Linkage of amino acid variation and evolution of human immunodeficiency virus type 1 gp120 envelope glycoprotein (subtype B) with usage of the second receptor.

To clarify the relationship between the amino acid variations of the gp120 of human immunodeficiency virus type 1 (HIV-1) and the chemokine receptors that are used as the second receptor for HIV, we evaluated amino acid site variation of gp120 between the X4 strains (use CXCR4) and the R5 strains (use CCR5) from 21 sequences of subtype B. Our analysis showed that residues 306 and 322 in the V3 loop and residue 440 in the C4 region were associated with usage of the second receptor. The polymorphism at residue 440 is clearly associated with the usage of the second receptor: The amino acid at position 440 was a basic amino acid in the R5 strains, and a nonbasic and smaller amino acid in the X4 strains, while the V3 loop of the X4 strains was more basic than that of the R5 strains.

Phase transition of triclinic hen egg-white lysozyme crystal associated with sodium binding.

A triclinic crystal of hen egg-white lysozyme obtained from a D2O solution at 313 K was transformed into a new triclinic crystal by slow release of solvent under a temperature-regulated nitrogen-gas stream. The progress of the transition was monitored by X-ray diffraction. The transition started with the appearance of strong diffuse streaks.

Stochastic formulation of sampling dynamics in generalized ensemble methods.

The fundamental relations of the sampling process in the multicanonical ensemble and simulated tempering have been studied in the expanded ensembles formalism. The simulated tempering is identified as the multicanonical sampling with the generalized weight determined by a Laplace transform of the temperature weight. The characteristic dynamics of both sampling methods has been verified in the stochastic formulation of the sampling process.

1H NMR spectra of branched-chain cyclomaltohexaoses (alpha-cyclodextrins).

The 1H NMR spectra of seven branched alpha-cyclodextrins (alpha-CDs) were observed and analyzed in detail. They were compared with spectra of alpha-CD and amylose. Although these branched alpha-CDs consist only of alpha-D-glucose with the same alpha-(1-->4) O-glucosyl binding, aside from one exception, differences in chemical shifts of corresponding signals were significantly large.

Fluorescent modification of N-terminal amino group in peptides for complete sequence analysis using PSD method in MALDI-TOF-MS.

In the sequence analyses of peptides from nucleoprotein in influenza virus, it was very difficult to obtain the sufficient numbers of fragment ions using the post-source decay (PSD) method in MALDI-TOF-MS. Fluorescent modification of the N-terminal amino group in the target peptides was introduced for the PSD measurement. The fluorescently-labelled peptides gave sufficient fragment ions in the PSD spectrum, which leads to the complete sequence analysis of peptide.

Aromatic residues located close to the active center are essential for the catalytic reaction of flap endonuclease-1 from hyperthermophilic archaeon Pyrococcus horikoshii.

Flap endonuclease-1 (FEN-1) possessing 5'-flap endonuclease and 5'-->3' exonuclease activity plays important roles in DNA replication and repair. In this study, the kinetic parameters of mutants at highly conserved aromatic residues, Tyr33, Phe35, Phe79, and Phe278-Phe279, in the vicinity of the catalytic centers of FEN-1 were examined. The substitution of these aromatic residues with alanine led to a large reduction in kcat values, although these mutants retained Km values similar to that of the wild-type enzyme.

Inositol 1,4,5-trisphosphate receptor contains multiple cavities and L-shaped ligand-binding domains.

Calcium concentrations are strictly regulated in all biological cells, and one of the key molecules responsible for this regulation is the inositol 1,4,5-trisphosphate receptor, which was known to form a homotetrameric Ca(2+) channel in the endoplasmic reticulum. The receptor is involved in neuronal transmission via Ca(2+) signaling and for many other functions that relate to morphological and physiological processes in living organisms. We analysed the three-dimensional structure of the ligand-free form of the receptor based on a single-particle technique using an originally developed electron microscope equipped with a helium-cooled specimen stage and an automatic particle picking system.

Role of Phe283 in enzymatic reaction of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp.1011: Substrate binding and arrangement of the catalytic site.

Cyclodextrin glycosyltransferase (CGTase) belonging to the alpha-amylase family mainly catalyzes transglycosylation and produces cyclodextrins from starch and related alpha-1,4-glucans. The catalytic site of CGTase specifically conserves four aromatic residues, Phe183, Tyr195, Phe259, and Phe283, which are not found in alpha-amylase. To elucidate the structural role of Phe283, we determined the crystal structures of native and acarbose-complexed mutant CGTases in which Phe283 was replaced with leucine (F283L) or tyrosine (F283Y).

A 21-amino acid peptide from the cysteine cluster II of the family D DNA polymerase from Pyrococcus horikoshii stimulates its nuclease activity which is Mre11-like and prefers manganese ion as the cofactor.

Family D DNA polymerase (PolD) is a new type of DNA polymerase possessing polymerization and 3'-5' exonuclease activities. Here we report the characterization of the nuclease activity of PolD from Pyrococcus horikoshii. By site-directed mutagenesis, we verified that the putative Mre11-like nuclease domain in the small subunit (DP1), predicted according to computer analysis and structure inference reported previously, is the catalytic domain.

Distinct domain functions regulating de novo DNA synthesis of thermostable DNA primase from hyperthermophile Pyrococcus horikoshii.

DNA primases are essential components of the DNA replication apparatus in every organism. Reported here are the biochemical characteristics of a thermostable DNA primase from the thermophilic archaeon Pyrococcus horikoshii, which formed the oligomeric unit L(1)S(1) and synthesized long DNA primers 10 times more effectively than RNA primers. The N-terminal (25KL) and C-terminal halves (20KL) of the large subunit (L) play distinct roles in regulating de novo DNA synthesis of the small catalytic subunit (S).

Topology representing network enables highly accurate classification of protein images taken by cryo electron-microscope without masking.

In single-particle analysis, a three-dimensional (3-D) structure of a protein is constructed using electron microscopy (EM). As these images are very noisy in general, the primary process of this 3-D reconstruction is the classification of images according to their Euler angles, the images in each classified group then being averaged to reduce the noise level. In our newly developed strategy of classification, we introduce a topology representing network (TRN) method.

Determination of multicanonical weight based on a stochastic model of sampling dynamics.

Based on the stochastic interpretation of the sampling process modeled by a Langevin equation, we present an effective iteration scheme to determine the weight in multicanonical molecular dynamics. Our method enables an automatic determination of the weight producing a uniform energy sampling via an iterative cancellation of the deterministic force in a Langevin equation. The deterministic force has been calculated from the energy trajectory by identifying the moments of the transition probability of a Fokker-Planck equation associated with a Langevin equation.

X-ray structural analysis of the ligand-recognition mechanism in the dual-affinity labeling of c-type lysozyme with 2',3'-epoxypropyl beta-glycoside of N-acetyllactosamine.

In spite of the belonging to the same c-type lysozyme family, hen egg-white lysozyme (HEWL) was much less susceptible to the dual-affinity labeling with 2',3'-epoxypropyl beta-glycoside of N-acetyllactosamine (Galbeta1,4GlcNAc-Epo) than human lysozyme (HL). The three-dimensional structures of the HEWL labeled with single Galbeta1,4GlcNAc-Epo and the Glu102-mutant HL labeled with double Galbeta1,4GlcNAc-Epo were determined by X-ray crystallography at resolutions of 1.85 and 2.

Crystal structure of a full-length LysR-type transcriptional regulator, CbnR: unusual combination of two subunit forms and molecular bases for causing and changing DNA bend.

The LysR-type transcriptional regulator (LTTR) proteins are one of the most common transcriptional regulators in prokaryotes. Here we report the crystal structure of CbnR, which is one of the LTTRs derived from Ralstonia eutropha NH9. This is the first crystal structure of a full-length LTTR.

Effect of pH on the aggregate formation of a non-amyloid component (1-13).

The formation of aggregates including amyloid fibrils in the peptide fragment of non-amyloid-beta component (NAC(1-13)) was investigated under a variety of solution conditions. Two types of sample preparation method from neutral and acidic conditions were examined. Electron microscopy observation showed amorphous aggregates in the sample at pH 4.

Structural genomics of membrane proteins.

A program on the structural genomics of membrane proteins has started at the BIRC, AIST, involving other academic institutions and industrial companies. Emphasis is being put on the development of techniques for the structural determination of membrane proteins of biological importance and ligand-receptor interactions by means of electron microscopy, X-ray diffraction, NMR, and computer simulation. Most efforts at the present stage, however, are being directed to finding suitable expression and purification systems and crystallization conditions for such proteins.

Dynamical origin of uniform sampling in multicanonical ensemble.

The stochastic model describing the sampling process in multicanonical ensemble has been derived by considering the sampling process as an overdamped Brownian motion on the free energy surface. The essential dynamics of the multicanonical sampling has been characterized by a Langevin equation in a piecewise multivalleyed free energy landscape, modulated by a temperature-dependent curvature. Based on the stochastic model we showed that the multicanonical weight can be determined by interpolating maximum probability energy points of the canonical samplings at different temperatures.

Crystallographic analysis of the thermal motion of the inclusion complex of cyclomaltoheptaose (beta-cyclodextrin) with hexamethylenetetramine.

The crystal structure of the inclusion complex of cyclomaltoheptaose (beta-cyclodextrin) with hexamethylenetetramine was determined at temperatures of 123, 173, 223, and 293 K. The rigid-body motion of the host and guest molecules was evaluated by means of the TLS method that represents the molecular motion in terms of translation, libration, and screw motion. In increasing the temperature from 123 to 293 K, the amplitude of the rigid body vibration of the host molecule was increased from 1.

Novel observation of a circular dichroism band originating from amyloid fibril.

A peptide fragment of a non-amyloid-beta component (NAC(1-13)) was studied by CD and electron microscopy. Typical amyloid fibrils were observed by EM in a solution of NAC(1-13). In addition to the beta-structural CD band in the far-UV region, a novel CD band near 285 nm was observed in a peptide solution of NAC(1-13).

Estimation of haplotype frequencies, linkage-disequilibrium measures, and combination of haplotype copies in each pool by use of pooled DNA data.

Inference of haplotypes is important for many genetic approaches, including the process of assigning a phenotype to a genetic region. Usually, the population frequencies of haplotypes, as well as the diplotype configuration of each subject, are estimated from a set of genotypes of the subjects in a sample from the population. We have developed an algorithm to infer haplotype frequencies and the combination of haplotype copies in each pool by using pooled DNA data.

Helicase and nuclease activities of hyperthermophile Pyrococcus horikoshii Dna2 inhibited by substrates with RNA segments at 5'-end.

Dna2 protein plays an important role in Okazaki fragment maturation on the lagging strand and also participates in DNA repair in Eukarya. Herein, we report the first biochemical characterization of a Dna2 homologue from Archaea, the hyperthermophile Pyrococcus horikoshii (Dna2Pho). Dna2Pho has both a RecB-like nuclease motif and seven conserved helicase motifs similar to Dna2 from Saccharomyces cerevisiae.

pH-dependent aggregate forms and conformation of Alzheimer amyloid beta-peptide (12-24).

The conformational transition to a beta-structure and the aggregation process of Alzheimer amyloid beta-peptide (12-24) [abbreviated as A beta(12-24)] were studied. The influence of sample dissolution methods for the aggregate structure was examined by electron microscopy (EM). The difference in the width of the aggregate of A beta(12-24) depended on the pH immediately after sample dissolution.

Three-dimensional structure of the inclusion complex between phloridzin and beta-cyclodextrin.

The inclusion of phloridzin into beta-cyclodextrin was studied as a model of molecular recognition in membranes. Effects on 1H NMR spectra and NOE correlational peaks between phloridzin and beta-cyclodextrin were observed in the complex. Strong NOEs were observed between hydrogens of a phenol group in phloridzin and beta-cyclodextrin.

NMR and CD studies on the interaction of Alzheimer beta-amyloid peptide (12-28) with beta-cyclodextrin.

Polymerization of the amyloid beta-peptide (Abeta) has been identified as a major feature of the pathogenesis of Alzheimer's disease (AD). Inhibition of the formation of these toxic polymers of Abeta has emerged as an approach for developing therapeutics for AD. NMR and CD spectra were used to investigate the interaction between cyclodextrin and Abeta(12-28) peptide, which was reported to be an important region for forming amyloid fibrils.