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Biomedical subjects

R W Janes

Publications and source records attributed to R W Janes.

At least 19 recordsLinked to original sources
PubMed2002

Resistance-associated point mutations of organophosphate insensitive acetylcholinesterase, in the olive fruit fly Bactrocera oleae.

A 2.2-kb full length cDNA containing an ORF encoding a putative acetylcholinesterase (AChE) precursor of 673 amino acid residues was obtained by a combined degenerate PCR and RACE strategy from an organophosphate-susceptible Bactrocera oleae strain. A comparison of cDNA sequences of individual insects from susceptible and resistant strains, coupled with an enzyme inhibition assay with omethoate, indicated a novel glycine-serine substitution (G488S), at an amino acid residue which is highly conserved across species (G396 of Torpedocalifornica AChE), as a likely cause of AChE insensitivity. This mutation was also associated with a 35-40% reduction in AChE catalytic efficiency. The I199V substitution, which confers low levels of resistance in Drosophila, was also present in B. oleae (I214V) and in combination with G488S produced up to a 16-fold decrease in insecticide sensitivity. This is the first agricultural pest where resistance has been associated with an alteration in AChE, which arises from point mutations located within the active site gorge of the enzyme.

Acetylcholinesterase↗
PubMed2001

Synchrotron radiation circular dichroism spectroscopy: vacuum ultraviolet irradiation does not damage protein integrity.

Synchrotron radiation circular dichroism (SRCD) spectroscopy is an emerging technique for sensitive determination of protein secondary structures and for monitoring of conformational changes. An important issue for its adoption as a useful technique is whether the high-intensity low-wavelength vacuum ultraviolet radiation in the SRCD chemically damages proteins. In this paper, using horse myoglobin as a test sample, it is shown that extensive irradiation in the SRCD does not produce any change in the chemical nature of the protein as detected by either SDS gel electrophoresis or mass spectrometry. In addition, no changes in the protein secondary structure are detectable from the SRCD spectra after extensive exposure to the SRCD beam.

Circular Dichroism↗
PubMed2001

Synchrotron radiation circular dichroism spectroscopy of proteins: secondary structure, fold recognition and structural genomics.

Recent developments in instrumentation and bioinformatics show that the technique of synchrotron radiation circular dichroism spectroscopy can provide novel information on protein secondary structures and folding motifs, and has the potential to play an important role in structural genomics studies, both as a means of target selection and as a high-throughput, low-sample-requiring screening method. This is possible because of the additional information content in the low-vacuum ultraviolet wavelength data obtainable with intense synchrotron radiation light sources, compared with that present in spectra from conventional lab-based circular dichroism instruments.

Circular Dichroism↗
PubMed2001

1-Methylindole-3-carboxaldehyde oxime derivatives.

1-Methylindole-3-carboxaldehyde oxime, C10H10N2O, (I), and (E)-5-methoxy-1-methylindole-3-carboxaldehyde oxime, C11H12N2O2, (II), were examined structurally to ascertain the geometry of the hydroxyimino function relative to the indole core. Oxime (I) exhibits cis geometry and there are two molecules in the asymmetric unit. In contrast, oxime (II) exhibits trans geometry and has four molecules in the asymmetric unit, with the geometry of the 5-methoxy group in one molecule differing from that in the other three. Both crystal structures are maintained by hydrogen bonding with no pi-stacking of the indole moiety present.

Crystallography, X-Ray↗
PubMed2000

Solution structure of alpha-conotoxin SI.

The nuclear magnetic resonance solution structure of alpha-conotoxin SI has been determined at pH 4.2. The 36 lowest energy structures show that alpha-conotoxin SI exists in a single major solution conformation and is stabilized by six hydrogen bonds. Comparisons are made between the SI solution structure and the solution and crystal structures of alpha-conotoxin GI. Surprisingly, a high degree of similarity between the backbone conformations of the GI crystal and the SI solution structures is seen in the region of lowest sequence homology, namely residues Gly-8 to Ser-12. This similarity is more surprising when considering that in SI a proline replaces the Arg-9 found in GI. The correspondence in conformation in this region provides the definitive evidence that it is the loss of the arginine basic charge at residue 9 which determines the differences in toxicity between GI and SI, rather than any changes in conformation induced by the cyclic proline residue.

Amino Acid Sequence↗
PubMed1999

Screening of a library of phage-displayed peptides identifies human bcl-2 as a taxol-binding protein.

A random library of phage displayed peptides was screened for binding to a biotinylated derivative of paclitaxel (Taxol). Affinity-selected peptides were analyzed for similarity to human proteins. There was no significant similarity between the paclitaxel-selected peptides and tubulin. However, a subset of the peptides was identified that exhibits significant similarity to a non-conserved region of the anti-apoptotic human protein Bcl-2: ELISA assays confirmed binding of paclitaxel to Bcl-2, and circular dichroism spectroscopy demonstrated that a substantial conformational change accompanies this binding. In vivo, treatment with paclitaxel has been shown to lead to Bcl-2 inactivation with concomitant phosphorylation of residues in a disordered, regulatory loop region of the protein. Similarity between paclitaxel-selected peptides and this loop region implicate these residues in drug binding, and suggest that the apoptotic action of paclitaxel may involve the binding of paclitaxel to Bcl-2. These results demonstrate that peptides displayed on the surface of bacteriophage particles can mimic the ligand-binding properties of disordered regions of proteins.

Amino Acid Sequence↗
PubMed1999

Tryptophans in membrane proteins. X-ray crystallographic analyses.

While tryptophans are generally found in low abundance in soluble proteins, in many integral membrane proteins they comprise a significantly higher proportion of the amino acid composition. Now that crystal structures are available for a number of membrane proteins, it has been possible to examine the distribution and disposition of the tryptophans within these structures. The tryptophan locations with respect to the lipid bilayer (along the direction normal to the membrane surface) are strikingly non-uniform in nearly all of the membrane proteins examined. They tend to cluster at the interface between the polar head group region and the hydrophobic interior, in a relatively uniform layer just below the surface. In many cases, their distributions with respect to the extra- and intra-cellular surfaces tend to be asymmetric. These observations provide evidence for possible structural roles for tryptophans in transmembrane sheets and helices, where they may play a part in the stabilization of the transmembrane segments and perhaps in the orientation and bilayer insertion processes.

Bacteria↗
PubMed1996

A model for Batten disease protein CLN3: functional implications from homology and mutations.

In an attempt to understand the molecular nature of Batten disease, we have examined the amino acid sequence of the affected CLN3 gene product (The International Batten Disease Consortium (1995) Cell 82, 949-957) and the site-specific mutations which give rise to the biological defect. Homology searches and molecular modeling have led to the development of a model for the folding and disposition of the protein, possibly within a mitochondrial membrane. High homology with a yeast protein of unknown function suggests a strong evolutionary conservation of function. We speculate that a possible role for the protein may be in chaperoning the folding/unfolding or assembly/ disassembly of other proteins, specifically subunit c of the mitochondrial ATP synthase complex.

Amino Acid Sequence↗
PubMed1996

A molecular model for human Big-Endothelin-1 (Big ET-1).

A molecular model has been developed for human Big Endothelin-1, which is the immediate precursor to the potent vasoconstrictor polypeptide endothelin-1 and the target of the highly specific endothelin converting enzyme. This model is produced by a threading algorithm protocol and is consistent with all the currently available structural and biochemical data for this molecule.

Algorithms↗
PubMed1995

Comparison of the structures of the endothelin A receptor antagonists BQ123 and N-methyl leucine BQ123 with the crystal structure of the C-terminal tail of endothelin-1.

The functionally important regions of the cyclic pentapeptide endothelin A receptor antagonist BQ123 are shown to correlate with the structure of the C-terminal tail of endothelin-1, as found in the recently-determined X-ray crystal structure. Residues 18 and 21 of endothelin-1 are spatially juxtaposed such that they superpose extremely well with D-Asp and D-Trp of the antagonist, consistent with the residues on this surface of the endothelin helix being important for binding. This study provides new information on the three-dimensional nature of the endothelin A receptor binding site which may prove useful for rational drug design.

Amino Acid Sequence↗
PubMed1995

A comparison of X-ray and NMR structures for human endothelin-1.

Direct comparisons between the recently solved X-ray and NMR structures of human endothelin-1 with respect to secondary structure, RMS deviations, surface accessibilities, and side-chain conformers indicate important differences in conformation, especially in the C-terminus, but also in the central loop region, that are important for defining the specificity of binding. These differences are larger than seen for other X-ray and NMR structures that have been compared. Comparisons between the X-ray structure and the NMR NOE constraints highlight the regions of flexibility and environment-induced diversity in the endothelin structures.

Computer Graphics↗
PubMed1995

The crystal structure of human endothelin-1 and how it relates to receptor binding.

Several different three-dimensional structures have recently been proposed for human endothelin-1 (ET-1) based on x-ray crystallographic, NMR spectroscopic, and modeling studies. All differ considerably in the regions that are critical for receptor binding, i.e., the central loop and the C-terminal tail, which is helical in the crystal structure but not helical in any of the other structures. In this study we examined the various ET structures in light of binding and vasoactivity data available for naturally occurring isoforms and synthetic mutants of ET. These studies strongly support the crystal structure alone as being biologically relevant with respect to receptor binding. Furthermore, molecular modeling studies based on the crystal structure of ET-1 have produced models for the ET-2 and ET-3 isoforms that suggest geometric properties for the receptor selectivity pocket of the ET(A) receptor. From these studies, it appears that the crystal structure may prove useful for aiding our understanding of the nature of the receptor/ligand binding sites and for providing a basis for the design of new agonists and antagonists.

Binding Sites↗
PubMed1994

The crystal structure of human endothelin.

The three-dimensional structure of the vasoactive polypeptide endothelin, the most potent vasoconstrictor yet identified, has been determined by X-ray crystallography to 2.18 A resolution. This intermediate-sized structure was solved by molecular replacement techniques using a fragment of an NMR-derived model for initial phasing of the data. However, comparisons of the final X-ray structure with the many diverse models derived from NMR data indicate some important differences, especially in the carboxy-terminal region of the molecule: the entire carboxy terminal tail (residues 16-21) is helical in the crystal structure, but not in any of the NMR structures. This may be a functionally significant difference as this region is crucial for receptor binding and vasoactivity.

Amino Acid Sequence↗
PubMed1993

Preliminary crystallization and X-ray analysis of orthorhombic human endothelin.

Human endothelin (ET-1) is a highly potent, ubiquitous, endogenous 21 amino acid residue polypeptide, which acts as a vaso- and bronchoconstrictor. Crystals of the active, uncomplexed form of ET-1 have been grown from aqueous solutions. These crystals, which are long hexagonal prisms, diffract to 2.98 A and are apparently of the space group P222(1) with cell dimensions a = 33.5 A, b = 57.9 A, c = 59.6 A. The six molecules in the asymmetric unit are related by non-crystallographic symmetry operations, which result in a pseudo 6(1) appearance to the data.

Crystallography, X-Ray↗