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This study employs a combination of spectroscopic techniques, including absorption, photoluminescence (PL), time-resolved PL kinetics, and transient absorption measurements, to elucidate the dynamics of excited states of oleic acid (OA)-capped colloidal PbS quantum dots (QDs) in toluene with a mean size of approximately 3 nm. PL decay profiles were properly treated employing either fitting or fitting-free models to extract average decay lifetimes and lifetime distributions. Our findings highlight the profound impact of factors such as particle concentration, size, surface treatment, and the surrounding environment on the optical properties of PbS QDs. We observed a notable blue shift in both absorption and emission spectra, along with shorter decay lifetimes, when the concentration of the particles was reduced. This effect was attributed to the dynamics of concentration-selective ligand desorption on the surface of PbS QDs. An observed distinct peak in the spectral dispersion of average lifetimes was linked to an external origin, which was proved by the addition of varying OA volumes, evidently affecting these features. This study also explores how the PL emission of PbS QD suspensions varies with experimental parameters, such as excitation pulse duration and laser power. A gradual reduction in average lifetimes was demonstrated under the modulation of pulse duration from 15 μs to 150 ns, illustrating a transition from continuous-wave (cw) excitation to quasi-pulsed excitation. This transition was shown to be accompanied by a noticeable shift in the lifetime distributions towards shorter durations, as a consequence of various onset components in the QD ensemble. Comparing with truly pulsed excitation, we experimentally confirmed that the amplitude-average lifetime under cw excitation matches the intensity-average lifetime under pulsed excitation. Lastly, we noted linear and nonlinear behaviors in the PL amplitude as a function of excitation power, with the average lifetime remaining nearly constant under pulsed excitation conditions.

The LutR protein represses the transcription of genes encoding enzymes for the utilization of l-lactate in Bacillus subtilis through binding to a specific DNA region. In this study, we employed oligonucleotide probes modified by viscosity-sensitive tetramethylated thiophene-BODIPY fluorophores to investigate the impact of selected metabolites on the LutR-DNA complex. Our goal was to identify the effector molecule whose binding alters the protein-DNA affinity, thereby enabling gene transcription. The designed DNA probes exhibited distinctive responses to the binding and release of the protein, characterized by significant alterations in fluorescence lifetime. Through this method, we have identified l-lactate as the sole metabolite exerting a substantial modulating effect on the protein-DNA interaction and thus confirmed its role as an effector molecule. Moreover, we showed that our approach was able to follow conformation changes affecting affinity, which were not captured by other methods commonly used to study the protein-DNA interaction, such as electro-mobility shift assays and florescence anisotropy binding studies. This work underlines the potential of environment-sensitive fluorophore-linked nucleotide modifications, i.e. dC TBdp , for studying the dynamics and subtle changes of protein-DNA interactions., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

A modified 2'-deoxycytidine triphosphate derivative (dC TO TP) bearing a thiazole orange moiety tethered via an oligoethylene glycol linker was designed and synthesized. The nucleotide was incorporated into DNA by DNA polymerases in vitro as well as in live cells. Upon incorporation of dC TO TP into DNA, the thiazole orange moiety exhibited a fluorescence lifetime that differed significantly from the non-incorporated (i.e. free and non-covalently intercalated) forms of dC TO TP. When dC TO TP was delivered into live U-2 OS cells using a synthetic nucleoside triphosphate transporter, it allowed us to distinguish and monitor cells that were actively synthesizing DNA in real time, from the very first moments after the treatment. We anticipate that this probe could be used to study chromatin organization and dynamics., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Molecules of fluorescent proteins (FPs) exhibit distinct optical directionality. This optical directionality is characterized by transition dipole moments (TDMs), and their orientation with respect to the molecular structures. Although our recent observations of FP crystals allowed us to determine the mean TDM directions with respect to the framework of representative FP molecules, the dynamics of TDM orientations within FP molecules remain to be ascertained. Here we describe the results of our investigations of the dynamics of TDM directions in the fluorescent proteins eGFP, mTurquoise2 and mCherry, through time-resolved fluorescence polarization measurements and microsecond time scale all-atom molecular dynamics (MD) simulations. The investigated FPs exhibit initial fluorescence anisotropies ( r 0 ) consistent with significant differences in the orientation of the excitation and emission TDMs. However, based on MD data, we largely attribute this observation to rapid (sub-nanosecond) fluorophore motions within the FP molecular framework. Our results allow improved determinations of orientational distributions of FP molecules by polarization microscopy, as well as more accurate interpretations of fluorescence resonance energy transfer (FRET) observations.

Electron transfer within and between proteins is a fundamental biological phenomenon, in which efficiency depends on several physical parameters. We have engineered a number of horse heart cytochrome c single-point mutants with cysteine substitutions at various positions of the protein surface. To these cysteines, as well as to several native lysine side chains, the photoinduced redox label 8-thiouredopyrene-1,3,6-trisulfonate (TUPS) was covalently attached. The long-lived, low potential triplet excited state of TUPS, generated with high quantum efficiency, serves as an electron donor to the oxidized heme c . The rates of the forward (from the label to the heme) and the reverse (from the reduced heme back to the oxidized label) electron transfer reactions were obtained from multichannel and single wavelength flash photolysis absorption kinetic experiments. The electronic coupling term and the reorganization energy for electron transfer in this system were estimated from temperature-dependent experiments and compared with calculated parameters using the crystal and the solution NMR structure of the protein. These results together with the observation of multiexponential kinetics strongly support earlier conclusions that the flexible arm connecting TUPS to the protein allows several shortcut routes for the electron involving through space jumps between the label and the protein surface.

Fluorescent molecules are like antennas: The rate at which they absorb light depends on their orientation with respect to the incoming light wave, and the apparent intensity of their emission depends on their orientation with respect to the observer. However, the directions along which the most important fluorescent molecules in biology, fluorescent proteins (FPs), absorb and emit light are generally not known. Our optical and X-ray investigations of FP crystals have now allowed us to determine the molecular orientations of the excitation and emission transition dipole moments in the FPs mTurquoise2, eGFP, and mCherry, and the photoconvertible FP mEos4b. Our results will allow using FP directionality in studies of molecular and biological processes, but also in development of novel bioengineering and bioelectronics applications., Competing Interests: Competing interest statement: J.L. is a coowner of Innovative Bioimaging, L.L.C., which, however, did not contribute to the current work., (Copyright © 2020 the Author(s). Published by PNAS.)

The absorption cross section (ACS) of silicon nanocrystals (Si NCs) in single-layer and multilayer structures with variable thickness of oxide barriers is determined via a photoluminescence (PL) modulation technique that is based on the analysis of excitation intensity-dependent PL kinetics under modulated pumping. We clearly demonstrate that roughly doubling the barrier thickness (from ca. 1 to 2.2 nm) induces a decrease of the ACS by a factor of 1.5. An optimum separation barrier thickness of ca. 1.6 nm is calculated to maximize the PL intensity yield. This large variation of ACS values with barrier thickness is attributed to a modulation of either defect population states or of the efficiency of energy transfer between confined NC layers. An exponential decrease of the ACS with decreasing temperature down to 120 K can be explained by smaller occupation number of phonons and expansion of the band gap of Si NCs at low temperatures. This study clearly shows that the ACS of Si NCs cannot be considered as independent on experimental conditions and sample parameters.

Room temperature transient absorption spectroscopy with nanosecond resolution was used to study quenching of the chlorophyll triplet states by carotenoids in two light-harvesting complexes of the dinoflagellate Amphidinium carterae: the water soluble peridinin-chlorophyll protein complex and intrinsic, membrane chlorophyll a-chlorophyll c2-peridinin protein complex. The combined study of the two complexes facilitated interpretation of a rather complicated relaxation observed in the intrinsic complex. While a single carotenoid triplet state was resolved in the peridinin-chlorophyll protein complex, evidence of at least two different carotenoid triplets was obtained for the intrinsic light-harvesting complex. Most probably, each of these carotenoids protects different chlorophylls. In both complexes the quenching of the chlorophyll triplet states by carotenoids occurs with a very high efficiency (~100%), and with transfer times estimated to be in the order of 0.1ns or even faster. The triplet-triplet energy transfer is thus much faster than formation of the chlorophyll triplet states by intersystem crossing. Since the triplet states of chlorophylls are formed during the whole lifetime of their singlet states, the apparent lifetimes of both states are the same, and observed to be equal to the carotenoid triplet state rise time (~5ns)., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The photocycle of photoactive yellow protein was studied by kinetic absorption spectroscopy from below 100ns to seconds, at moderately alkaline pH, in the presence of high concentrations of various salts. Chemometric analysis combined with multiexponential fit of the flash-induced difference spectra provided evidence for five intermediates, including a spectrally silent form before the final recovery of the parent state, but only three with significantly distinct spectra. The calculated intermediate spectra constituted the input for the following spectrotemporal model fit using a sufficiently complex photocycle scheme with reversible transitions. This yielded the rate coefficients of the molecular transitions, the final spectra and the kinetics of the intermediates. Except for the transition between the two red shifted (early) intermediates (pR1 and pR2) and the final photocycle step, all reactions appeared to be reversible. Kosmotropic and chaotropic cosolutes had a systematic effect on the molecular rate coefficients. The largest effect, associated presumably with the exposure of the hydrophobic interior of the protein, accompanies the transition between the second red-shifted and the first blue-shifted intermediate (pR2 and pB1, respectively), i.e. it coincides with the chromophore protonation. The dependence of the rate coefficients on the Hofmeister cosolutes suggests that the conformational change of photoactive yellow protein leading eventually to the most unfolded signaling state takes place in several steps, and starts already with the relaxation after the chromophore isomerization in the microsecond time domain., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The purified photosynthetic reaction center protein (RC) from Rhodobacter sphaeroides R-26 purple bacteria was bound to porous silicon microcavities (PSiMc) either through silane-glutaraldehyde (GTA) chemistry or via a noncovalent peptide cross-linker. The characteristic resonance mode in the microcavity reflectivity spectrum red shifted by several nanometers upon RC binding, indicating the protein infiltration into the porous silicon (PSi) photonic structure. Flash photolysis experiments confirmed the photochemical activity of RC after its binding to the solid substrate. The kinetic components of the intraprotein charge recombination were considerably faster (τ(fast) = 14 (±9) ms, τ(slow) = 230 (±28) ms with the RC bound through the GTA cross-linker and only τ(fast) = 27 (±3) ms through peptide coating) than in solution (τ(fast) = 120 (±3) ms, τ(slow) = 1387 (±2) ms), indicating the effect of the PSi surface on the light-induced electron transfer in the protein. The PSi/RC complex was found to oxidize the externally added electron donor, mammalian cytochrome c, and the cytochrome oxidation was blocked by the competitive RC inhibitor, terbutryne. This fact indicates that the specific surface binding sites on the PSi-bound RC are still accessible to external cofactors and an electronic interaction with redox components in the aqueous environment is possible. This new type of biophotonic material is considered to be an excellent model for new generation applications at the interface of silicon-based electronics and biological redox systems designed by nature.

In the present work we demonstrate that FT-IR spectroscopy is a powerful tool for the time resolved and noninvasive measurement of multi-substrate/product interactions in complex metabolic networks as exemplified by the oscillating glycolysis in yeast extract. We found that many of the glycolytic intermediates can be identified with FT-IR spectroscopy. For this, we have constructed a spectral library of most of the glycolytic intermediates and obtained the kinetics of single components in spectra from glycolysing yeast extract by the use of mathematical fitting procedures. The results are in good agreement with the known phase relationships of oscillatory glycolysis. They provide the basis for future application of this method to investigate the energy metabolism of living cells.