Evgeny A. Shirshin

The purple Trp288Ala mutant of Synechocystis OCP persistently quenches phycobilisome fluorescence and tightly interacts with FRP.

In Cyanobacteria, the Orange Carotenoid Protein (OCP) and Fluorescence Recovery Protein (FRP) are central to the photoprotective mechanism consisting in regulated quenching of phycobilisome (PBs) fluorescence. Due to a transient and flexible nature of the light-activated red quenching form, OCPR, which is obtained from the stable dark-adapted orange form, OCPO, by photoconversion, the detailed mechanism of photoprotection remains unclear. Here we demonstrate that our recently described W288A mutant of the Synechocystis OCP (hereinafter called OCPW288A) is a fully functional analogue of the OCPR form which is capable of constitutive PBs fluorescence quenching in vitro with no need of photoactivation. This PBs quenching effect is abolished in the presence of FRP, which interacts with OCPW288A with micromolar affinity and an apparent stoichiometry of 1:1, unexpectedly, implying dissociation of the FRP dimers. This establishes OCPW288A as a robust model system providing novel insights into the interplay between OCP and FRP to regulate photoprotection in cyanobacteria.

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 μmolphotonsm⁻²s⁻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.

Two-photon autofluorescence lifetime imaging of human skin papillary dermis in vivo : assessment of blood capillaries and structural proteins localization

The papillary dermis of human skin is responsible for its biomechanical properties and for supply of epidermis with chemicals. Dermis is mainly composed of structural protein molecules, including collagen and elastin, and contains blood capillaries. Connective tissue diseases, as well as cardiovascular complications have manifestations on the molecular level in the papillary dermis (e.g. alteration of collagen I and III content) and in the capillary structure. In this paper we assessed the molecular structure of internal and external regions of skin capillaries using two-photon fluorescence lifetime imaging (FLIM) of endogenous compounds. It was shown that the capillaries are characterized by a fast fluorescence decay, which is originated from red blood cells and blood plasma. Using the second harmonic generation signal, FLIM segmentation was performed, which provided for spatial localization and fluorescence decay parameters distribution of collagen I and elastin in the dermal papillae. It was demonstrated that the lifetime distribution was different for the inner area of dermal papillae around the capillary loop that was suggested to be due to collagen III. Hence, we propose a generalized approach to two-photon imaging of the papillary dermis components, which extends the capabilities of this technique in skin diagnosis.

Laser fluorimetry of proteins containing one and two tryptophan residues

The true molecular photophysical parameters’ values of tryptophan residues in single-tryptophan-containing and two-tryptophan-containing proteins (by the example of human serum albumin and bovine serum albumin) have been determined for the first time with the use of the authors’ suggested algorithm. The algorthm is based on the simultaneous use of nonlinear and kinetic-laser fluorimetry. The obtained result opens up new approaches in the monitoring of living systems and other objects, containing single fluorophores and localized pairs of fluorophores.

Native fluorescence spectroscopy of blood plasma of rats with experimental diabetes: identifying fingerprints of glucose-related metabolic pathways.

Abstract. We present the results of a native fluorescence spectroscopy study of blood plasma of rats with experimental diabetes. It was shown that the fluorescence emission band shape at 320 nm excitation is the most indicative of hyperglycemia in the blood plasma samples. We provide the interpretation of this fact based on the changes in reduced nicotinamide adenine dinucleotide phosphate concentration due to glucose-related metabolic pathways and protein fluorescent cross-linking formation following nonenzymatic glycation.

Interaction of the signaling state analog and the apoprotein form of the orange carotenoid protein with the fluorescence recovery protein

Photoprotection in cyanobacteria relies on the interplay between the orange carotenoid protein (OCP) and the fluorescence recovery protein (FRP) in a process termed non-photochemical quenching, NPQ. Illumination with blue-green light converts OCP from the basic orange state (OCPO) into the red-shifted, active state (OCPR) that quenches phycobilisome (PBs) fluorescence to avoid excessive energy flow to the photosynthetic reaction centers. Upon binding of FRP, OCPR is converted to OCPO and dissociates from PBs; however, the mode and site of OCPR/FRP interactions remain elusive. Recently, we have introduced the purple OCPW288A mutant as a competent model for the signaling state OCPR (Sluchanko et al., Biochim Biophys Acta 1858:1–11, 2017). Here, we have utilized fluorescence labeling of OCP at its native cysteine residues to generate fluorescent OCP proteins for fluorescence correlation spectroscopy (FCS). Our results show that OCPW288A has a 1.6(±0.4)-fold larger hydrodynamic radius than OCPO, supporting the hypothesis of domain separation upon OCP photoactivation. Whereas the addition of FRP did not change the diffusion behavior of OCPO, a substantial compaction of the OCPW288A mutant and of the OCP apoprotein was observed. These results show that sufficiently stable complexes between FRP and OCPW288A or the OCP apoprotein are formed to be detected by FCS. 1:1 complex formation with a micromolar apparent dissociation constant between OCP apoprotein and FRP was confirmed by size-exclusion chromatography. Beyond the established OCP/FRP interaction underlying NPQ cessation, the OCP apoprotein/FRP interaction suggests a more general role of FRP as a scaffold protein for OCP maturation.

The photocycle of orange carotenoid protein conceals distinct intermediates and asynchronous changes in the carotenoid and protein components

The 35-kDa Orange Carotenoid Protein (OCP) is responsible for photoprotection in cyanobacteria. It acts as a light intensity sensor and efficient quencher of phycobilisome excitation. Photoactivation triggers large-scale conformational rearrangements to convert OCP from the orange OCPO state to the red active signaling state, OCPR, as demonstrated by various structural methods. Such rearrangements imply a complete, yet reversible separation of structural domains and translocation of the carotenoid. Recently, dynamic crystallography of OCPO suggested the existence of photocycle intermediates with small-scale rearrangements that may trigger further transitions. In this study, we took advantage of single 7 ns laser pulses to study carotenoid absorption transients in OCP on the time-scale from 100 ns to 10 s, which allowed us to detect a red intermediate state preceding the red signaling state, OCPR. In addition, time-resolved fluorescence spectroscopy and the assignment of carotenoid-induced quenching of different tryptophan residues derived thereof revealed a novel orange intermediate state, which appears during the relaxation of photoactivated OCPR to OCPO. Our results show asynchronous changes between the carotenoid- and protein-associated kinetic components in a refined mechanistic model of the OCP photocycle, but also introduce new kinetic signatures for future studies of OCP photoactivity and photoprotection.

The effect of lead cations on the fluorescence characteristics of bovine serum albumin in aqueous solution

The effect of lead (heavy metal) cations on the fluorescence characteristics and photophysical parameters (fluorescence intensity and anisotropy, absorption cross section, excited state lifetime, and rates of singlet-triplet conversion and reversible photobleaching) of tryptophan in an aqueous solution of bovine serum albumin (two-tryptophan protein) is studied and compared with the effect in the aqueous solution of tryptophan. It is demonstrated that the effect of lead on the fluorescence characteristics of the protein is manifested at a molar concentration ratio of metal cations and protein macromolecules of greater than 10 and related to the dynamic quenching of the excited state, protein aggregation, and an increase in the rate of singlet-triplet conversion (the effect of a heavy atom) in tryptophan molecules.

Localized donor-acceptor pairs of fluorophores: Determination of the energy transfer rate by nonlinear fluorimetry

A laser fluorescence method has been developed for determining the energy transfer rate. It is based on the model of collective states of localized donor-acceptor pairs of fluorophores proposed in our previous work and involves nonlinear fluorimetry (other photophysical parameters of the donor and acceptor are simultaneously determined). The proposed approach has been tested on mRFP1 fluorescent (red) protein.

Tyrosine fluorescence probing of conformational changes in tryptophan-lacking domain of albumins

We addressed the possibility of using tyrosine (Tyr) fluorescence for monitoring conformational changes of proteins which are undetectable via tryptophan (Trp) fluorescence. The model objects, human (HSA) and bovine (BSA) serum albumins, contain one and two Trp residues, respectively, while Tyr is more uniformly distributed over their structure. The results of the investigation of albumins interaction with ethanol using intrinsic Trp and Tyr steady-state and time-resolved picosecond fluorescence indicated the presence of an intermediate at 10% (v/v) of ethanol in solution, that was supported by the results of extrinsic fluorescence measurements with the Nile Red dye. Based on the comparison of HSA and BSA Trp and Tyr fluorescence, it was suggested that conformational changes at low ethanol concentration are located in the domain III of albumins, which lacks tryptophan residues. The sensitivity of Tyr fluorescence to domain III alterations was further verified by studying albumins interaction with GdnHCl.