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G N Phillips

Publications and source records attributed to G N Phillips.

At least 19 recordsLinked to original sources
PubMed1992

Structure of tropomyosin at 9 angstroms resolution.

We have used molecular replacement followed by a highly parameterized refinement to determine the structure of tropomyosin crystals to a resolution to 9 A. The shape, coiled-coil structure and interactions of the molecules in the crystals have been determined. These crystals have C2 symmetry with a = 259.7 A, b = 55.3 A, c = 135.6 A and beta = 97.2 degrees. Because of the unusual distribution of intensity in X-ray diffraction patterns from these crystals, it was possible to solve the rotation problem by inspection of qualitative aspects of the diffraction data and to define unequivocally the general alignment of the molecules along the (332) and (3-32) directions of the unit cell. The translation function was then solved by a direct search procedure, while electron microscopy of a related crystal form indicated the probable location of molecular ends in the asymmetric unit, as well as the anti-parallel arrangement. The structural model we have obtained is much clearer than that obtained previously with crystals of extraordinarily high solvent content and shows the two alpha-helices of the coiled coil over most of the length of the molecules and establishes the coiled-coil pitch at 140(+/- 10) A. Moreover, the precise value of the coiled-coil pitch varies along the molecule, probably in response to local variations in the amino acid sequence, which we have determined by sequencing the appropriate cDNA. The crystals are constructed from layers of tropomyosin filaments. There are two molecules in the crystallographic asymmetric unit and the molecules within a layer are bent into an approximately sinusoidal profile. Molecules in consecutive layers in the crystal lie at an angle relative to one another as found in crystalline arrays of actin and myosin rod. There are three classes of interactions between tropomyosin molecules in the spermine-induced crystals and these give some insights into the molecular interactions between coiled-coil molecules that may have implications for assemblies such as muscle thick filaments and intermediate filaments. In interactions within a layer, the geometry of coiled-coil contacts is retained, whereas in contacts between molecules in adjacent layers the coiled-coil geometry varies and these interactions instead appear to be dominated by the repeating pattern of charged zones along the molecule.

Amino Acid Sequence↗
PubMed1992

A novel site-directed mutant of myoglobin with an unusually high O2 affinity and low autooxidation rate.

Mutants of sperm whale myoglobin were constructed at position 29 (B10 in helix notation) to examine the effects of distal pocket size on the rates of ligand binding and autooxidation. Leu29 was replaced with Ala, Val, and Phe using the synthetic gene and Escherichia coli expression system of Springer and Sligar (Springer, B. A., and Sligar, S. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8961-8965). Structures of the ferric forms of Val29 and Phe29, and the oxy form of Phe29 myoglobin were determined to 1.7 A by x-ray crystallography. The ferric mutant proteins are remarkably isomorphous with the wild type protein except in the immediate vicinity of residue 29. Thus, the protein structure in the distal pocket of myoglobin can accommodate either a large "hole" (i.e. Ala or Val) or a large side chain (i.e. Phe) at position 29 without perturbation of tertiary structure. Phe29 oxymyoglobin is also identical to the native oxy protein in terms of overall structure and interactions between the bound O2 and His64, Val68, Phe43, and Ile107. The distance between the nearest side chain atom of residue 29 and the second atom of the bound oxygen molecule is 3.2 A in the Phe29 protein and 4.9 A in native myoglobin. The equilibrium constants for O2 binding to Ala29, Val29, and Leu29 (native) myoglobin are the same, approximately 1.0 x 10(6) M-1 at 20 degrees C, whereas that for the Phe29 protein is markedly greater, 15 x 10(6) M-1. This increase in affinity is due primarily to a 10-fold decrease in the O2 dissociation rate constant for the Phe29 mutant and appears to be the result of stabilizing interactions between the negative portion of the bound O2 dipole and the partially positive edge of the phenyl ring. Increasing the size of residue 29 causes large decreases in the rate of autooxidation of myoglobin: k(ox) = 0.24, 0.23, 0.055, and 0.005 h-1 for Ala29, Val29, Leu29 (native), and Phe29 myoglobin, respectively, in air at 37 degrees C. Thus, the Leu29----Phe mutation produces a reduced protein that is remarkably stable and is expressed in E. coli as 100% MbO2. The selective pressure to conserve Leu29 at the B10 position probably represents a compromise between reducing the rate of autooxidation and maintaining a large enough O2 dissociation rate constant to allow rapid oxygen release during respiration.

Amino Acid Sequence↗
PubMed1992

What is the pitch of the alpha-helical coiled coil?

The alpha-helical, coiled-coil protein motif is increasingly recognized in a variety of functional classes of proteins. The pitch of a coiled coil, or rate of winding of the alpha-helices around each other, is a key determinant of both intra- and intermolecular interactions. Experimental measurements of the pitch of parallel two-stranded coiled coils of muscle proteins, and examination of the recently determined structure of another two-stranded coiled coil, the GCN4 transcription factor protein, suggest that the pitch has an average value of about 140 A. This value is consistent with the observed number of residues per turn in alpha-helices of globular proteins, the determinant of the interhelical packing within the coiled-coil motif. An understanding of the structural determinants of this value for the pitch and possible variations will be important in defining the interactions of coiled-coil proteins with other macromolecules.

Protein Structure, Secondary↗
PubMed1992

Diffuse x-ray scattering from tropomyosin crystals.

Diffuse scattering analyses are emerging as a technique to extract additional dynamic information from x-ray diffraction data. In fact, when examined carefully, most protein crystals show significant diffuse scattering in addition to the usual Bragg diffraction. This diffuse scattering contains information about the disorder in the crystal that cannot be obtained from the Bragg diffraction data. Diffraction from tropomyosin crystals shows characteristic diffuse scattering streaks that are directly related to motion of the molecules. The structure of tropomyosin to 15 A resolution shows that the limited molecular contacts between molecules allow large conformational fluctuations of up to 8 A amplitude. Models for the three-dimensional motion of tropomyosin have been tested by comparing their predicted diffuse scattering patterns with the experimental data. From the parameters of the successful simulations, we were able to determine the amplitudes, directions, and distances over which the atomic displacements are correlated.

Biophysical Phenomena↗
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↗
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↗
PubMed1981

Spikes and fimbriae: alpha-helical proteins form surface projections on microorganisms.

Two basic alpha-helical plans appear to characterize portions of the proteins projecting from the surface of many microorganisms. Structure A is related to that of the antibody-binding protein A of Staphylococcus aureus, in which a single polypeptide chain doubles back to form an Anti-parallel domain; the other, structure M, is related to the M proteins of group A streptococci, in which Multiple parallel alpha-helical chains interlock in a coiled-coil conformation. A projection about 100 A long may be organized on either plan, but very long projections are likely to be of the M pattern. The alpha-helical motif may account for the length of these spikes, with other functions such as anchoring or antiphagocytic activity located in domains of different structure.

Bacterial Proteins↗