Photoinduced electron transfer reactions of pyranine with benzoquinone and titanium dioxide

The fluorescence quenching of pyranine by benzoquinone in acetonitrile medium was studied using steady‐state and time‐resolved fluorescence techniques. The quenching process was characterized by a Stern–Volmer plot, which displayed a linear aspect. From the linear plot, the bimolecular quenching rate constant was obtained. The obtained rate constants are within diffused controlled limits. The results show that benzoquinone can efficiently quench the fluorescence of pyranine with dynamic quenching rate constants in the order of 10¹⁰ M^–1 s^–1, suggesting that the pyranine can act as a good electron donor for photoinduced electron transfer in artificial photosynthesis and organic solar cells. In addition, the electron injection dynamics of a pyranine/titanium dioxide semiconductor film was also investigated and electron injection from the excited state pyranine into the conduction band of titanium dioxide is suggested. These preliminary results hold promise for the possibility of using pyranine in dye‐sensitized solar cells. Copyright © 2016 John Wiley & Sons, Ltd.     

Beta-lactoglobulin assembles into amyloid through sequential aggregated intermediates

We have investigated the aggregation and amyloid fibril formation of bovine β-lactoglobulin variant A, with a focus on the early stages of aggregation. We used noncovalent labeling with thioflavin T and 1-anilino-8-naphthalenesulfonate to follow the conformational changes occurring in β-lactoglobulin during aggregation using time resolved luminescence. 1-Anilino-8-naphthalenesulfonate monitored the involvement of the hydrophobic core/calyx of β-lactoglobulin in the aggregation process. Thioflavin T luminescence monitored the formation of amyloid. The luminescence lifetime distributions of both probes showed changes that could be attributed to conformational changes occurring during and following aggregation. To correlate the luminescence measurements with the degree of aggregation and the morphology of the aggregates, we also measured dynamic light scattering and atomic force microscopy images. We evaluated the relative stability of the intermediates with an assay that is sensitive to aggregation reversibility. Our results suggest that initial aggregation during the first 5 days occurred with partial disruption of the characteristic calyx in β-lactoglobulin. As the globular aggregates grew from days 5 to 16, the calyx was completely disrupted and the globular aggregates became more stable. After this second phase of aggregation, conversion into a fibrillar form occurred, marking the growth phase, and still more changes in the luminescence signals were observed. Based on these observations, we propose a three-step process by which monomer is converted first into weakly associated aggregates, which rearrange into stable aggregates, which eventually convert into protofibrils that elongate in the growth phase.     

Mechanistic role of ergosterol in membrane rigidity and cycloheximide resistance in Saccharomyces cerevisiae

Mutants of Saccharomyces cerevisiae defective in the late steps of ergosterol biosynthesis are viable but accumulate structurally altered sterols within the plasma membrane. Despite the significance of pleiotropic abnormalities in the erg mutants, little is known about how sterol alterations mechanically affect the membrane structure and correlate with individual mutant phenotypes. Here we demonstrate that the membrane order and occurrence of voids are determinants of membrane rigidity and hypersensitivity to a drug. Among five ergΔ mutants, the erg2Δ mutant exhibited the most marked sensitivity to cycloheximide. Notably, measurement of time-resolved anisotropy indicated that the erg2Δ mutation decreased the membrane order parameter (S), and dramatically increased the rotational diffusion coefficient (D_w) of 1-[4-(trimethylamino)pheny]-6-phenyl-1,3,5-hexatriene (TMA-DPH) in the plasma membrane by 8-fold, providing evidence for the requirement of ergosterol for membrane integrity. The IC₅₀ of cycloheximide was closely correlated with S/D_w in these strains, suggesting that the membrane disorder and increasing occurrence of voids within the plasma membrane synergistically enhance passive diffusion of cycloheximide across the membrane. Exogenous ergosterol partially restored the membrane properties in the upc2-1erg2Δ strain. In this study, we describe the ability of ergosterol to adjust the dynamic properties of the plasma membrane, and consider the relevance of drug permeability.     

Ultrafast electron transfer in the recognition of different DNA sequences by a DNA-binding protein with different dynamical conformations

Ultrafast electron transfer (ET) phenomenon in protein and protein–DNA complex is very much crucial and often leads to the regulation of various kinds of redox reactions in biological system. Although, the conformation of the protein in protein–DNA complex is concluded to play the key role in the ET process, till date very little evidences exist in the literature. λ-repressor–operator DNA interaction, particularly O_R1 and O_R2, is a key component of the λ-genetic switch and is a model system for understanding the chemical principles of the conformation-dependent ET reaction, governed by differential protein dynamics upon binding with different DNA target sequences. Here, we have explored the photoinduced electron transfer from the tryptophan moieties of the protein λ-repressor to two operators DNA of different sequences (O_R1 and O_R2) using picosecond-resolved fluorescence spectroscopy. The enhanced flexibility and different conformation of the C-terminal domain of the repressor upon complexation with O_R1 DNA compared to O_R2 DNA are found to have pronounced effect on the rate of ET. We have also observed the ET phenomenon from a dansyl chromophore, bound to the lysine residue, distal from the DNA-binding domain of the protein to the operator DNA with a specific excitation at 299?nm wavelength. The altered ET dynamics as a consequence of differential protein conformation upon specific DNA sequence recognition may have tremendous biological implications.     

Microsecond subdomain folding in dihydrofolate reductase

The characterization of microsecond dynamics in the folding of multisubdomain proteins has been a major challenge in understanding their often complex folding mechanisms. Using a continuous-flow mixing device coupled with fluorescence lifetime detection, we report the microsecond folding dynamics of dihydrofolate reductase (DHFR), a two-subdomain α/β/α sandwich protein known to begin folding in this time range. The global dimensions of early intermediates were monitored by Förster resonance energy transfer, and the dynamic properties of the local Trp environments were monitored by fluorescence lifetime detection. We found that substantial collapse occurs in both the locally connected adenosine binding subdomain and the discontinuous loop subdomain within 35 μs of initiation of folding from the urea unfolded state. During the fastest observable ∼ 550 μs phase, the discontinuous loop subdomain further contracts, concomitant with the burial of Trp residue(s), as both subdomains achieve a similar degree of compactness. Taken together with previous studies in the millisecond time range, a hierarchical assembly of DHFR—in which each subdomain independently folds, subsequently docks, and then anneals into the native conformation after an initial heterogeneous global collapse—emerges. The progressive acquisition of structure, beginning with a continuously connected subdomain and spreading to distal regions, shows that chain entropy is a significant organizing principle in the folding of multisubdomain proteins and single-domain proteins. Subdomain folding also provides a rationale for the complex kinetics often observed.     

The time course of non-photochemical quenching in phycobilisomes of Synechocystis sp. PCC6803 as revealed by picosecond time-resolved fluorimetry

As high-intensity solar radiation can lead to extensive damage of the photosynthetic apparatus, cyanobacteria have developed various protection mechanisms to reduce the effective excitation energy transfer (EET) from the antenna complexes to the reaction center. One of them is non-photochemical quenching (NPQ) of the phycobilisome (PB) fluorescence. In Synechocystis sp. PCC6803 this role is carried by the orange carotenoid protein (OCP), which reacts to high-intensity light by a series of conformational changes, enabling the binding of OCP to the PBs reducing the flow of energy into the photosystems. In this paper the mechanisms of energy migration in two mutant PB complexes of Synechocystis sp. were investigated and compared. The mutant CK is lacking phycocyanin in the PBs while the mutant ΔPSI/PSII does not contain both photosystems. Fluorescence decay spectra with picosecond time resolution were registered using a single photon counting technique. The studies were performed in a wide range of temperatures — from 4 to 300 K. The time course of NPQ and fluorescence recovery in darkness was studied at room temperature using both steady-state and time-resolved fluorescence measurements. The OCP induced NPQ has been shown to be due to EET from PB cores to the red form of OCP under photon flux densities up to 1000 μmol photons m^− 2 s^− 1. The gradual changes of the energy transfer rate from allophycocyanin to OCP were observed during the irradiation of the sample with blue light and consequent adaptation to darkness. This fact was interpreted as the revelation of intermolecular interaction between OCP and PB binding site. At low temperatures a significantly enhanced EET from allophycocyanin to terminal emitters has been shown, due to the decreased back transfer from terminal emitter to APC. The activation of OCP not only leads to fluorescence quenching, but also affects the rate constants of energy transfer as shown by model based analysis of the decay associated spectra. The results indicate that the ability of OCP to quench the fluorescence is strongly temperature dependent. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

The site of regulation of light capture in Symbiodinium: Does the peridinin–chlorophyll a–protein detach to regulate light capture?

Dinoflagellates from the genus Symbiodinium form symbiotic associations with cnidarians including corals and anemones. The photosynthetic apparatuses of these dinoflagellates possess a unique photosynthetic antenna system incorporating the peridinin–chlorophyll a–protein (PCP). It has been proposed that the appearance of a PCP-specific 77 K fluorescence emission band around 672–675 nm indicates that high light treatment results in PCP dissociation from intrinsic membrane antenna complexes, blocking excitation transfer to the intrinsic membrane-bound antenna complexes, chlorophyll a–chlorophyll c₂–peridinin–protein-complex (acpPC) and associated photosystems (Reynolds et al., 2008 Proc Natl Acad Sci USA 105:13674–13678).We have tested this model using time-resolved fluorescence decay kinetics in conjunction with global fitting to compare the time-evolution of the PCP spectral bands before and after high light exposure. Our results show that no long-lived PCP fluorescence emission components appear either before or after high light treatment, indicating that the efficiency of excitation transfer from PCP to membrane antenna systems remains efficient and rapid even after exposure to high light. The apparent increased relative emission at around 675 nm was, instead, caused by strong preferential exciton quenching of the membrane antenna complexes associated with acpPC and reaction centers. This strong non-photochemical quenching (NPQ) is consistent with the activation of xanthophyll-associated quenching mechanisms and the generally-observed avoidance in nature of long-lived photoexcited states that can lead to oxidative damage. The acpPC component appears to be the most strongly quenched under high light exposure suggesting that it houses the photoprotective exciton quencher.     

Microsecond Barrier-Limited Chain Collapse Observed by Time-Resolved FRET and SAXS

It is generally held that random-coil polypeptide chains undergo a barrier-less continuous collapse when the solvent conditions are changed to favor the fully folded native conformation. We test this hypothesis by probing intramolecular distance distributions during folding in one of the paradigms of folding reactions, that of cytochrome c. The Trp59-to-heme distance was probed by time-resolved Förster resonance energy transfer in the microsecond time range of refolding. Contrary to expectation, a state with a Trp59–heme distance close to that of the guanidinium hydrochloride (GdnHCl) denatured state is present after ~ 27 μs of folding. A concomitant decrease in the population of this state and an increase in the population of a compact high-FRET (Förster resonance energy transfer) state (efficiency > 90%) show that the collapse is barrier limited. Small-angle X-ray scattering (SAXS) measurements over a similar time range show that the radius of gyration under native favoring conditions is comparable to that of the GdnHCl denatured unfolded state. An independent comprehensive global thermodynamic analysis reveals that marginally stable partially folded structures are also present in the nominally unfolded GdnHCl denatured state. These observations suggest that specifically collapsed intermediate structures with low stability in rapid equilibrium with the unfolded state may contribute to the apparent chain contraction observed in previous fluorescence studies using steady-state detection. In the absence of significant dynamic averaging of marginally stable partially folded states and with the use of probes sensitive to distance distributions, barrier-limited chain contraction is observed upon transfer of the GdnHCl denatured state ensemble to native-like conditions.     

Time-resolved fluorescence of 2-aminopurine in DNA duplexes in the presence of the EcoP15I Type III restriction–modification enzyme

EcoP15I is a Type III DNA restriction and modification enzyme of Escherichia coli. We show that it contains two modification (Mod) subunits for sequence-specific methylation of DNA and one copy of a restriction endonuclease (Res) subunit for cleavage of DNA containing unmethylated target sequences. Previously the Mod₂ dimer in the presence of cofactors was shown to use nucleotide flipping to gain access to the adenine base targeted for methylation (Reddy and Rao, J. Mol. Biol. 298 (2000) 597–610.). Surprisingly the Mod₂ enzyme also appeared to flip a second adenine in the target sequence, one which was not subject to methylation. We show using fluorescence lifetime measurements of the adenine analogue, 2-aminopurine, that only the methylatable adenine undergoes flipping by the complete Res₁Mod₂ enzyme and that this occurs even in the absence of cofactors. We suggest that this is due to activation of the Mod₂ core by the Res subunit.