Possibility of two-dimensional ordering of cryptochrome 4a from European robin.

Cryptochrome (Cry) in some species could act as a quantum senser to detect the inclination angle of geomagnetic field, the function of which attributes the magnetic sensitivity of spins of unpaired electrons in radical pair (RP) in CRY generated by blue light irradiation. However, the effect of blue light on the structure and molecular behavior of Cry has not been well investigated. We conducted the size exclusion chromatography (SEC) and small-angle X-ray scattering (SAXS) analyses to inspect the molecular structure and behavior of cryptochrome 4a (ErCry4a) from European robin, a representative magnetosensory animal.

Fibrillization Process of Human Amyloid-Beta Protein (1-40) under a Molecular Crowding Environment Mimicking the Interior of Living Cells Using Cell Debris.

Molecular crowding environments play a crucial role in understanding the mechanisms of biological reactions. Inside living cells, a diverse array of molecules coexists within a volume fraction ranging from 10% to 30% /. However, conventional spectroscopic methods often face difficulties in selectively observing the structures of particular proteins or membranes within such molecularly crowded environments due to the presence of high background signals.

Magnetic field effects on the structure and molecular behavior of pigeon iron-sulfur protein.

Pigeon iron-sulfur (Fe-S) cluster assembly 1 homolog (clISCA1) is a target protein for research into the biomagnetoreception mechanism, as the clCRY4/clISCA1 oligomer, a complex composed of the columnar clISCA1 oligomer and the magnetosensor candidate protein cryptochrome-4 (clCRY4) oligomer, tends to orient itself along weak magnetic fields, such as geomagnetic fields, under blue light. To obtain insight into the magnetic orientation mechanism of the clCRY4/clISCA1 oligomer, we inspected magnetic field effects on the structure and molecular behavior of clISCA1 by small angle X-ray scattering analysis. The results indicated that the clISCA1 protomer took the Fe-S cluster-bound globular form and unbound rod-like form.

Expression and characterization of l-arabinose isomerase from Geobacillus stearothermophilus for improved activity under acidic condition.

A low-calorie sugar-substituting sweetener, d-tagatose, can be produced by l-arabinose isomerase (l-AI) from the substrate d-galactose. However, this process suffers from a Maillard reaction when performed at alkaline pH and high temperature. For industrial applications, therefore, a reaction under slightly acidic conditions is desirable to minimize the Maillard reaction.

Catalytic mechanism and evolutionary characteristics of thioredoxin from Halobacterium salinarum NRC-1.

Thioredoxin (TRX) is an important antioxidant against oxidative stress. TRX from the extremely halophilic archaeon Halobacterium salinarum NRC-1 (HsTRX-A), which has the highest acidic residue content [(Asp + Glu)/(Arg + Lys + His) = 9.0] among known TRXs, was chosen to elucidate the catalytic mechanism and evolutionary characteristics associated with haloadaptation.

Expression, Folding, and Activation of Halophilic Alkaline Phosphatase in Non-Halophilic Brevibacillus choshinensis.

Halophilic enzymes contain a large number of acidic amino acids and marginal large hydrophobic amino acids, which make them highly soluble even under strongly hydrophobic conditions. This characteristic of halophilic enzymes provides potential for their industrial application. However, halophilic enzymes easily degrade when used for industrial applications compared with enzymes from other extremophiles because of their instability in low-salt environments.

Observation of Protein and Lipid Membrane Structures in a Model Mimicking the Molecular-Crowding Environment of Cells Using Neutron Scattering and Cell Debris.

The interior of living cells is a molecular-crowding environment, where large quantities of various molecules coexist. Investigations into the nature of this environment are essential for an understanding of both the elaborate biological reactions and the maintenance of homeostasis occurring therein. The equilibrium states of biological macromolecular systems are affected by molecular-crowding environments unmatched by in vitro diluted environments; knowledge about crowding effects is still insufficient due to lack of relevant experimental studies.

The non-glycosylated N-terminal domain of human thrombopoietin is a molten globule under native conditions.

Human thrombopoietin (hTPO) is a primary hematopoietic growth factor that regulates megakaryocytopoiesis and platelet production. The non-glycosylated form of 1-163 residues of hTPO (hTPO ) including the N-terminal active site domain (1-153 residues) is a candidate for treating thrombocytopenia. However, the autoantigenicity level of hTPO is higher than that of the full-length glycosylated hTPO (ghTPO ).

Extended structure of pleiotropic DNA repair-promoting protein PprA from Deinococcus radiodurans.

Pleiotropic protein promoting DNA repair A (PprA) is a key protein facilitating the extreme radiation resistance of Deinococcus radiodurans. PprA is a unique protein to the genus Deinococcus and exists as an oligomer ranging from a tetramer to an ∼100-mer depending on protein concentrations. Here, the X-ray crystal structure of PprA was determined to clarify how PprA confers radiation resistance.

Hydration Structures of the Human Protein Kinase CK2α Clarified by Joint Neutron and X-ray Crystallography.

Casein kinase 2 (CK2) has broad phosphorylation activity against various regulatory proteins, which are important survival factors in eukaryotic cells. To clarify the hydration structure and catalytic mechanism of CK2, we determined the crystal structure of the alpha subunit of human CK2 containing hydrogen and deuterium atoms using joint neutron (1.9 Å resolution) and X-ray (1.

Improved substrate specificity for D-galactose of L-arabinose isomerase for industrial application.

L-Arabinose isomerase isolated from Geobacillus stearothermophilus (GSAI) was modified to improve its substrate specificity for D-galactose for the production of D-tagatose, a potential reduced-energy sweetener. Among the selected residues, mutation at residue 18 produced a mutant strain, H18T, which exhibited increased activity for D-galactose compared with the wild-type (WT) enzyme. Analysis of the substrate specificity of H18T showed a 45.

Expression and characterization of the Renilla luciferase with the cumulative mutation.

Luciferase from Renilla reniformis (RLuc) is a good research tool as a reporter protein and bioimaging probes, yielding blue light using the substrate coelenterazine. However, the applications are limited since RLuc is unstable under various conditions. Therefore, an attempt was made to increase RLuc thermostability.

Stabilization of luciferase from Renilla reniformis using random mutations.

We expressed luciferase (RLuc) from Renilla reniformis in Escherichia coli RLuc was purified using a Ni-NTA column and subsequently characterized. It was unstable in acidic solutions and at 30°C. To increase the stability of RLuc, the Rluc gene was randomly mutated using error-prone polymerase chain reaction.

An insight into the thermodynamic characteristics of human thrombopoietin complexation with TN1 antibody.

Human thrombopoietin (hTPO) primarily stimulates megakaryocytopoiesis and platelet production and is neutralized by the mouse TN1 antibody. The thermodynamic characteristics of TN1 antibody-hTPO complexation were analyzed by isothermal titration calorimetry (ITC) using an antigen-binding fragment (Fab) derived from the TN1 antibody (TN1-Fab). To clarify the mechanism by which hTPO is recognized by TN1-Fab the conformation of free TN1-Fab was determined to a resolution of 2.

Nucleoside Diphosphate Kinase from Psychrophilic Pseudoalteromonas sp. AS-131 Isolated from Antarctic Ocean.

Nucleoside diphosphate kinase isolated from psychrophilic Pseudoalteromonas sp. AS-131 (ASNDK) was expressed in Escherichia coli and purified to homogeneity. Comparing to mesophilic NDK isolated from Pseudomonas aeruginosa, ASNDK exhibited highly elevated thermolability: E.

Structure of a highly acidic β-lactamase from the moderate halophile Chromohalobacter sp. 560 and the discovery of a Cs(+)-selective binding site.

Environmentally friendly absorbents are needed for Sr(2+) and Cs(+), as the removal of the radioactive Sr(2+) and Cs(+) that has leaked from the Fukushima Nuclear Power Plant is one of the most important problems in Japan. Halophilic proteins are known to have many acidic residues on their surface that can provide specific binding sites for metal ions such as Cs(+) or Sr(2+). The crystal structure of a halophilic β-lactamase from Chromohalobacter sp.

Structural characteristics of alkaline phosphatase from the moderately halophilic bacterium Halomonas sp. 593.

Alkaline phosphatase (AP) from the moderate halophilic bacterium Halomonas sp. 593 (HaAP) catalyzes the hydrolysis of phosphomonoesters over a wide salt-concentration range (1-4 M NaCl). In order to clarify the structural basis of its halophilic characteristics and its wide-range adaptation to salt concentration, the tertiary structure of HaAP was determined by X-ray crystallography to 2.

Structure and function of ∆1-tetrahydrocannabinolic acid (THCA) synthase, the enzyme controlling the psychoactivity of Cannabis sativa.

∆1-Tetrahydrocannabinolic acid (THCA) synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into THCA, the precursor of the primary psychoactive agent ∆1-tetrahydrocannabinol in Cannabis sativa. The enzyme was overproduced in insect cells, purified, and crystallized in order to investigate the structure-function relationship of THCA synthase, and the tertiary structure was determined to 2.75Å resolution by X-ray crystallography (R(cryst)=19.

Reduction of salt-requirement of halophilic nucleoside diphosphate kinase by engineering S-S bond.

Nucleoside diphosphate kinase (HsNDK) from extremely halophilic haloarchaeon, Halobacterium salinarum, requires salt at high concentrations for folding. A D148C mutant, in which Asp148 was replaced with Cys, was designed to enhance stability and folding in low salt solution by S-S bond. It showed increased thermal stability by about 10 °C in 0.

A structural mechanism for dimeric to tetrameric oligomer conversion in Halomonas sp. nucleoside diphosphate kinase.

Nucleoside diphosphate kinase (NDK) is known to form homotetramers or homohexamers. To clarify the oligomer state of NDK from moderately halophilic Halomonas sp. 593 (HaNDK), the oligomeric state of HaNDK was characterized by light scattering followed by X-ray crystallography.

Promoter-associated noncoding RNA from the CCND1 promoter.

More than 90% of the human genome have been found to be transcribed and most of the transcripts are noncoding (nc) RNAs (Willingham et al., Science 309:1570-1573, 2005; ENCODE-consortium, Science 306:636-640, 2004; Carninci et al., Science 309:1559-1563, 2005; Bertone et al.

Loop lengths of G-quadruplex structures affect the G-quadruplex DNA binding selectivity of the RGG motif in Ewing's sarcoma.

The G-quadruplex nucleic acid structural motif is a target for designing molecules with potential anticancer properties. To achieve therapeutic selectivity by targeting the G-quadruplex, the molecules must be able to differentiate between the DNA of different G-quadruplexes. We recently reported that the Arg-Gly-Gly repeat (RGG) of the C-terminus in Ewing's sarcoma protein (EWS), which is a group of dominant oncogenes that arise due to chromosomal translocations, is capable of binding to G-quadruplex telomere DNA and RNA via arginine residues and stabilize the G-quadruplex DNA form in vitro.

Identification of Ewing's sarcoma protein as a G-quadruplex DNA- and RNA-binding protein.

The Ewing's sarcoma (EWS) oncogene contains an N-terminal transcription activation domain and a C-terminal RNA-binding domain. Although the EWS activation domain is a potent transactivation domain that is required for the oncogenic activity of several EWS fusion proteins, the normal role of intact EWS is poorly characterized because little is known about its nucleic acid recognition specificity. Here we show that the Arg-Gly-Gly (RGG) domain of the C-terminal in EWS binds to the G-rich single-stranded DNA and RNA fold in the G-quadruplex structure.

TLS and PRMT1 synergistically coactivate transcription at the survivin promoter through TLS arginine methylation.

TLS (Translocated in LipoSarcoma), also termed FUS, is a multifunctional protein implicated in diverse cellular events such as maintaining genome integrity and regulating gene expression. We have focused on the role of TLS as a coregulator in transcriptional regulation. In the process of investigating TLS-binding proteins, we found that PRMT1 (protein arginine methyltransferase 1) was in complex with TLS.

Id4, a new candidate gene for senile osteoporosis, acts as a molecular switch promoting osteoblast differentiation.

Excessive accumulation of bone marrow adipocytes observed in senile osteoporosis or age-related osteopenia is caused by the unbalanced differentiation of MSCs into bone marrow adipocytes or osteoblasts. Several transcription factors are known to regulate the balance between adipocyte and osteoblast differentiation. However, the molecular mechanisms that regulate the balance between adipocyte and osteoblast differentiation in the bone marrow have yet to be elucidated.