Temperature studies of Raman spectra in MnBi2Te4 and MnSb2Te4 magnetic topological insulators

Raman spectra of magnetic topological crystalline insulators in a wide temperature range including the magnetic ordering region are studied in detail. The anharmonicity parameters and Grüneisen mode parameters of Raman-active phonons in the studied crystals have been determined. It has been shown that the temperature dependence of the frequency of the (~48 cm–1) phonon in MnBi2Te4 coincides within ±0.1 cm–1 with the standard anharmonic model disregarding the spin–phonon coupling. The polarization dependences of Raman spectra in the MnSb2Te4 crystals indicate that Sb and Mn atoms are strongly mixed in them unlike the isostructural MnBi2Te4 crystals.This work was supported by the Azerbaijan Ministry of Science and Education (program “Development of the Preparation Technology of Multifunctional Convertors Based on Nanostructures”). E.V.C. acknowledges the s-upport of St. Petersburg State University (project no. 94031444).Peer reviewe

Soluble Cyanobacterial Carotenoprotein as a Robust Antioxidant Nanocarrier and Delivery Module

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 from Anabaena sp. PCC 7120 (termed AnaCTDH) transiently interacts with liposomes to efficiently extract carotenoids via carotenoid-mediated homodimerization, yielding violet–purple protein samples. We characterize the spectroscopic properties of the obtained pigment–protein complexes and the thermodynamics of liposome–protein carotenoid transfer and demonstrate the delivery of carotenoid echinenone from AnaCTDH into liposomes with an efficiency of up to 70 ± 3%. Most importantly, we show efficient carotenoid delivery to membranes of mammalian cells, which provides protection from reactive oxygen species (ROS). Incubation of neuroblastoma cell line Tet21N in the presence of 1 μM AnaCTDH binding echinenone decreased antimycin A ROS production by 25% (p < 0.05). The described carotenoprotein may be considered as part of modular systems for the targeted antioxidant delivery.BMBF, 01DJ15007, Carotenoidbindende photoschaltbare Proteine: Lichtinduzierte Dynamik und Anwendungen in modernen mikroskopischen Verfahre

Solution structure of human steroidogenic acute regulatory protein STARD1 studied by small-angle X-ray scattering

Intracellular cholesterol transfer to mitochondria, a bottleneck of adrenal and gonadal steroidogenesis, relies on the functioning of the steroidogenic acute regulatory protein (StAR, STARD1), for which many disease-associated mutations have been described. Despite significant progress in the field, the exact mechanism of cholesterol binding and transfer by STARD1 still remains debatable and often considers significant structural rearrangements to achieve ligand binding. The crystal structure of STARD1, obtained recently at medium resolution, suggests that this protein has the same fold as other members of the START family. However, hydrodynamic properties and solution conformation of STARD1 are insufficiently characterized, partially due to poor solubility of this protein. Here, we used our recent protocol to obtain stable and soluble STARD1 and analyzed its hydrodynamic properties and solution conformation using a previously inapplicable small-angle X-ray scattering (SAXS). The SAXS data obtained exclusively from a monodisperse fraction of the monomeric protein suggest that, apart from movements of the flexible Ω1-loop, STARD1 unlikely undergoes significant spontaneous rearrangements proposed earlier as a gating mechanism for cholesterol binding. The consistency with the previously reported solution NMR structure of STARD6 suggests similarity of hydrodynamic behavior of other STARD-containing proteins

The Role of the Photon Counting Loss Effect in Time-Resolved Measurements of Fluorescence Anisotropy

Determining the rate of rotation of molecules from their fluorescence anisotropy decay curves is a powerful method for studying molecular systems in biological applications. The single photon count detection systems used for this have a nonlinear dependence of the photon counting rate on the fluorescence intensity flux (photon counting loss effect), which can lead to a number of artifacts. Using metal complexes of phthalocyanines as a test sample, we have shown that such a nonlinearity can cause distortions in the determination of the fluorescence anisotropy lifetime and the asymptotic fluorescence anisotropy. We also assessed the dependence of the described phenomena on temperature and estimated the manifestations of the photon counting loss effect in the case of photobleaching of the fluorophores

OCP–FRP protein complex topologies suggest a mechanism for controlling high light tolerance in cyanobacteria

In cyanobacteria, high light photoactivates the orange carotenoid protein (OCP) that binds to antennae complexes, dissipating energy and preventing the destruction of the photosynthetic apparatus. At low light, OCP is efficiently deactivated by a poorly understood action of the dimeric fluorescence recovery protein (FRP). Here, we engineer FRP variants with defined oligomeric states and scrutinize their functional interaction with OCP. Complemented by disulfide trapping and chemical crosslinking, structural analysis in solution reveals the topology of metastable complexes of OCP and the FRP scaffold with different stoichiometries. Unable to tightly bind monomeric FRP, photoactivated OCP recruits dimeric FRP, which subsequently monomerizes giving 1:1 complexes. This could be facilitated by a transient OCP–2FRP–OCP complex formed via the two FRP head domains, significantly improving FRP efficiency at elevated OCP levels. By identifying key molecular interfaces, our findings may inspire the design of optically triggered systems transducing light signals into protein–protein interactions

Solution Structure and Conformational Flexibility in the Active State of the Orange Carotenoid Protein: Part I. Small-Angle Scattering

Orange carotenoid proteins (OCPs) are photoswitchable macromolecules playing an important role in nonphotochemical quenching of excess energy in cyanobacterial light harvesting. Upon absorption of a blue photon (450–500 nm), OCPs undergo a structural change from the ground state OCPO to the active state OCPR, but high-resolution structures of the active state OCPR are not yet available. Here, we use small-angle scattering methods combined with simulation tools to determine low-resolution structures of the active state at low protein concentrations via two approaches: first, directly by in situ illumination of wild-type OCP achieving a turnover to the active state of >90% and second, by using the mutant OCPW288A anticipated to mimic the active state structure. Data fits assuming the shape of an ellipsoid yield three ellipsoidal radii of about 9, 29, and 51 ± 1 Å, in the case of the ground state OCPO. In the active state, however, the molecule becomes somewhat narrower with the two smaller radii being 9 and only 19 ± 3 Å, while the third dimension of the ellipsoid is significantly elongated to 85–92 ± 5 Å. Reconstitutions of the active state structure corroborate that OCPR is significantly elongated compared to the ground state OCPO and characterized by a separation of the N-terminal and C-terminal domains with unfolded N-terminal extension. By direct comparison of small-angle scattering data, we directly show that the mutant OCPW288A can be used as a structural analogue of the active state OCPR. The small-angle experiments are repeated for OCPO and the mutant OCPW288A at high protein concentrations of 50–65 mg/mL required for neutron spectroscopy investigating the molecular dynamics of OCP (see accompanying paper). The results reveal that the OCPO and OCPW288A samples for dynamics experiments are preferentially dimeric and widely resemble the structures of the ground and active states of OCP, respectively. This enables us to properly characterize the molecular dynamics of both states of OCP in the accompanying paper

Light-controlled carotenoid transfer between water-soluble proteins related to cyanobacterial photoprotection

International audienceCarotenoids are lipophilic pigments with multiple biological functions from coloration to vision and photoprotection. Still, the number of water-soluble carotenoid-binding proteins described to date is limited, and carotenoid transport and carotenoprotein maturation processes are largely underexplored. Recent studies revealed that CTDHs, which are natural homologs of the C-terminal domain (CTD) of the Orange Carotenoid Protein (OCP), a photoswitch involved in cyanobacterial photoprotection, are able to bind carotenoids, with absorption shifted far into the red region of the spectrum. Despite the recent discovery of their participation in carotenoid transfer processes, the functional roles of the diverse family of CTDHs are not well understood. Here we characterized CTDH carotenoproteins from Anabaena variabilis (AnaCTDH) and Thermosynechococcus elongatus (TeCTDH) and examined their ability to participate in carotenoid transfer processes with a set of OCP-derived proteins. This revealed that carotenoid transfer occurs in several directions guided by different affinities for carotenoid and specific protein-protein interactions. We show that CTDHs have higher carotenoid affinity compared to the CTD of OCP from Synechocystis, which results in carotenoid translocation from the CTD into CTDH via a metastable heterodimer intermediate. Activation of OCP by light, or mutagenesis compromising the OCP structure, provides AnaCTDH with an opportunity to extract carotenoid from the full-length OCP, either from Synechocystis or Anabaena. These previously unknown reactions between water-soluble carotenoproteins demonstrate multidirectionality of carotenoid transfer, allowing for efficient and reversible control over the carotenoid-mediated protein oligomerization by light, which gives insights into the physiological regulation of OCP activity by CTDH and suggests multiple applications. This article is protected by copyright. All rights reserved

Probing cytochrome c in living mitochondria with surface-enhanced Raman spectroscopy

Selective study of the electron transport chain components in living mitochondria is essential for fundamental biophysical research and for the development of new medical diagnostic methods. However, many important details of inter- and intramembrane mitochondrial processes have remained in shadow due to the lack of non-invasive techniques. Here we suggest a novel label-free approach based on the surface-enhanced Raman spectroscopy (SERS) to monitor the redox state and conformation of cytochrome c in the electron transport chain in living mitochondria. We demonstrate that SERS spectra of living mitochondria placed on hierarchically structured silver-ring substrates provide exclusive information about cytochrome c behavior under modulation of inner mitochondrial membrane potential, proton gradient and the activity of ATP-synthetase. Mathematical simulation explains the observed enhancement of Raman scattering due to high concentration of electric near-field and large contact area between mitochondria and nanostructured surfaces

Probing cytochrome <em>c</em> in living mitochondria with surface-enhanced Raman spectroscopy

Selective study of the electron transport chain components in living mitochondria is essential for fundamental biophysical research and for the development of new medical diagnostic methods. However, many important details of inter- and intramembrane mitochondrial processes have remained in shadow due to the lack of non-invasive techniques. Here we suggest a novel label-free approach based on the surface-enhanced Raman spectroscopy (SERS) to monitor the redox state and conformation of cytochrome c in the electron transport chain in living mitochondria. We demonstrate that SERS spectra of living mitochondria placed on hierarchically structured silver-ring substrates provide exclusive information about cytochrome c behavior under modulation of inner mitochondrial membrane potential, proton gradient and the activity of ATP-synthetase. Mathematical simulation explains the observed enhancement of Raman scattering due to high concentration of electric near-field and large contact area between mitochondria and nanostructured surfaces

Solution Structures of Two Different FRP-OCP Complexes as Revealed via SEC-SANS

Photosynthetic organisms have established photoprotective mechanisms in order to dissipate excess light energy into heat, which is commonly known as non-photochemical quenching. Cyanobacteria utilize the orange carotenoid protein (OCP) as a high-light sensor and quencher to regulate the energy flow in the photosynthetic apparatus. Triggered by strong light, OCP undergoes conformational changes to form the active red state (OCPR). In many cyanobacteria, the back conversion of OCP to the dark-adapted state is assisted by the fluorescence recovery protein (FRP). However, the exact molecular events involving OCP and its interaction with FRP remain largely unraveled so far due to their metastability. Here, we use small-angle neutron scattering combined with size exclusion chromatography (SEC-SANS) to unravel the solution structures of FRP-OCP complexes using a compact mutant of OCP lacking the N-terminal extension (∆NTEOCPO) and wild-type FRP. The results are consistent with the simultaneous presence of stable 2:2 and 2:1 FRP-∆NTEOCPO complexes in solution, where the former complex type is observed for the first time. For both complex types, we provide ab initio low-resolution shape reconstructions and compare them to homology models based on available crystal structures. It is likely that both complexes represent intermediate states of the back conversion of OCP to its dark-adapted state in the presence of FRP, which are of transient nature in the photocycle of wild-type OCP. This study demonstrates the large potential of SEC-SANS in revealing the solution structures of protein complexes in polydisperse solutions that would otherwise be averaged, leading to unspecific results