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R M Cooke

Publications and source records attributed to R M Cooke.

34 records · Page 2Linked to original sources
PubMed1990

Structure-function relationships in epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha).

The solution structures of the homologous growth factors human epidermal growth factor (hEGF) and human transforming growth factor-alpha (hTGF-alpha), as determined by high resolution NMR and various computational methods, are described. Knowledge of these structures and the sequences of other homologous proteins leads to predictions about growth factor residues which may be involved in the receptor/ligand interface. Recent experiments designed to check these predictions are described briefly. These involve site-specific mutagenesis, receptor binding assays and high resolution NMR studies.

Amino Acid Sequence↗
PubMed1990

Structure-function analysis of epidermal growth factor: site directed mutagenesis and nuclear magnetic resonance.

The role of leucine-47 in determining the structure and activity of human epidermal growth factor was examined using site-directed mutagenesis. Wild type protein and four variants in which Leu47 was replaced by valine, glutamate, aspartate and alanine were produced from yeast. 1H NMR experiments demonstrated that substitution of Leu47 had little effect on the protein structure. The observed reduction in receptor binding affinity caused by the substitutions could thus be attributed to perturbation of a residue directly involved in receptor interactions.

Binding, Competitive↗
PubMed1990

A 1H NMR study of the solution conformation of the neuropeptide galanin.

The conformations of the neuropeptide galanin in water and trifluoroethanol solutions have been examined by 1H NMR spectroscopy. Analysis of two-dimensional NMR experiments enabled the assignment of virtually all the 1H resonances of galanin in trifluoroethanol solution and many of the 1H resonances in aqueous solution. Interpretation of the NMR data in structural terms suggests that in trifluoroethanol galanin is predominantly helical while in water it does not adopt a fixed conformation.

Amino Acid Sequence↗
PubMed1990

Structure function relationships in EGF, TGF-alpha and IGFI.

The solution structures of the homologous growth factors hEGF and hTGF-alpha, have been determined independently from high resolution nuclear magnetic resonance (NMR) data. A model of the insulin-like growth factor structure based on insulin coordinates (Blundell et al. (1978) Proc natn. Acad. Sci. U.S.A. 75, 180-184), has also been refined using molecular dynamics simulations with NMR-determined restraints. Knowledge of these structures, together with known sequences of other homologous proteins and experiments with site-specific residue changes, allows predictions to be made about growth factor residues which might be involved in the receptor-ligand interfaces.

Animals↗
PubMed1989

A high-resolution 1H-NMR study of human transforming growth factor alpha. Structure and pH-dependent conformational interconversion.

The 500-MHz and 600-MHz 1H-NMR spectra of recombinant human transforming growth factor alpha have been recorded at pH values of 3.8, 6.5 and 9.4. Analysis of various two-dimensional spectra has enabled sequence-specific assignments to be made and the secondary structure to be identified. Information on the tertiary fold has also been obtained from observed nuclear Overhauser effects and titration of histidine residues. The overall fold of the protein is very similar to that of epidermal growth factor, as might be expected from the sequence similarity. However, the structure of transforming growth factor alpha at pH 3.8 is found to show interesting differences from those at the two higher pHs and from that of epidermal growth factor.

Epidermal Growth Factor↗
PubMed1989

The solution structures of epidermal growth factor and transforming growth factor alpha.

The structures of human epidermal growth factor (EGF) and human transforming growth factor alpha (TGF alpha) have been determined in solution using nuclear magnetic resonance techniques. The features of each structure are described and similarities and differences between them are discussed. The structures are combined with information from sequence homologies to produce a model of the receptor-recognition sites of EGF and TGF alpha, which can be tested in a site-directed mutagenesis programme. The model assists in explaining previous observations of sequence-activity relationships. The TGF alpha and EGF structures also serve as models for homologous modules in other extracellular proteins.

Amino Acid Sequence↗
PubMed1987

NMR studies of the heme pocket conformations of monomeric hemoglobins from Glycera dibranchiata. Implications for ligand binding.

Two-dimensional 1H-NMR methods have been used to assign side-chain resonances for the tryptophan residues and for several amino acids located in the heme pockets of the carbon monoxide complexes of the major monomeric hemoglobins from Glycera dibranchiata. The NMR spectra reveal a high degree of conservation of the heme pocket structure in the different hemoglobins. However some conformational differences are evident and residues at positions B10 and G8 on the distal side of the heme pocket are not conserved. From the present NMR studies it appears that the monomeric G. dibranchiata hemoglobin examined by X-ray crystallography [Padlan, E. A. & Love, W. (1974) J. Biol. Chem. 249, 4067-4078] corresponds to HbC. Except that the orientation of the heme in solution is the reverse of that reported in the crystal structure, there is a close correspondence between the heme pocket structure in the crystal and in solution. The proximal histidine coordination geometry is almost identical in the CO complexes of the three monomeric hemoglobins studied. Distal residues are strongly implicated in determining the observed kinetic differences in ligand binding reactions. In particular, steric crowding of the ligand binding site in hemoglobin A is probably a major factor in the slower kinetics of this component.

Amino Acid Sequence↗
PubMed1987

Structural consequences of heme isomerism in monomeric hemoglobins from Glycera dibranchiata.

Two-dimensional 1H-NMR methods have been used to assign heme and amino acid proton resonances in both isomeric states of the carbon monoxide complexes of two Glycera dibranchiata monomeric hemoglobins, HbA and HbB. For each hemoglobin, there are small differences in heme pocket structure in the two isomeric forms. The largest structural perturbations associated with heme isomerism involve residues close to pyrrole rings I and II. The positions relative to the heme of phenylalanine CD1 and the proximal histidine ligand are almost unaffected by heme isomerism. These residues probably play a key role in determining the location of the heme within the heme pocket.

Animals↗
PubMed1986

A high resolution 1H NMR study of the solution structure of human epidermal growth factor.

500 MHz 1H NMR studies of human epidermal growth factor are described. The backbone resonances of the 1-48 derivative of hEGF have been assigned using two-dimensional techniques. Analysis of the type and magnitude of the observed sequential nuclear Overhauser effects and the NH-alpha CH spin-spin coupling constants allowed prediction of the secondary structure. Aspects of the tertiary structure are also identified. A pair of antiparallel beta-sheets involving residues 18-23 and 28-34 is a dominant feature of the solution structure.

Chemical Phenomena↗
PubMed1986

Monte Carlo sampling for generalized knowledge dependence with application to human reliability.

A general discussion of knowledge dependence in risk calculations shows that the assumption of independence underlying standard Monte Carlo simulation in uncertainty analysis is frequently violated. A model is presented for performing Monte Carlo simulation when the variabilities of the component failure probabilities are either negatively or positively coupled. The model is applied to examples in human reliability analysis and the results are compared to the results of Sandia Laboratories as published in the Peer Review Study and to recalculations using more recent methods of uncertainty analysis.

Humans↗
PubMed1985

Conformational disorder of the distal leucine in monomeric Glycera hemoglobins and implications for oxygen binding.

1H NMR studies of the carbon monoxide complexes of the major monomeric hemoglobins from Glycera dibranchiata show that distal leucine is conserved at position E7. The observed ring current shifts and nuclear Overhauser enhancements indicate conformational disorder of the leucine E7 side chain. The conformational substates interconvert rapidly on the NMR time scale. The rapid conformational fluctuations of leucine E7 may play a fundamental role in governing diffusion of ligands to the heme.

Animals↗
PubMed1983

Selective transcription of a cloned cauliflower mosaic virus DNA fragment in vitro by soybean RNA polymerase II in the presence of dinucleotide primers.

Transcription of a cloned cauliflower mosaic virus (CaMV) DNA fragment (plasmid pCa 8) was studied at a low enzyme: DNA ratio. Preincubation with purine nucleoside triphosphates leads to essentially random transcription, while in the presence of a dinucleoside monophosphate and a purine nucleoside triphosphate in the preincubation medium certain combinations prime preferential transcription of the eucaryotic moiety of the chimeric plasmid. Characterisation of transcription primed by the most efficient combination, ApG + ATP, shows that a low enzyme: DNA ratio is absolutely essential for selective initiation. Interestingly the presence of the eucaryotic insertion is essential for the transcription of vector sequences. Analysis of RNA primed by ApG + ATP and of short chains synthesised in the presence of the GTP analogue 3'-OMeGTP shows a high degree of selectivity of transcription initiation sites. Hybridisation of primed RNA to restriction fragments of pCa8 shows that initiation occurs within a limited region of the inserted CaMV fragment.

Adenosine Triphosphate↗
PubMedDate not supplied

The solution structure of human epidermal growth factor.

The epidermal growth factors (EGFs) are powerful mitogens for a wide variety of cells in culture; human EGF (hEGF), known as urogastrone, also inhibits gastric acid secretion in vivo. The transforming growth factors (TGF-alpha) are related to the EGF family both in sequence and activity and EGF-like sequences are often observed in a wide range of functionally unrelated proteins. Attempts to examine the structure of EGF by diffraction methods have not yet succeeded because of difficulties with crystallization. We report here a three-dimensional structure of a biologically active derivative (residues 1-48) of the 53-residue human EGF. An analysis of high resolution 1H nuclear magnetic resonance (NMR) spectra was used together with a combination of distance geometry, restrained energy minimization and restrained molecular dynamics methods. The three-dimensional structure provides a basis for understanding the properties of EGFs and for predicting the structures of homologous sequences in other proteins.

Chemical Phenomena↗