Transferase and hydrolytic activities of the laminarinase from rhodothermus marinus and its M133A, M133C, and M133W mutants

Comparative studies of the transglycosylation and hydrolytic activities have been performed on the Rhodothermus marinus β-1,3-glucanase (laminarinase) and its M133A, M133C, and M133W mutants. The M133C mutant demonstrated near 20% greater rate of transglycosylation activity in comparison with the M133A and M133W mutants that was measured by NMR quantitation of nascent β(1-4) and β(1-6) linkages. To obtain kinetic probes for the wild-type enzyme and Met-133 mutants, p-nitrophenyl β-laminarin oligosaccharides of degree of polymerisation 2–8 were synthesized enzymatically. Catalytic efficiency values, k _cat/K _m, of the laminarinase catalysed hydrolysis of these oligosaccharides suggested possibility of four negative and at least three positive binding subsites in the active site. Comparison of action patterns of the wild-type and M133C mutant in the hydrolysis of the p-nitrophenyl-β-D-oligosac- charides indicated that the increased transglycosylation activity of the M133C mutant did not result from altered subsite affinities. The stereospecificity of the transglycosylation reaction also was unchanged in all mutants; the major transglycosylation products in hydrolysis of p-nitrophenyl laminaribioside were β-glucopyranosyl-β-1,3-D-glucopy- ranosyl-β-1,3-D-glucopyranose and β-glucopyranosyl-β-1, 3-D-glucopyranosyl-β-1,3-D-glucpyranosyl-β-1,3-D- glucopyranoxside. In a memoriam of Dr. Kirill N. Neustroev. All we, his friends and colleagues, mourn for his sudden death. He was a bright and talented scientist, brilliant manager and good friend.     

Modular toxin from the lynx spider Oxyopes takobius: Structure of spiderine domains in solution and membrane‐mimicking environment

We have recently demonstrated that a common phenomenon in evolution of spider venom composition is the emergence of so‐called modular toxins consisting of two domains, each corresponding to a “usual” single‐domain toxin. In this article, we describe the structure of two domains that build up a modular toxin named spiderine or OtTx1a from the venom of Oxyopes takobius. Both domains were investigated by solution NMR in water and detergent micelles used to mimic membrane environment. The N‐terminal spiderine domain OtTx1a‐AMP (41 amino acid residues) contains no cysteines. It is disordered in aqueous solution but in micelles, it assumes a stable amphiphilic structure consisting of two α‐helices separated by a flexible linker. On the contrary, the C‐terminal domain OtTx1a‐ICK (59 residues) is a disulfide‐rich polypeptide reticulated by five S–S bridges. It presents a stable structure in water and its core is the inhibitor cystine knot (ICK) or knottin motif that is common among single‐domain neurotoxins. OtTx1a‐ICK structure is the first knottin with five disulfide bridges and it represents a good reference for the whole oxytoxin family. The affinity of both domains to membranes was measured with NMR using titration by liposome suspensions. In agreement with biological tests, OtTx1a‐AMP was found to show high membrane affinity explaining its potent antimicrobial properties.     

Optimal Schedules of Light Exposure for Rapidly Correcting Circadian Misalignment

Jet lag arises from a misalignment of circadian biological timing with the timing of human activity, and is caused by rapid transmeridian travel. Jet lag''s symptoms, such as depressed cognitive alertness, also arise from work and social schedules misaligned with the timing of the circadian clock. Using experimentally validated mathematical models, we develop a new methodology to find mathematically optimal schedules of light exposure and avoidance for rapidly re-entraining the human circadian system. In simulations, our schedules are found to significantly outperform other recently proposed schedules. Moreover, our schedules appear to be significantly more robust to both noise in light and to inter-individual variations in endogenous circadian period than other proposed schedules. By comparing the optimal schedules for thousands of different situations, and by using general mathematical arguments, we are also able to translate our findings into general principles of optimal circadian re-entrainment. These principles include: 1) a class of schedules where circadian amplitude is only slightly perturbed, optimal for dim light and for small shifts 2) another class of schedules where shifting occurs along the shortest path in phase-space, optimal for bright light and for large shifts 3) the determination that short light pulses are less effective than sustained light if the goal is to re-entrain quickly, and 4) the determination that length of daytime should be significantly shorter when delaying the clock than when advancing it.     

Semax,An ACTH(4-10) Analogue with Nootropic Properties,Activates Dopaminergic and Serotoninergic Brain Systems in Rodents

Corticotrophin (ACTH) and its analogues, particularly Semax (Met-Glu-His-Phe-Pro-Gly-Pro), demonstrate nootropic activity. Close functional and anatomical links have been established between melanocortinergic and monoaminergic brain systems. The aim of present work was to investigate the effects of Semax on neurochemical parameters of dopaminergic- and serotonergic systems in rodents. The tissue content of 5-hydroxyindoleacetic acid (5-HIAA) in the striatum was significantly increased (+25%) 2 h after Semax administration. The extracellular striatal level of 5-HIAA gradually increased up to 180% within 1–4 h after Semax (0.15 mg/kg, ip) administration. This peptide alone failed to alter the tissue and extracellular concentrations of dopamine and its metabolites. Semax injected 20 min prior d-amphetamine dramatically enhanced the effects of the latter on the extracellular level of dopamine and on the locomotor activity of animals. Our results reveal the positive modulatory effect of Semax on the striatal serotonergic system and the ability of Semax to enhance both the striatal release of dopamine and locomotor behavior elicited by d-amphetamine. Special issue dedicated to Dr. Simo S. Oja.     

CRISPR RNA binding and DNA target recognition by purified Cascade complexes from Escherichia coli

Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated Cas proteins comprise a prokaryotic RNA-guided adaptive immune system that interferes with mobile genetic elements, such as plasmids and phages. The type I-E CRISPR interference complex Cascade from Escherichia coli is composed of five different Cas proteins and a 61-nt-long guide RNA (crRNA). crRNAs contain a unique 32-nt spacer flanked by a repeat-derived 5′ handle (8 nt) and a 3′ handle (21 nt). The spacer part of crRNA directs Cascade to DNA targets. Here, we show that the E. coli Cascade can be expressed and purified from cells lacking crRNAs and loaded in vitro with synthetic crRNAs, which direct it to targets complementary to crRNA spacer. The deletion of even one nucleotide from the crRNA 5′ handle disrupted its binding to Cascade and target DNA recognition. In contrast, crRNA variants with just a single nucleotide downstream of the spacer part bound Cascade and the resulting ribonucleotide complex containing a 41-nt-long crRNA specifically recognized DNA targets. Thus, the E. coli Cascade-crRNA system exhibits significant flexibility suggesting that this complex can be engineered for applications in genome editing and opening the way for incorporation of site-specific labels in crRNA.     

Localized Surface Plasmon Resonance in Gold Nanocluster Arrays on Opaque Substrates

Plasmonics - We report on the investigation of the localized surface plasmon resonance (LSPR) in periodical Au nanostructures. The arrays of Au nanoclusters and dimers were fabricated on Si and...     

Structural dynamics of ribosomal RNA during decoding on the ribosome

Decoding is a multistep process by which the ribosome accurately selects aminoacyl-tRNA (aa-tRNA) that matches the mRNA codon in the A site. The correct geometry of the codon-anticodon complex is monitored by the ribosome, resulting in conformational changes in the decoding center of the small (30S) ribosomal subunit by an induced-fit mechanism. The recognition of aa-tRNA is modulated by changes of the ribosome conformation in regions other than the decoding center that may either affect the architecture of the latter or alter the communication of the 30S subunit with the large (50S) subunit where the GTPase and peptidyl transferase centers are located. Correct codon-anticodon complex formation greatly accelerates the rates of GTP hydrolysis and peptide bond formation, indicating the importance of crosstalk between the subunits and the role of the 50S subunit in aa-tRNA selection. In the present review, recent results of the ribosome crystallography, cryoelectron microscopy (cryo-EM), genetics, rapid kinetics and biochemical approaches are reviewed which show that the dynamics of the structure of ribosomal RNA (rRNA) play a crucial role in decoding.     

Cavin4 interacts with Bin1 to promote T-tubule formation and stability in developing skeletal muscle

The cavin proteins are essential for caveola biogenesis and function. Here, we identify a role for the muscle-specific component, Cavin4, in skeletal muscle T-tubule development by analyzing two vertebrate systems, mouse and zebrafish. In both models, Cavin4 localized to T-tubules, and loss of Cavin4 resulted in aberrant T-tubule maturation. In zebrafish, which possess duplicated cavin4 paralogs, Cavin4b was shown to directly interact with the T-tubule–associated BAR domain protein Bin1. Loss of both Cavin4a and Cavin4b caused aberrant accumulation of interconnected caveolae within the T-tubules, a fragmented T-tubule network enriched in Caveolin-3, and an impaired Ca²⁺ response upon mechanical stimulation. We propose a role for Cavin4 in remodeling the T-tubule membrane early in development by recycling caveolar components from the T-tubule to the sarcolemma. This generates a stable T-tubule domain lacking caveolae that is essential for T-tubule function.