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B Rees

Publications and source records attributed to B Rees.

At least 19 recordsLinked to original sources
PubMed1998

Transmembrane signaling across the ligand-gated FhuA receptor: crystal structures of free and ferrichrome-bound states reveal allosteric changes.

FhuA protein facilitates ligand-gated transport of ferrichrome-bound iron across Escherichia coli outer membranes. X-ray analysis at 2.7 A resolution reveals two distinct conformations in the presence and absence of ferrichrome. The monomeric protein consists of a hollow, 22-stranded, antiparallel beta barrel (residues 160-714), which is obstructed by a plug (residues 19-159). The binding site of ferrichrome, an aromatic pocket near the cell surface, undergoes minor changes upon association with the ligand. These are propagated and amplified across the plug, eventually resulting in substantially different protein conformations at the periplasmic face. Our findings reveal the mechanism of signal transmission and suggest how the energy-transducing TonB complex senses ligand binding.

Allosteric Regulation↗
PubMed1996

Conformational flexibility of tRNA: structural changes in yeast tRNA(Asp) upon binding to aspartyl-tRNA synthetase.

The availability of several X-ray structures at atomic resolution of tRNA(Asp) from yeast, both in its free state and complexed with its cognate tRNA-synthetase, enables a detailed examination of the conformational changes due to interaction with the enzyme. Although the molecule conserves its general L shape, its conformation undergoes important modifications. They may be described as a bending of the two arms which brings the 3' acceptor end and the anticodon part closer together, completed by a drastic change of the anticodon loop, which puts the anticodon bases in a more exposed position, facilitating their interaction with the synthetase. The packing interactions in the crystals are also discussed. Finally, the results of protection studies by chemical probes in solution are discussed in view of the RNA-protein contacts observed in the crystals.

Aspartate-tRNA Ligase↗
PubMed1996

32P and beta emitting radionuclides in microbiology and cell biology.

Microbiology and cell biology researchers make extensive use of beta emitting nuclides. Because of the short half-lives or difficulty of direct measurement some researchers do not view radionuclide use with proper care and respect. Radionuclide use in microbiology and cell biology research is unique. Review of the isotopes and processes may be useful to the health physicist and research alike.

Animals↗
PubMed1995

Crystal structure of histidyl-tRNA synthetase from Escherichia coli complexed with histidyl-adenylate.

The crystal structure at 2.6 A of the histidyl-tRNA synthetase from Escherichia coli complexed with histidyl-adenylate has been determined. The enzyme is a homodimer with a molecular weight of 94 kDa and belongs to the class II of aminoacyl-tRNA synthetases (aaRS). The asymmetric unit is composed of two homodimers. Each monomer consists of two domains. The N-terminal catalytic core domain contains a six-stranded antiparallel beta-sheet sitting on two alpha-helices, which can be superposed with the catalytic domains of yeast AspRS, and GlyRS and SerRS from Thermus thermophilus with a root-mean-square difference on the C alpha atoms of 1.7-1.9 A. The active sites of all four monomers are occupied by histidyl-adenylate, which apparently forms during crystallization. The 100 residue C-terminal alpha/beta domain resembles half of a beta-barrel, and provides an independent domain oriented to contact the anticodon stem and part of the anticodon loop of tRNA(His). The modular domain organization of histidyl-tRNA synthetase reiterates a repeated theme in aaRS, and its structure should provide insight into the ability of certain aaRS to aminoacylate minihelices and other non-tRNA molecules.

Adenosine↗
PubMed1995

The class II aminoacyl-tRNA synthetases and their active site: evolutionary conservation of an ATP binding site.

Previous sequence analyses have suggested the existence of two distinct classes of aminoacyl-tRNA synthetase. The partition was established on the basis of exclusive sets of sequence motifs (Eriani et al. [1990] Nature 347:203-306). X-ray studies have now well defined the structural basis of the two classes: the class I enzymes share with dehydrogenases and kinases the classic nucleotide binding fold called the Rossmann fold, whereas the class II enzymes possess a different fold, not found elsewhere, built around a six-stranded antiparallel beta-sheet. The two classes of synthetases catalyze the same global reaction that is the attachment of an amino acid to the tRNA, but differ as to where on the terminal adenosine of the tRNA the amino acid is placed: class I enzymes act on the 2' hydroxyl whereas the class II enzymes prefer the 3' hydroxyl group. The three-dimensional structure of aspartyl-tRNA synthetase from yeast, a typical class II enzyme, is described here, in relation to its function. The crucial role of the sequence motifs in substrate binding and enzyme structure is high-lighted. Overall these results underline the existence of an intimate evolutionary link between the aminoacyl-tRNA synthetases, despite their actual structural diversity.

Adenosine Triphosphate↗
PubMed1995

Angiotensin and bradykinin peptides in the TGR(mRen-2)27 rat.

The transgenic TGR(mRen-2)27 rat, in which the Ren-2 mouse renin gene is transfected into the genome of the Sprague-Dawley rat, develops severe hypertension at a young age that responds to inhibitors of angiotensin-converting enzyme and to antagonists of the type 1 angiotensin II (Ang II) receptor. Despite this evidence that the hypertension is Ang II dependent, TGR(mRen-2)27 rats have suppressed renal renin and renin mRNA content, and there is controversy concerning the plasma levels of renin and Ang II in these rats. We investigated the effect of the transgene on circulating and tissue levels of angiotensin and bradykinin peptides in 6-week-old male homozygous TGR(mRen-2)27 rats. Systolic blood pressure of TGR(mRen-2)27 rats was 212 +/- 4 mm Hg (mean +/- SEM, n = 25) compared with 108 +/- 2 mm Hg (n = 29) for age- and sex-matched Sprague-Dawley rats. Compared with control rats, TGR(mRen-2)27 rats had increased plasma levels of active renin (4.5-fold), prorenin (300-fold), and Ang II (fourfold) as well as tissue levels of Ang II (twofold to fourfold in kidney, adrenal, heart, aorta, brown adipose tissue, and lung and 18-fold in brain). Plasma angiotensinogen levels were reduced to 73% of control, and plasma aldosterone levels were increased fourfold. Plasma angiotensin-converting enzyme was reduced to 64% of control. Compared with control rats, TGR(mRen-2)27 rats had increased bradykinin levels in brown adipose tissue (1.9-fold) and lung (1.6-fold).(ABSTRACT TRUNCATED AT 250 WORDS)

Aldosterone↗
PubMed1995

Electrostatic potential in aminoacylation by aspartyl-tRNAs synthetase.

Based upon the X-ray structures of complexes between tRNAAsp and aspRS including ATP or Asp-AMP, several electrostatic potentials were calculated by solving the Poisson-Boltzmann equation. The potentials indicate clearly that a Mg2+ ion is essential for binding of ATP and that aspartate is identified electrostatically. The alpha-carboxyl group is forced to contact with the alpha-phosphorus atom of ATP, suggesting its inversion to form an Asp-AMP. When the cognate tRNA is bound to the aspRS:Asp-AMP complex, the 3'-hydroxyl group is located in an electrostatically favorable position to transfer the amino acid as a class II aminoacylation.

Acylation↗
PubMed1994

X-ray structure at 1.55 A of toxin gamma, a cardiotoxin from Naja nigricollis venom. Crystal packing reveals a model for insertion into membranes.

The crystal structure of toxin gamma from Naja nigricollis has been solved and refined to 1.55 A resolution. The final R-factor, computed with all X-ray data available, is 17.9%. The three-dimensional structure is characterized by a core formed by two beta-sheets organized in three extended loops. It is similar to that of cardiotoxin V4II from Naja mossambica mossambica, with the exception of the hydrophobic loop I. The flexibility and variability of the loops contrast sharply with the rigidity of the molecular core and its high degree of structural conservation among the cardiotoxin family. The most flexible loop II adopts different conformations in the three monomers forming the crystal asymmetric unit. These monomers form a trimer around an approximate 3-fold axis, with conserved hydrophobic side-chains on the outside and hydrophilic residues in the central channel or involved in interactions with the other molecules. The trimer thus resembles a membrane protein with a central channel that could allow the passage of small ions. It is proposed as a model for the insertion of cardiotoxin into a membrane.

Amino Acid Sequence↗
PubMed1994

The active site of yeast aspartyl-tRNA synthetase: structural and functional aspects of the aminoacylation reaction.

The crystal structures of the various complexes formed by yeast aspartyl-tRNA synthetase (AspRS) and its substrates provide snapshots of the active site corresponding to different steps of the aminoacylation reaction. Native crystals of the binary complex tRNA-AspRS were soaked in solutions containing the two other substrates, ATP (or its analog AMPPcP) and aspartic acid. When all substrates are present in the crystal, this leads to the formation of the aspartyl-adenylate and/or the aspartyl-tRNA. A class II-specific pathway for the aminoacylation reaction is proposed which explains the known functional differences between the two classes while preserving a common framework. Extended signature sequences characteristic of class II aaRS (motifs 2 and 3) constitute the basic functional unit. The ATP molecule adopts a bent conformation, stabilized by the invariant Arg531 of motif 3 and a magnesium ion coordinated to the pyrophosphate group and to two class-invariant acidic residues. The aspartic acid substrate is positioned by a class II invariant acidic residue, Asp342, interacting with the amino group and by amino acids conserved in the aspartyl synthetase family. The amino acids in contact with the substrates have been probed by site-directed mutagenesis for their functional implication.

Acylation↗
PubMed1993

Yeast tRNA(Asp) recognition by its cognate class II aminoacyl-tRNA synthetase.

Aminoacyl-RNA synthetases can be divided into two classes according to structural features inferred from sequence alignments. This classification correlates almost perfectly with the attachment of the amino acid to the 2'-OH (class I) or 3'-OH (class II) group of the terminal adenosine. Six subgroups of higher homology can be inferred from sequence analysis. The five aminoacyl-tRNA synthetases whose crystal structures are known (MetRS, TyrRS and GlnRS in class I, SerRS and AspRS in class II) belong to different subgroups. Two of them, GlnRS and AspRS, have been cocrystallized with their cognate tRNA. AspRS, like six other members of class II, is an alpha 2 dimer. Yeast tRNA(Asp) exhibits five identity determinants: the three anticodon bases, the discriminator base G73 and the base pair G10-U25. We report here that the refined crystal structure of AspRS complexed with tRNA(Asp) at 2.9 A resolution reveals three regions of contact, each involving a domain of AspRS and at least one identity determinant of tRNA(Asp). The mode of binding of the acceptor stem of tRNA(Asp) by AspRS can be generalized to class II aminoacyl-tRNA synthetases, whereas the deciphering of the anticodon, which involves a large conformational change of the loop and the formation of a bulge, is more specific to the aspartic system.

Amino Acid Sequence↗
PubMed1993

Crystallographic analysis of the interaction between cyclosporin A and the Fab fragment of a monoclonal antibody.

The structure of the complex between cyclosporin A and the Fab fragment of a monoclonal antibody has been established by crystallographic analysis to 2.65 A resolution. The structure has been solved by molecular replacement using a composite Fab model. The current R-factor after refinement is 0.179 between 8 and 2.65 A resolution. The antibody is one among three known structures with long H3 loops. This loop conformation is observed for the first time in the presence of the antigen. Residues from all six hypervariable loops interact with cyclosporin A. However, the 17 residues long loop H3 is the main contributor to the buried combining site area and to the van der Waals contacts made with cyclosporin A, with 52 and 63%, respectively, of the total contribution.

Amino Acid Sequence↗
PubMed1993

Yeast aspartyl-tRNA synthetase: a structural view of the aminoacylation reaction.

The refinement of the crystal structure of a binary complex formed by yeast AspRS and tRNA(Asp) provided a detailed understanding of the recognition of tRNA by an aminoacyl-tRNA synthetase. The crystal structures of several complexes containing ATP, alone or with aspartic acid, were also determined and refined. These studies led to a complete description of the active site of the enzyme and to the elucidation of the location and interactions of the various substrates. Based on these structural results, a class II-specific pathway for the aminoacylation reaction can be proposed.

Acylation↗
PubMed1992

A conformation of cyclosporin A in aqueous environment revealed by the X-ray structure of a cyclosporin-Fab complex.

The conformation of the immunosuppressive drug cyclosporin A (CsA) in a complex with a Fab molecule has been established by crystallographic analysis to 2.65 angstrom resolution. This conformation of CsA is similar to that recently observed in the complex with the rotamase cyclophilin, its binding protein in vivo, and totally different from its conformation in an isolated form as determined from x-ray and nuclear magnetic resonance analysis. Because the surfaces of CsA interacting with cyclophilin or with the Fab are not identical, these results suggest that the conformation of CsA observed in the bound form preexists in aqueous solution and is not produced by interaction with the proteins.

Amino Acid Isomerases↗
PubMed1991

Class II aminoacyl transfer RNA synthetases: crystal structure of yeast aspartyl-tRNA synthetase complexed with tRNA(Asp).

The crystal structure of the binary complex tRNA(Asp)-aspartyl tRNA synthetase from yeast was solved with the use of multiple isomorphous replacement to 3 angstrom resolution. The dimeric synthetase, a member of class II aminoacyl tRNA synthetases (aaRS's) exhibits the characteristic signature motifs conserved in eight aaRS's. These three sequence motifs are contained in the catalytic site domain, built around an antiparallel beta sheet, and flanked by three alpha helices that form the pocket in which adenosine triphosphate (ATP) and the CCA end of tRNA bind. The tRNA(Asp) molecule approaches the synthetase from the variable loop side. The two major contact areas are with the acceptor end and the anticodon stem and loop. In both sites the protein interacts with the tRNA from the major groove side. The correlation between aaRS class II and the initial site of aminoacylation at 3'-OH can be explained by the structure. The molecular association leads to the following features: (i) the backbone of the GCCA single-stranded portion of the acceptor end exhibits a regular helical conformation; (ii) the loop between residues 320 and 342 in motif 2 interacts with the acceptor stem in the major groove and is in contact with the discriminator base G and the first base pair UA; and (iii) the anticodon loop undergoes a large conformational change in order to bind the protein. The conformation of the tRNA molecule in the complex is dictated more by the interaction with the protein than by its own sequence.

Aspartate-tRNA Ligase↗
PubMed1990

Cardiotoxin VII4 from Naja mossambica mossambica. The refined crystal structure.

The crystal structure of cardiotoxin VII4 from Naja mossambica mossambica was refined to 2.5 A resolution. Fifty ordered solvent sites were localized and included in the refinement. The final R factor is 0.197 (lambda/(2sin theta) less than 5 A; F greater than 3 sigma). The three-dimensional structure is characterized by two beta-sheets. Of particular interest is the two-stranded beta-sheet in the N-terminal region. This shows a large right-handed twist and, though strongly connected to the core of the molecule, and in particular to the C-terminal end, protrudes out of the bulk of the molecule. The segment of four amino acid residues connecting the two strands of this sheet is particularly exposed. It contains an invariant proline residue that has probably an important structural role, and is completely hydrophobic. Two other conserved hydrophobic zones were identified; the largest extends over the second and third loops, on one side only of the molecule. All side-chains of invariant hydrophobic character (except proline residues) belong to one of these three zones. Also discussed are the dimeric assembly and the rather loose packing in the crystal. The three-dimensional structure is compared with that of short and long alpha-neurotoxins. Comparison with two-dimensional nuclear magnetic resonance results on the 68% homologous cardiotoxin CT X IIb shows an excellent overall agreement. A few differences are probably genuine.

Amino Acid Sequence↗
PubMed1989

The fat content of suckled breast milk: a new approach to its assessment.

Using a modification of the "interrupted feed" technique (Woolridge et al., 1982, Early Hum. Dev., 6, 265-272), the authors devised an equation relating milk fat content at any point during a feed from a breast to the volume consumed since the start of the feed. Hence, an equation was obtained to estimate the mean fat content (MF) of milk consumed during a feed from the fat content of small samples of fore-milk (Fb) and hind-milk (Fe): MF = 0.57Fb + 0.43Fe The authors argue that their approach yields a closer approximation to the true value of MF than previously described estimators of MF.

Adult↗