecules were driven electrophoretically from a nanoslit into a nanochannel to confine and dynamically elongate them beyond their equilibrium length for repeated detection via laser-induced fluorescence spectroscopy. A single-molecule analysis algorithm was developed to analytically model bursts of fluorescence and determine the folding conformation of each stretched molecule. This technique achieved a molecular length resolution of 114 nm and an analysis time of around 20 ms per molecule, which enabled the sensitive investigation of several aspects of the physical behavior of DNA in a nanochannel. λ-bacteriophage DNA was used to study the dependence of stretching on the applied device bias, the effect of conformation on speed, and the amount of DNA fragmentation in the device. A mixture of λ-bacteriophage with the fragments of its own HindIII digest, a standard DNA ladder, was sized by length as well as by fluorescence intensity, which also allowed the characterization of DNA speed in a nanochannel as a function of length over two and a half orders of magnitude.     

A fluorescence study of the effects of pH and Mg2+ on the conformation of fructose 1,6-diphosphatase from spinach chloroplasts.

2-p-Toluidino-naphthalene-6-sulfonate is a sensitive fluorescent reporter group which can be used for the detection of the conformation of fructose 1,6-diphosphatase from spinach chloroplasts. When fructose 1,6-diphosphatase was added to a dilute solution of 2-p-toluidino-naphthalene-6-sulfonate at pH 9.0, the fluorescence intensity gradually increased. At this pH, the enzyme activity decreased at the same rate. However, at neutral pH (7.5), this time-dependent fluorescence change was not observed. In the presence of Mg2+, which is an activator of the enzyme, the fluorescence intensity was increased instantly and did not change for 30 min in the pH range 8.0--9.0. From the concentration dependence of the fluorescence intensity, the dissociation constant for Mg2+ was determined, Kdis = 3 mM. The effects of pH and Mg2+ on the conformation and activity of chloroplast fructose 1,6-diphosphatase are discussed.     

Applying the increase in rate constants of cooperative proteolysis to the determination of transition curves of protein denaturation

The transition curves of soybean glycinin egg lysozyme, bovine serum albumin (BSA) denaturation under the action of increasing urea concentration were monitored by determination of the increase in rate constants of cooperative enzymatic proteolysis. The results were compared with those obtained using intrinsic fluorescence and the number of accessible tyrosine. It was shown that the determination of the changes of proteolysis rate permits to disclose conformation changes not detected by other methods.     

Kinetics of actin unfolding induced by guanidine hydrochloride.

The kinetics of actin unfolding induced by guanidine hydrochloride has been studied. On the basis of obtained experimental data a new kinetic pathway of actin unfolding was proposed. We have shown that the transition from native to inactivated actin induced by guanidine hydrochloride (GdnHCl) passes through essential unfolding of the protein. This means that inactivated actin should be considered as the off-pathway species rather than an intermediate conformation between native and completely unfolded states of actin, as has been assumed earlier. The rate constants of the transitions that give rise to the inactivated actin were determined. At 1.0-2.0 M GdnHCl the value of the rate constant of the transition from native to essentially unfolded actin exceeds that of the following step of inactivated actin formation. It leads to the accumulation of essentially unfolded macromolecules early in the unfolding process, which in turn causes the minimum in the time dependencies of tryptophan fluorescence intensity, parameter A, characterizing the intrinsic fluorescence spectrum position, and tryptophan fluorescence anisotropy.     

Fluorescence lifetime distributions in proteins.

The fluorescence lifetime value of tryptophan residues varies by more than a factor of 100 in different proteins and is determined by several factors, which include solvent exposure and interactions with other elements of the protein matrix. Because of the variety of different elements that can alter the lifetime value and the sensitivity to the particular environment of the tryptophan residue, it is likely that non-unique lifetime values result in protein systems. The emission decay of most proteins can be satisfactorily described only using several exponential components. Here it is proposed that continuous lifetime distributions can better represent the observed decay. An approach based on protein dynamics is presented, which provides fluorescence lifetime distribution functions for single tryptophan residue proteins. First, lifetime distributions for proteins interconverting between two conformations, each characterized by a different lifetime value, are derived. The evolution of the lifetime values as a function of the interconversion rate is studied. In this case lifetime distributions can be obtained from a distribution of rates of interconversion between the two conformations. Second, the existence of a continuum of energy substates within a given conformation was considered. The occupation of a particular energy substate at a given temperature is proportional to the Boltzmann factor. The density of energy states of the potential well depends upon the width of the well, which determines the degree of freedom the residue can move in the conformational space. Lifetime distributions can be obtained by association of each energy substate with a different lifetime value and assuming that the average conformation can change as the energy of the substate is increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trifluoroethanol-induced unfolding of concanavalin A: equilibrium and time-resolved optical spectroscopic studies

Conformational structure changes in concanavalin A (Con A), a legume lectin protein which is composed of 18 beta-strands, induced by dissolving in 50% trifluoroethanol (TFE) were monitored at neutral and low pH by far- and near-UV circular dichroism (CD), fluorescence, and FTIR under equilibrium conditions. Stopped-flow studies using CD and fluorescence as well as FTIR, at low and high protein concentration, respectively, were carried out to follow the time-dependent conformation changes occurring after rapid mixing of the protein with TFE. Equilibrium CD results show that, upon addition of TFE, low-concentration Con A transforms to a highly alpha-helical conformation at both neutral and low pH. However, at neutral pH under high protein concentration conditions, aggregation and precipitation are eventually detected with FTIR, indicating that a final beta-structure is attained. Stopped-flow fluorescence shows the existence of an unfolding intermediate for pH 2.0 and 4.5, which could be related to the dissociation of the dimer form. However, evidence for an intermediate is not obtained at pH 6.7, where the native protein is a tetramer. Stopped-flow FTIR is consistent with these results, indicating formation of a H(+)-stabilized intermediate alpha-helical conformation before aggregation develops. Con A in TFE provides an example of an intermediate with non-native secondary structure appearing on the unfolding pathway. On the basis of the kinetic results obtained, an unfolding mechanism is proposed and some stable intermediates are identified. In turn, the slow structural change of Con A induced by TFE provides a useful model system for study of protein unfolding due to its accessibility with several spectroscopic and kinetic tools.