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Red blood cell (RBC) aggregation and deformation are governed by the molecular processes occurring on the membrane. Since several social important diseases are accompanied by alterations in RBC aggregation and deformability, it is important to develop a diagnostic parameter of RBC membrane structural integrity and stability. In this work, we propose membrane microviscosity assessed by time-resolved fluorescence anisotropy of the lipophilic PKH26 fluorescent probe as a diagnostic parameter. We measured the fluorescence decay curves of the PKH26 probe in the RBC membrane to establish the optimal parameters of the developed fluorescence assay. We observed a complex biphasic profile of the fluorescence anisotropy decay characterized by two correlation times corresponding to the rotational diffusion of free PKH26, and membrane-bounded molecules of the probe. The developed assay allowed us to estimate membrane microviscosity ηm in the range of 100–500 cP depending on the temperature, which paves the way for assessing RBC membrane properties in clinical applications as predictors of blood microrheological abnormalities.

Found in many organisms, water-soluble carotenoproteins are prospective antioxidant nanocarriers for biomedical applications. Yet, the toolkit of characterized carotenoproteins is rather limited: such proteins are either too specific binders of only few different carotenoids, or their ability to transfer carotenoids to various acceptor systems is unknown. Here, by focusing on a recently characterized recombinant ~27-kDa Carotenoid-Binding Protein from Bombyx mori (BmCBP) [Slonimskiy et al., International Journal of Biological Macromolecules 214 (2022): 664-671], we analyze its carotenoid-binding repertoire and potential as a carotenoid delivery module. We show that BmCBP forms productive complexes with both hydroxyl- and ketocarotenoids - lutein, zeaxanthin, astaxanthin, canthaxanthin and a smaller antioxidant, aporhodoxanthinone, but not with β-carotene or retinal, which defines its broad ligand specificity toward xanthophylls valuable to human health. Moreover, His-tagged BmCBP apoform is capable of cost-efficient and scalable enrichment of xanthophylls from various crude methanolic herbal extracts. Upon carotenoid binding, BmCBP remains monomeric and shows a remarkable ability to dynamically shuttle carotenoids to biological membrane models and to unrelated carotenoproteins, which in particular makes from the cyanobacterial Orange Carotenoid Protein a blue-light controlled photoswitch. Furthermore, administration of BmCBP loaded by zeaxanthin stimulates fibroblast growth, which is attractive for cell- and tissue-based assays.

Found in many organisms, soluble carotenoproteins are considered as antioxidant nanocarriers for biomedical applications, although the structural basis for their carotenoid transfer function, a prerequisite for rational bioengineering, is largely unknown. We report crystal structures of the Carotenoid-Binding Protein from Bombyx mori (BmCBP) in apo- and zeaxanthin (ZEA)-bound forms. We use spectroscopy and calorimetry to characterize how ZEA and BmCBP mutually affect each other in the complex, identify key carotenoid-binding residues, confirm their roles by crystallography and carotenoid-binding capacity of BmCBP mutants and reconstitute BmCBP complexes with biomedically-relevant xanthophylls lutein, zeaxanthin, canthaxanthin and astaxanthin. By cost-effectively and scalably solubilizing xanthophylls from various crude herbal extracts, His-tagged BmCBP remains monomeric and forms a dynamic nanocontainer delivering carotenoids to liposomes and to other carotenoid-binding proteins, which in particular makes the Orange Carotenoid Protein, a promising optogenetic tool, photoactive. Furthermore, BmCBP(ZEA) administration stimulates fibroblast growth, which paves the way for its biomedical applications.

STARD3, a steroidogenic acute regulatory lipid transfer protein, was identified as a key xanthophyll-binding protein in the human retina. STARD3 and its homologs in invertebrates are known to bind and transport carotenoids, but this lacks structural elucidation. Here, we report high-resolution crystal structures of the apo- and zeaxanthin (ZEA)-bound carotenoid-binding protein from silkworm Bombyx mori (BmCBP). Having a STARD3-like fold, BmCBP features novel elements, including the Ω1-loop that, in the apoform, is uniquely fixed on the α4-helix by an R173-D279 salt bridge. We exploit absorbance, Raman and dichroism spectroscopy, and calorimetry to describe how ZEA and BmCBP mutually affect each other in the complex. We identify key carotenoid-binding residues, confirm their roles by ZEA-binding capacity and X-ray structures of BmCBP mutants, and also demonstrate that markedly different carotenoid-binding capacities of BmCBP and human STARD3 stem from differences in the structural organization of their carotenoid-binding cavity.

Cell migration is one of the most important processes in which the cytoskeleton plays a main role. The cytoskeleton network is formed by tubulin microtubules, actin filaments, and intermediate filaments (IFs). While the structure and functions of the two aforementioned proteins have been extensively investigated during the last decades, vimentin IFs structure and their role in cell migration and adhesion remain unclear. Here, we investigated polarity determination in rat fibroblasts with either a knocked out vim gene or with a mutation that blocks filament formation on the stage of unit-length filaments (ULFs). Structured illumination microscopy has demonstrated the difference in the morphology of IFs in wild-type fibroblasts and of ULFs in mutant fibroblasts. We have developed an approach to measure cell stiffness separately on the trailing and leading edges using atomic force microscopy. Young's modulus values on the leading and trailing edge of migrating rat fibroblasts differ approximately by two times, being larger on the leading edge. The knockout of the vim gene leads to having comparable values of Young's moduli on both edges. Vimentin-null cells change the direction of migration more frequently than those expressing wild-type or mutated vimentin. Our results have shown the principle role of vimentin, not only in the form of IFs, but also as ULFs, in the determination of the polarity and the directionality of fibroblast migration.

To counteract oxidative stress, antioxidants including carotenoids are highly promising, yet their exploitation is drastically limited by the poor bioavailability and fast photodestruction, whereas current delivery systems are far from being efficient. Here we demonstrate that the recently discovered nanometer-sized water-soluble carotenoprotein fromAnabaena(termed CTDH) transiently interacts with liposomes to efficiently extract carotenoids via carotenoid-mediated homodimerization, yielding violet-purple protein samples amenable to lyophilization and long-term storage. We characterize spectroscopic properties of the pigment-protein complexes and thermodynamics of liposome-protein carotenoid transfer and demonstrate the highly efficient delivery of echinenone form CTDH into liposomes. Most importantly, we show carotenoid delivery to membranes of mammalian cells, which provides protection from reactive oxygen species. The described carotenoprotein may be considered as part of modular systems for the targeted antioxidant delivery.Significance statementCarotenoids are excellent natural antioxidants but their delivery to vulnerable cells is challenging due to their hydrophobic nature and susceptibility to degradation. Thus, systems securing antioxidant stability and facilitating targeted delivery are of great interest for the design of medical agents. In this work, we have demonstrated that soluble cyanobacterial carotenoprotein can deliver echinenone into membranes of liposomes and mammalian cells with almost 70 % efficiency, which alleviates the induced oxidative stress. Our findings warrant the robustness of the protein-based carotenoid delivery for studies of carotenoid activities and effects on cell models.

An increase in growth rates of children suffering from growth hormone deficiency (GHD) subjected to recombinant growth hormone treatment (rGHT) was shown to be accompanied by acceleration of metabolic processes that may stimulate oxygen consumption in various organs and tissues. Therefore, oxygen-transporting properties of RBC should undergo considerable changes during the rGHT. The aim of this study was to examine the effects of rGHT on erythrocyte shape and hemoglobin state in GHD children. The level of oxyhemoglobin (Oxy-Hb) in RBC was analyzed by Raman spectroscopy. The RBC count, mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV) and other parameters were calculated. The blood of eleven treatment-naive prepubertal children with GHD (aged 3–9, median 5.7 years) was examined and compared with control group (aged 5–7; median 6.0 years) at three time points: 0, 3 and 12 months of rGHT. Before rGHT, the MI in GHD children was higher (median 0.48 vs 0.14 p=0.0018) and the RBC count was lower (median 4.20 vs 4.96 10 12 cells/L p=0.0022) than in control group. After the treatment, cell count in GHD patients did not differ significantly from the control group, but Oxy-Hb level became higher (median 0.64 vs 0.41 p=0.0075). During rGHT, MCV decreased (median 80.3 vs 83.2μm 3 p=0.0231). Morphological and functional characteristics of erythrocytes in GHD children were shown to differ significantly from the healthy control group. A twelve-month rGHT partially improved some of the studied parameters but Oxy-Hb level and echinocyte count remained high.

In our previous communication, we have reported that virions of plant Potyvirus Potato Virus A (PVA) have a peculiar structure characterized by high content of disordered regions in intravirus coat protein (CP). In this report, we describe unusual properties of the PVA CP. With the help of a number of physicochemical methods, we have observed that the PVA CP just released from the virions by heating at 60–70 °C undergoes association into oligomers and transition to β- (and even cross-β-) conformation. Transition to β-structure on heating has been recently reported for a number of viral and non-viral proteins. The PVA CP isolated by LiCl method was also transformed into cross-β-structure on heating to 60 °C. Using the algorithms for protein aggregation prediction, we found that the aggregation-prone segments should be located in the central region of a PVA CP molecule. Possibly this transition mimics some functions of PVA CP in the virus life cycle in infected plants.

Orange carotenoid protein (OCP) is the photoactive protein that is responsible for high light tolerance in cyanobacteria. We studied the kinetics of the OCP photocycle by monitoring changes in its absorption spectrum, intrinsic fluorescence, and fluorescence of the Nile red dye bound to OCP. It was demonstrated that all of these three methods provide the same kinetic parameters of the photocycle, namely, the kinetics of OCP relaxation in darkness was biexponential with a ratio of two components equal to 2:1 independently of temperature. Whereas the changes of the absorption spectrum of OCP characterize the geometry and environment of its chromophore, the intrinsic fluorescence of OCP reveals changes in its tertiary structure, and the fluorescence properties of Nile red indicate the exposure of hydrophobic surface areas of OCP to the solvent following the photocycle. The results of molecular-dynamics studies indicated the presence of two metastable conformations of 3′-hydroxyechinenone, which is consistent with characteristic changes in the Raman spectra. We conclude that rotation of the β-ionylidene ring in the C-terminal domain of OCP could be one of the first conformational rearrangements that occur during photoactivation. The obtained results suggest that the photoactivated form of OCP represents a molten globule-like state that is characterized by increased mobility of tertiary structure elements and solvent accessibility.

Conversion of the rod-like tobacco mosaic virus (TMV) virions into “ball-like particles” by thermal denaturation at 90–98 °C had been described by R.G. Hart in 1956. We have reported recently that spherical particles (SPs) generated by thermal denaturation of TMV at 94–98 °C were highly stable, RNA-free, and water-insoluble. The SPs were uniform in shape but varied widely in size (53–800 nm), which depended on the virus concentration. Here, we describe some structural characteristics of SPs using circular dichroism, fluorescence spectroscopy, and Raman spectroscopy. It was found that the structure of SPs protein differs strongly from that of the native TMV and is characterized by coat protein subunits transition from mainly (about 50%) α-helical structure to a structure with low content of α-helices and a significant fraction of β-sheets. The SPs demonstrate strong reaction with thioflavin T suggesting the formation of amyloid-like structures.