Koit Mauring

Sharp‐line structure in the fluorescence and excitation spectra of greening etiolated leaves

Vibrationally resolved sharp‐line optical fluorescence and excitation spectra are observed for etiolated and greening barley leaves at T = 5 K. Conditions necessary for obtaining fine‐structured spectra in the case of chromophores in biological cells are discussed. The observed fine‐line spectra confirm earlier results that the inactive protochlorophyll F630 and initial chlorophyll(ide) forms are present as monomeric pigment molecules weakly bound to proteins, while the active holochrome F655 is an aggregated protochlorophyllide‐protein complex

Site-selection optical spectra of bacteriochlorophyll and bacteriopheophytin in frozen solutions

Abstract Site-selection spectra of title compounds in different frozen solvents at 5K have been obtained: fluorescence spectra on selective excitation in vibronic as well as in the O-O absorption region and excitation spectra with narrow-band fluorescence recording. Frequencies of vibrational modes active in fluorescence and excitation spectra of bacteriochlorophyll a (BChl) and its metal-free derivative have been determined from these fine-line spectra. Most favourable vibronic line-to-background intensity ratios have been found in non-polar aprotic glassy environments (triethylamine, di-iso-amylether). The intensity of zero-phonon lines in microcrystallic protic matrices was low, indicating strong electron-phonon coupling. The vibrational frequencies of the excited electronic state characteristic of axially (at Mg atom) mono- and disolvated BChl species have been identified. Narrow spectral holes of about 20% of the initial absorption could be burned with ≈10-4 quantum yield within the O-O band of BChl and bacteriopheophytin.

Spectral shift mechanisms of chlorophylls in liquids and proteins

Origins of non-excitonic spectral shifts of chlorophylls that can reach -1,000 cm(-1) in pigment-protein complexes are actively debated in literature. We investigate possible shift mechanisms, basing on absorption and fluorescence measurements in large number of liquids. Transition wavelength in solvent-free state was estimated (±2 nm) for chlorophyll a (Chl a, 647 nm), Chl b (624 nm), bacteriochlorophyll a (BChl a, 752 nm), and pheophytines. The dispersive-repulsive shift is a predominating mechanism. It depends on polarizability difference between the ground and the excited state Δα and the Lorenz-Lorentz function of refractive index of solvent (n). The approximate (± 2Å(3)) increase of polarizability Δα is close to 15Å(3) for S(1) bands of Chl a, BChl a, and BPheo a, slightly larger for Chl b (18Å(3)), and less for Pheo a (11Å(3)). The effect of solvent polarity, expressed in terms of static dielectric permittivity (ε) is relatively minor, but characteristic for different pigments and transitions. Remarkably, maximum influence of ε on S(1) band of BChl a is less (-20 ± 10 cm(-1)) than that for Chl a (-50 ± 10 cm(-1)), and not correlated with dipole moment changes on excitation Δμ (∼2D and 0.1 ± 0.1D, respectively). Hydrogen bonding in protic solvents produces red shifts in Chl a (-60 cm(-1)) and BChl a (-100 cm(-1)), but not in Chl b. Second axial ligand of BChl a has no influence on the S(1) band, whereas the S(2) transition suffers a -400 to -600 cm(-1) down shift. Aromatic character of solvent is responsible for a ∼-100 cm(-1) red shift of both Q transitions in BChl a. The S(1) bands in chlorophylls are relatively insensitive with respect to dielectric properties and specific solvation. Therefore, nontrivial mechanisms, yielding large site-energy shifts are expected in photosynthetic chlorophyll-proteins.

Control of the blue fluorescent protein with advanced evolutionary pulse shaping

We demonstrate optical coherent control of the two-photon fluorescence of the blue fluorescent protein (BFP), which is of interest in investigations of protein-protein interactions. In addition to biological relevance, BFP represents an interesting target for coherent control from a chemical perspective due to its many components of highly nonexponential fluorescence decay and low quantum yield resulting from excited state isomerization. Using a genetic algorithm with a multiplicative (rather than ratiometric) fitness parameter, we are able to control the ratio of BFP fluorescence to second-harmonic generation without a considerable drop in the maximized signal. The importance of linear chirp and power-scaling on the discrimination process is investigated in detail.

Control of Two-photon Fluorescence of Common Dyes and Conjugated Dyes

We present a comprehensive study of the selective excitation of two-photon fluorescence from various pairs of dyes and dyes in different conjugation states with tailored pulse shapes found with a genetic algorithm (GA). We investigate a number of biologically important dyes, and include dyes conjugated to trastuzumab (Herceptin®) and to a poly(amidoamine) dendrimer. We consider in detail the ability of tailored pulse shaping to discriminate dyes with significant spectral overlap. Our procedure for adaptive pulse shaping includes power-law and chirp-scaling checks to prevent trivial convergences. The GA uses a multiplicative fitness parameter in a graded search method that converges on pulse shapes that not only differentiate two-photon processes, but do so in a high signal regime. We consider the results in terms of not only the absolute maximum ratio of discrimination achieved, but also present the evolutionary course of the GA and compare the improvement to a quantitative measure of the noise level. We also implement a time-domain acousto-optic measurement of two-photon excitation cross-section spectra. The results show that the ability to discriminate dyes is determined almost entirely by their differences in two-photon excitation cross section.

A spectral hole-burning study of long-wavelength chlorophyll a forms in greening leaves at 5 K

Persistent holes have been burnt in the spectra of post‐etiolated maize leaves in the spectral interval 700–745 nm at 5 K. Narrow (< 1 cm−1) holes detectable both in fluorescence and excitation spectra correspond to the zero‐phonon purely electronic S1◀S0 transition of chlorophyll a rather than to the S1←S0 vibronic or S 1 ←S0 higher singlet transitions. The spectral composition of the inhomogeneously broadened absorption and fluorescence contours of long‐wavelength chlorophyll a forms in plants is discussed in terms of the contributions of phonon‐wings, vibronic satellites, purely electronic S1◀S0 transitions and its higher excitonic components.

Energy transfer in ethane-bisporphyrin dimers studied by fluorescence line narrowing and spectral hole burning

Abstract The quasi-line fluorescence excitation spectrum of 1,2-bis (2,3,7,8,12,13,17,18-octaethyl-21 H ,23 H -porphino) ethane at 4.8 K consists of two subbands with the splitting mean value of 51cm −1 , that are ascribed to the donor and the acceptor half of the homodimer. The donors fluorescence is quenched by an efficient energy transfer to the acceptor. The energy transfer rate of 10 11 s −1 , determined by spectral hole burning, has been compared with the calculated value and a conclusion of nonconsistency with the Forster energy transfer mechanism has been drawn.

Physicochemical properties of blue fluorescent protein determined via molecular dynamics simulation

Blue fluorescent protein (BFP) is a mutant of green fluorescent protein (GFP), where the chromophore has been modified to shift the emitted fluorescence into the blue spectral region. In this study, MD calculations were performed with the GROMACS simulation package and AMBER force field to investigate the dependence of BFPs physicochemical properties on temperature and applied pressure. The MD approach enabled us to calculate the compressibility of protein itself, separately from the nontrivial contribution of the hydration shell, which is difficult to achieve experimentally. The computed compressibility of BFP (3.94 x10(-5) MPa(-1)) is in agreement with experimental values of globular proteins. The center-of-mass diffusion coefficient of BFP and its dependence on temperature and pressure, which plays an important role in its application as a probe for intracellular liquid viscosity measurement, was calculated and found to be in good agreement with photobleaching recovery experimental data. We have shown that decreased temperature as well as applied pressure increases the water viscosity, but the concomitant decrease of the BFP diffusion coefficient behaves differently from Stokes-Einstein formula. It is shown that the number of hydrogen bonds around the protein grows with pressure, which explains the aforementioned deviation. Pressure also reduces root mean square (RMS) fluctuations, especially those of the most flexible residues situated in the loops. The analysis of the RMS fluctuations of the backbone Calpha atoms also reveals that the most rigid part of BFP is the center of the beta-barrel, in accord with temperature B factors obtained from the Protein Data Bank.

Fluorescence-detected triplet kinetics study of the specifically solvated chlorophyll a and protochlorophyll in frozen solutions

Abstract The triplet lifetimes (τ T ) of the title compounds have been measured in a number of solvent hosts by using an indirect triplet state detection through fluorescence fading kinetics at 77 K. Three types of complexes with solvent molecules, i.e. with a mono-(L 1 -type) and bisligated (L 2 -type) central magnesium atom and the latter with hydrogen bridges (L 2 H-type), have average τ T values of 2.5, 1.4 and 0.8 ms and 10, 4.5 and 2.5 ms for chlorophyll a (Chl a ) and protochlorophyll (PChl), respectively. Hydrogen-bonded L 1 H-type complexes of chl a occurring in tert-alcohols and perfluoroaniline have the same τ T as L 1 -type solvates. The solvent deuteration isotopic effects show that in L 2 H-complexes (e.g. in methanol) triplet quenching arises partially due to electronic energy transfer to the vibrational over tones of OH groups attached via Mg atom to the central part of Chl a macrocycle. The dependence of fluorescence band-shapes and maxima on the excitation wavelength indicates the presence of several types of pigment-solvent complexes in most matrices. Minor amounts (∼ 1 %) of L 2 - and L 2 H-type solvates on the background of dominating monoligated species could be selectively excited with a 457.9 nm Ar + laser line.

Energy Selection Is Not Correlated in the Qx and Qy Bands of a Mg-Porphyrin Embedded in a Protein

The Qx-Qy splitting observed in the fluorescence excitation spectra of Mg-mesoporphyrin-IX substituted horseradish peroxidase (MgMP-HRP) and of its complex with naphthohydroxamic acid (NHA) was studied by spectral hole burning techniques. The width of a hole directly burnt in the Qy band and that of a satellite hole indirectly produced in Qy as a result of hole burning in Qx was compared. We also studied the dependence of the satellite hole in the Qy band on the burning frequency used in the Qx band. Both the directly and indirectly burnt holes were very broad in the (higher energy) Qy band. The width of the satellite hole in the Qy band was equal to the entire width of the inhomogeneously broadened band, independently from the position of hole burning in Qx. This is indicative of a clear lack of correlation between the electronic transition energies of the Qx and Qy bands. A photoproduct was produced by laser irradiation of the MgMP-HRP/NHA complex and was identified as a species with lowered Q-splitting. Conversion of the photoproduct could be achieved by thermal activation measured in temperature-cycling experiments, with a characteristic temperature of 25 K. We attribute the phototransformation to a conformational change of MgMP.