DNA binding to trans- and cis-isomers of azobenzene containing cationic surfactant in 5 mM NaCl solution was investigated by the methods of dynamic light scattering (DLS), low-gradient viscometry (LGV), atomic force microscopy (AFM), circular dichroism (CD), gel electrophoresis (GE), flow birefringence (FB), UV-Vis spectrophotometry. Light-responsive conformational transitions of DNA in complex with photosensitive surfactant, changes in DNA optical anisotropy and persistent length, phase transition of DNA into nanoparticles induced by high surfactant concentration, as well as transformation of surfactant conformation under its binding to macromolecule were studied. Computer simulations of micelles formation for cis- and trans-isomers of azobenzene containing surfactant, as well as DNA-surfactant interaction, were carried out. Phase diagram for DNA-surfactant solutions was designed. The possibility to reverse the DNA packaging induced by surfactant binding with the dilution and light irradiation was shown.

the photoactive orange carotenoid protein (ocp) plays a key role in cyanobacterial photoprotection. In OCP, a single non-covalently bound keto-carotenoid molecule acts as a light intensity sensor, while the protein is responsible for forming molecular contacts with the light-harvesting antenna, the fluorescence of which is quenched by OCP. Activation of this physiological interaction requires signal transduction from the photoexcited carotenoid to the protein matrix. Recent works revealed an asynchrony between conformational transitions of the carotenoid and the protein. Intrinsic tryptophan (Trp) fluorescence has provided valuable information about the protein part of OCP during its photocycle. However, wild-type OCP contains five Trp residues, which makes extraction of site-specific information impossible. In this work, we overcame this problem by characterizing the photocycle of a fully photoactive OCP variant (OCP-3FH) with only the most critical tryptophan residue (Trp-288) in place. Trp-288 is of special interest because it forms a hydrogen bond to the carotenoid's keto-oxygen to keep OCP in its dark-adapted state. Using femtosecond pump-probe fluorescence spectroscopy we analyzed the photocycle of OCP-3FH and determined the formation rate of the very first intermediate suggesting that generation of the recently discovered S* state of the carotenoid in OCP precedes the breakage of the hydrogen bonds. Therefore, following Trp fluorescence of the unique photoactive OCP-3FH variant, we identified the rate of the H-bond breakage and provided novel insights into early events accompanying photoactivation of wild-type OCP. The regulation of excitation energy flows in photosynthetic organisms plays a crucial role for improving biomass production in changing environmental conditions 1. Carotenoids as essential photosynthetic pigments can contribute to light-harvesting or act as excitation quenchers depending on the structural organization of antennas and the carotenoid state 2 , resulting in excitation energy transfer (EET) to or from chlorophylls, respectively. EET can be conducted by several mechanisms: transfer to (or from) the carotenoid S 1 excited level 3 , exciton coupling 4,5 and charge transfer 6,7. Although functional roles of carotenoids in light-harvesting antennas are clear, there are still debates considering the involvement of specific EET mechanisms in these roles. The situation is additionally complicated by non-Condon nature of the pigment interaction with regard to wave-like EET process and the occurrence of excitation energy coherence 8,9. Regulation of light-harvesting depends on pH 10 , carotenoid content 11 , lipid content 12 , protein-protein interactions 13 , and other factors 14 , thus highlighting the central role

Here, we propose a possible photoactivation mechanism of a 35-kDa blue light-triggered photoreceptor, the Orange Carotenoid Protein (OCP), suggesting that the reaction involves the transient formation of a protonated ketocarotenoid (oxocarbenium cation) state. Taking advantage of engineering an OCP variant carrying the Y201W mutation, which shows superior spectroscopic and structural properties, it is shown that the presence of Trp201 augments the impact of one critical H-bond between the ketocarotenoid and the protein. This confers an unprecedented homogeneity of the dark-adapted OCP state and substantially increases the yield of the excited photoproduct S*, which is important for the productive photocycle to proceed. A 1.37 Å crystal structure of OCP Y201W combined with femtosecond time-resolved absorption spectroscopy, kinetic analysis, and deconvolution of the spectral intermediates, as well as extensive quantum chemical calculations incorporating the effect of the local electric field, highlighted the role of charge-transfer states during OCP photoconversion.

Development of CAR‐T therapy led to immediate success in the treatment of B cell leukemia. Manufacturing of therapy‐competent functional CAR‐T cells needs robust protocols for ex vivo/in vitro expansion of modified T‐cells. This step is challenging, especially if non‐viral low‐efficiency delivery protocols are used to generate CAR‐T cells. Modern protocols for CAR‐T cell expansion are imperfect since non‐specific stimulation results in rapid outgrowth of CAR‐negative T cells, and removal of feeder cells from mixed cultures necessitates additional purification steps. To develop a specific and improved protocol for CAR‐T cell expansion, cell‐derived membrane vesicles are taken advantage of, and the simple structural demands of the CAR‐antigen interaction. This novel approach is to make antigenic microcytospheres from common cell lines stably expressing surface‐bound CAR antigens, and then use them for stimulation and expansion of CAR‐T cells. The data presented in this article clearly demonstrate that this protocol produced antigen‐specific vesicles with the capacity to induce stronger stimulation, proliferation, and functional activity of CAR‐T cells than is possible with existing protocols. It is predicted that this new methodology will significantly advance the ability to obtain improved populations of functional CAR‐T cells for therapy.

The heterogeneity of metabolic reactions leads to a non-uniform distribution of temperature in different parts of the living cell. The demand to study normal functioning and pathological abnormalities of cellular processes requires the development of new visualization methods. Previously, we have shown that the 35-kDa photoswitchable Orange Carotenoid Protein (OCP) has a strong temperature dependency of photoconversion rates, and its tertiary structure undergoes significant structural rearrangements upon photoactivation, which makes this protein a nano-sized temperature sensor. However, the determination of OCP conversion rates requires measurements of carotenoid absorption, which is not suitable for microscopy. In order to solve this problem, we fused green and red fluorescent proteins (TagGFP and TagRFP) to the structure of OCP, producing photoactive chimeras. In such chimeras, electronic excitation of the fluorescent protein is effectively quenched by the carotenoid in OCP. Photoactivation of OCP-based chimeras triggers rearrangements of complex geometry, permitting measurements of the conversion rates by monitoring changes of fluorescence intensity. This approach allowed us to determine the local temperature of the microenvironment. Future directions to improve the OCP-based sensor are discussed.