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Biomedical subjects

G N Phillips

Publications and source records attributed to G N Phillips.

At least 73 records · Page 4Linked to original sources
PubMed1991

Assignment of the nucleotide binding sites and the mechanism of substrate inhibition of Escherichia coli adenylate kinase.

Site-directed mutagenesis of key amino acids of adenylate kinase has been used to suggest a new model for the location of the AMP and ATP binding sites. Phe-86 and Tyr-133, which are in close contact with the inhibitor Ap5A according to previous crystallographic results, have been independently changed to tryptophan and other amino acids. The Phe-86----Trp mutant had a 3- to 6-fold change in the Km for ATP and a 44-fold increase in the Km for AMP with a simultaneous loss of AMP substrate inhibition. Thus Phe-86 is probably in close contact with bound AMP. The Tyr-133----Trp mutant showed no large effects on enzyme kinetics and suggests that the previous assignment of Ap5A occupying natural adenosine binding sites is probably incorrect. A temperature-sensitive Leu-107----Gln mutant showed a 6-fold decrease in the Km for ATP and no effect on AMP binding, suggesting that this amino acid is near the ATP binding site. Changes in the fluorescence of single tryptophan-containing mutant enzymes provided specific information about AMP and ATP binding. The fluorescence results are consistent with the kinetic studies, and also suggest that AMP substrate inhibition is caused by the formation of an abortive complex that prevents the release of product.

Adenosine Monophosphate↗
PubMed1991

Structural studies of tropomyosin by cryoelectron microscopy and x-ray diffraction.

A comparison has been made between cryoelectron microscope images and the x-ray structure of one projection of the Bailey tropomyosin crystal. The computed transforms of the electron micrographs extend to a resolution of approximately 18 A compared with the reflections from x-ray crystallography which extend to 15 A. After correction of the images for lattice distortions and the contrast transfer function, the structure factors were constrained to the plane group (pmg) symmetry of this projection. Amplitude and phase data for five images were compared with the corresponding view from the three-dimensional x-ray diffraction data (Phillips, G.N., Jr., J.P. Fillers, and C. Cohen. 1986. J. Mol. Biol. 192: 111-131). The average R factor between the electron microscopy and x-ray amplitudes was 15%, with an amplitude-weighted mean phase difference of 4.8 degrees. The density maps derived from cryoelectron microscopy contain structural features similar to those from x-ray diffraction: these include the width and run of the filaments and their woven appearance at the crossover regions. Preliminary images obtained from frozen-hydrated tropomyosin/troponin cocrystals suggest that this approach may provide structural details not readily obtainable from x-ray diffraction studies.

Animals↗
PubMed1990

Crystal structure of myoglobin from a synthetic gene.

Crystals have been grown of myoglobin produced in Escherichia coli from a synthetic gene, and the structure has been solved to 1.9 A resolution. The space group of the crystals is P6, which is different from previously solved myoglobin crystal forms. The synthetic myoglobin is essentially identical to myoglobin isolated from sperm whale tissue, except for the retention of the initiator methionine at the N-terminus and the substitution of asparagine for aspartic acid at position 122. Superposition of the coordinates of native and synthetic sperm whale myoglobins reveals only minor changes in the positions of main chain atoms and reorientation of some surface side chains. Crystals of variants of the "synthetic" myoglobin have also been grown for structural analysis of the role of key amino acid residues in ligand binding and specificity.

Escherichia coli↗
PubMed1990

Comparison of the dynamics of myoglobin in different crystal forms.

Crystals have been grown of "sperm whale" myoglobin produced in Escherichia coli from a synthetic gene and the structure has been solved to 1.9 A resolution. Because of a remaining initiator methionine, this protein crystallizes in a different space group from native sperm whale myoglobin. The three-dimensional structure of the synthetic protein is essentially identical to the native sperm whale protein. However, the crystallographic B-factors for parts of the molecule are quite different in the two crystal forms, and provide a measure of the effect of different packing constraints on the flexibility of the protein. The effect of the packing forces is to reduce the mobility of the protein in the regions of contact and thereby introduce differences in mobilities between the two crystal forms. Discrepancies between mobilities calculated from molecular dynamics simulations and crystallography can be reduced by considering the data from both crystal forms.

Animals↗
PubMed1989

Structure of myoglobin-ethyl isocyanide. Histidine as a swinging door for ligand entry.

The structure of myoglobin(Fe II)-ethyl isocyanide has been solved at 1.68 A resolution by X-ray crystallography. The isocyano group of the ligand is distorted from the linear conformation observed in solution and in model compounds. Local changes in the protein conformation are also seen. The side-chain of Arg-CD3 moves out into the solvent, and the side-chain of His-E7 swings up and away from the ligand. Both of these side-chains show disorder indicative of dynamic behavior. These outward movements of His-E7 and Arg-CD3 side-chains clear a path from the solvent to the heme iron, suggesting a mechanism for ligand entry.

Animals↗
PubMed1989

Paleosols and their relevance to Precambrian atmospheric composition

Various paleosols have been reported from within the Witwatersrand and Ventersdorp Supergroups, South Africa. They were studied in an attempt to constrain the amount of oxygen available in the atmosphere during deposition of the gold- and uranium-bearing Witwatersrand conglomerates. The majority of these horizons do not have any physical characteristics of paleosols, and none of them have a chemistry consistent with weathering, suggesting they have been subjected to modification by later alteration processes. A similar chemistry, indicating overprinting of any original soil chemistry, has been reported from paleosols elsewhere (Elliot Lake region, Canada; Hekpoort basalt, South Africa), but it does not appear that significant cognizance has been taken of this fact when using these paleosols to determine the composition of the Precambrian atmosphere. It is concluded here that characteristics previously attributed to Precambrian weathering in an oxygen-deficient atmosphere are better explained by post-burial, hydrothermal alteration along lithological contacts.

Aerosols↗
PubMed1988

Crystallization and preliminary X-ray diffraction analysis of 11 S acetylcholinesterase.

The 11 S form of acetylcholinesterase from Electrophorus electricus was purified by affinity chromatography. The protein was crystallized from polyethylene glycol solutions. One crystal form proved suitable for x-ray diffraction studies. Preliminary x-ray analysis demonstrates that the space group of this crystal is F222. The unit cell dimensions are a = 141.0 +/- 0.2, b = 202.4 +/- 0.2, and c = 237.4 +/- 0.1 A. The diffraction is anisotropic, extending to at least 3.5 A along the a* and b* axes, but becoming weak beyond about 6 A along the c* axis. Crystal density measurements suggest that one complete 11 S tetramer occupies the asymmetric unit of the crystal.

Acetylcholinesterase↗
PubMed1988

Effects of alkaline hydrogen peroxide treatment of cotton and wheat straw on cellulose crystallinity and on composition and site and extent of disappearance of wheat straw cell wall phenolics and monosaccharides by sheep.

Effects of alkaline hydrogen peroxide (AHP) treatment on cellulose crystallinity and cell wall phenolic monomer and monosaccharide composition were measured using cotton and wheat straw (WS). Two WS treatments were used in this study, Type I WS, for which pH is not regulated during AHP treatment, and Type II WS, for which pH is regulated at 11.5 +/- .2 during AHP treatment. Wheat straw had a lower degree of cellulose crystallinity than cotton, but no differences occurred between treated and untreated substrates. Alkali-labile and nitrobenzene-extractable phenolic monomer concentrations were generally lower for Type I and Type II WS compared with untreated WS. Concentrations of glucose were higher and xylose and arabinose lower in Types I and II WS than in untreated WS. Disappearance of alkali-labile phenolic monomers and cell wall monosaccharides by wethers fed diets containing Type I (Exp. 1) or Type II (Exp. 2) AHP-treated WS were determined. Apparent digestibility of glucose and xylose before the duodenum, and of glucose, xylose and arabinose in the total tract, was greatest (P less than .05) when sheep were fed AHP-treated WS diets in both experiments. In Exp. 2, disappearance of alkali-labile phenolic monomers was greatest (P less than .05) before the duodenum and in the total tract when sheep were fed AHP-treated WS diets. Treatment of WS with AHP modified cell wall composition and increased cell wall monosaccharide digestion by sheep.

Animal Feed↗
PubMed1987

A new crystal form of tropomyosin. Preliminary X-ray diffraction analysis.

A new crystalline form of tropomyosin has been produced that diffracts to about 4 A resolution. The crystals are grown at room temperature by slowly lowering the concentration of spermine. This polyamine apparently neutralizes the acidic amino acid side-chains of tropomyosin and allows close side-by-side packing of molecules. The space group is C2, with unit cell dimensions a = 259.7 A, b = 55.3 A, c = 135.6 A, and beta = 97.2 degrees. The tropomyosin molecules appear to be bonded head-to-tail to form straight filaments that run along the crystallographic (332) direction in an arrangement closely related to thin crystalline sheets previously described.

Crystallization↗
PubMed1986

Tropomyosin crystal structure and muscle regulation.

The crystal structure of tropomyosin filaments has been solved to 15 A resolution by refinement of models against the diffraction data and heavy atom labeling of cysteine residues. These results confirm and extend earlier findings. The improved maps reveal the pitch of the coiled coil, the location of the cysteine residues, and the location and features of the overlapping molecular ends in the filaments. A correlation can now be made between regions of the amino acid sequence and key features of the molecule, such as contact sites in the lattice and departures from regularity along the coiled coil. The crystal shows remarkable dynamic features and the relative flexibility of different parts of the molecule as well as its anisotropic character have been determined. The structure and motions of tropomyosin in the crystal provide information on the structure of tropomyosin in muscle and its possible role in regulation. An atomic model of the molecule has been constructed, based on the low resolution X-ray results, together with the stereochemistry of alpha-helical coiled coils. In contrast to previous views, the molecule appears to display but one set of seven alpha-sites that permit weak linkages of the flexible tropomyosin filament to the actin helix. Correspondingly, we picture that in the "off" state of ATPase activity, the alpha-sites are not occupied; in the "on" state, they are only partly occupied; and in the "potentiated" state, they are more completely saturated. Control of contraction is therefore seen as a statistical mechanism requiring at least three distinct average conformations for the tropomyosin molecule on the actin helix.

Amino Acid Sequence↗
PubMed1985

Crystallization and preliminary x-ray investigation of the regulatory subunit of cAMP-dependent protein kinase.

Crystals of type I cAMP-dependent protein kinase regulatory subunit have been grown from solutions of ammonium sulfate. The crystals are square bipyramids, space group P4(1)2(1)2 (P4(3)2(1)2), with a = b = 106.9 +/- 0.6 A and c = 212.4 +/- 1.0 A. There are two dimers of the regulatory subunit/crystallographic asymmetric unit. The crystals are stable for 3-4 days in the x-ray beam and diffract to at least 3.5-A resolution.

Animals↗
PubMed1983

The structure of fibrinogen and fibrin: II. Architecture of the fibrin clot.

Our present low resolution model for fibrinogen based on electron microscopy and x-ray diffraction data has been described by Cohen et al. A unique aspect of the structural analysis of fibrous proteins is that the molecular packing in ordered arrays reflects biologically significant intermolecular interactions. We have shown that the orthogonal sheet microcrystals, which are closely related to fibrin, are made up of a highly regular arrangement of two-stranded protofibrils, and we have visualized aspects of both the substructure of the protofibrils as well as their packing to form the fibrin clot. By correlation of structural data with biochemical studies we have begun to identify certain functional regions of the fibrinogen model related to fibrin. Many aspects of fibrinogen's physiological activity remain to be related to its structure. As our present model is improved by higher resolution studies, we will see with increasing clarity molecular features critical for clot formation and fibrinolysis.

Computers↗