A Detailed View on the (Re)isomerization Dynamics in Microbial Rhodopsins Using Complementary Near-UV and IR Readouts.

Isomerization is a key process in many (bio)chemical systems. In microbial rhodopsins, the photoinduced isomerization of the all-trans retinal to the 13-cis isomer initiates a cascade of structural changes of the protein. The interplay between these changes and the thermal relaxation of the isomerized retinal is one of the crucial determinants for rhodopsin functionality.

A subgroup of light-driven sodium pumps with an additional Schiff base counterion.

Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identify a subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base.

Custom Design of a Humidifier Chamber for Crystallization.

Membrane proteins are indispensable for every living organism, yet their structural organization remains underexplored. Despite the recent advancements in single-particle cryogenic electron microscopy and cryogenic electron tomography, which have significantly increased the structural coverage of membrane proteins across various kingdoms, certain scientific methods, such as time-resolved crystallography, still mostly rely on crystallization techniques, such as lipidic cubic phase (LCP) or crystallization. In this study, we present an open-access blueprint for a humidity control chamber designed for LCP/ crystallization experiments using a Gryphon crystallization robot.

Mechanisms of inward transmembrane proton translocation.

Proton transport is indispensable for cell life. It is believed that molecular mechanisms of proton movement through different types of proton-conducting molecules have general universal features. However, elucidation of such mechanisms is a challenge.

Optogenetic cytosol acidification of mammalian cells using an inward proton-pumping rhodopsin.

Ion gradients are a universal form of energy, information storage and conversion in living cells. Advances in optogenetics inspire the development of novel tools towards control of different cellular processes with light. Rhodopsins are perspective tools for optogenetic manipulation of ion gradients in cells and subcellular compartments, controlling pH of the cytosol and intracellular organelles.

Flotillin-associated rhodopsin (FArhodopsin), a widespread paralog of proteorhodopsin in aquatic bacteria with streamlined genomes.

Microbial rhodopsins are found more than once in a single genome (paralogs) often have different functions. We screened a large dataset of open ocean single-amplified genomes (SAGs) for co-occurrences of multiple rhodopsin genes. Many such cases were found among Pelagibacterales (SAR11), HIMB59, and the Gammaproteobacteria SAGs.

Mirror proteorhodopsins.

Proteorhodopsins (PRs), bacterial light-driven outward proton pumps comprise the first discovered and largest family of rhodopsins, they play a significant role in life on the Earth. A big remaining mystery was that up-to-date there was no described bacterial rhodopsins pumping protons at acidic pH despite the fact that bacteria live in different pH environment. Here we describe conceptually new bacterial rhodopsins which are operating as outward proton pumps at acidic pH.

Structural insights into the effects of glycerol on ligand binding to cytochrome P450.

New antitubercular drugs are vital due to the spread of resistant strains. Carbethoxyhexyl imidazole (CHImi) inhibits cytochrome P450 CYP124, which is a steroid-metabolizing enzyme that is important for the survival of Mycobacterium tuberculosis in macrophages. The available crystal structure of the CYP124-CHImi complex reveals two glycerol molecules in the active site.

Fault Current Limitation in Electrical Power Networks Containing HTS Cable and HTS Fuse.

Numerical calculations of parameters of an electrical power network where an HTS fuse is used as a fault current limiting device have been done. The calculations were performed for networks containing different types of HTS cables as well. The design of HTS fuse was developed based on the numerical calculation for the network-rated parameters considering the special types 2G HTS tape characteristics.

Structural insights into light-driven anion pumping in cyanobacteria.

Transmembrane ion transport is a key process in living cells. Active transport of ions is carried out by various ion transporters including microbial rhodopsins (MRs). MRs perform diverse functions such as active and passive ion transport, photo-sensing, and others.

Structural basis for receptor selectivity and inverse agonism in S1P receptors.

The bioactive lysophospholipid sphingosine-1-phosphate (S1P) acts via five different subtypes of S1P receptors (S1PRs) - S1P. S1P is predominantly expressed in nervous and immune systems, regulating the egress of natural killer cells from lymph nodes and playing a role in immune and neurodegenerative disorders, as well as carcinogenesis. Several S1PR therapeutic drugs have been developed to treat these diseases; however, they lack receptor subtype selectivity, which leads to side effects.

Crystallization of Microbial Rhodopsins.

Microbial rhodopsins are light-sensitive transmembrane proteins, evolutionary adapted by various organisms like archaea, bacteria, simple eukaryote, and viruses to utilize solar energy for their survival. A complete understanding of functional mechanisms of these proteins is not possible without the knowledge of their high-resolution structures, which can be primarily obtained by X-ray crystallography. This technique, however, requires high-quality crystals, growing of which is a great challenge especially in case of membrane proteins.

E. coli Expression and Purification of Microbial and Viral Rhodopsins.

Microbial rhodopsins have become an indispensable tool for neurobiology. Of thousands of identified microbial rhodopsins, a minute fraction has been studied so far and they have shown remarkable functional diversity suggesting more great promises that this large family holds. Effective production of recombinant microbial and viral rhodopsins is a prerequisite for the success of functional and structural studies of these proteins.

Mechanisms of membrane protein crystallization in 'bicelles'.

Despite remarkable progress, mainly due to the development of LCP and 'bicelle' crystallization, lack of structural information remains a bottleneck in membrane protein (MP) research. A major reason is the absence of complete understanding of the mechanism of crystallization. Here we present small-angle scattering studies of the evolution of the "bicelle" crystallization matrix in the course of MP crystal growth.

True-atomic-resolution insights into the structure and functional role of linear chains and low-barrier hydrogen bonds in proteins.

Hydrogen bonds are fundamental to the structure and function of biological macromolecules and have been explored in detail. The chains of hydrogen bonds (CHBs) and low-barrier hydrogen bonds (LBHBs) were proposed to play essential roles in enzyme catalysis and proton transport. However, high-resolution structural data from CHBs and LBHBs is limited.

Engineering the Catalytic Properties of Two-Domain Laccase from Ac-993.

Laccases catalyze the oxidation of substrates with the concomitant reduction of oxygen to water. Recently, we found that polar residues located in tunnels leading to Cu2 and Cu3 ions control oxygen entrance (His 165) and proton transport (Arg 240) of two-domain laccase (2D) from (SgfSL). In this work, we have focused on optimizing the substrate-binding pocket (SBP) of SgfSL while simultaneously adjusting the oxygen reduction process.

Crystal structure of human 14-3-3ζ complexed with the noncanonical phosphopeptide from proapoptotic BAD.

Several signaling pathways control phosphorylation of the proapoptotic protein BAD and its phosphorylation-dependent association with 14-3-3 proteins in the cytoplasm. The stability of the 14-3-3/BAD complex determines the cell fate: unphosphorylated BAD escapes from 14-3-3, migrates to the mitochondria and initiates apoptosis. While the 14-3-3/BAD interaction represents a promising drug target, it lacks structural characterization.

The Voltage Dependent Sidedness of the Reprotonation of the Retinal Schiff Base Determines the Unique Inward Pumping of Xenorhodopsin.

The new class of microbial rhodopsins, called xenorhodopsins (XeRs), extends the versatility of this family by inward H pumps. These pumps are an alternative optogenetic tool to the light-gated ion channels (e.g.

Structure-based insights into evolution of rhodopsins.

Rhodopsins, most of which are proton pumps generating transmembrane electrochemical proton gradients, span all three domains of life, are abundant in the biosphere, and could play a crucial role in the early evolution of life on earth. Whereas archaeal and bacterial proton pumps are among the best structurally characterized proteins, rhodopsins from unicellular eukaryotes have not been well characterized. To fill this gap in the current understanding of the proton pumps and to gain insight into the evolution of rhodopsins using a structure-based approach, we performed a structural and functional analysis of the light-driven proton pump LR (Mac) from the pathogenic fungus Leptosphaeria maculans.