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G N Phillips

Publications and source records attributed to G N Phillips.

At least 37 records · Page 2Linked to original sources
PubMed1997

Characterization of recombinant soybean leghemoglobin a and apolar distal histidine mutants.

The cDNA for soybean leghemoglobin a (Lba) was cloned from a root nodule cDNA library and expressed in Escherichia coli. The crystal structure of the ferric acetate complex of recombinant wild-type Lba was determined at a resolution of 2.2 A. Rate constants for O2, CO and NO binding to recombinant Lba are identical with those of native soybean Lba. Rate constants for hemin dissociation and auto-oxidation of wild-type Lba were compared with those of sperm whale myoglobin. At 37 degrees C and pH 7, soybean Lba is much less stable than sperm whale myoglobin due both to a fourfold higher rate of auto-oxidation and to a approximately 600-fold lower affinity for hemin. The role of His61(E7) in regulating oxygen binding was examined by site-directed mutagenesis. Replacement of His(E7) with Ala, Val or Leu causes little change in the equilibrium constant for O2 binding to soybean Lba, whereas the same mutations in sperm whale myoglobin cause 50 to 100-fold decreases in K(O2). These results show that, at neutral pH, hydrogen bonding with His(E7) is much less important in regulating O2 binding to the soybean protein. The His(E7) to Phe mutation does cause a significant decrease in K(O2) for Lba, apparently due to steric hindrance of the bound ligand. The rate constants for O2 dissociation from wild-type and native Lba decrease significantly with decreasing pH. In contrast, the O2 dissociation rate constants for mutants with apolar E7 residues are independent of pH, suggesting that hydrogen bonding to the distal histidine residue in the native protein is enhanced under acid conditions. All of these results support the hypothesis that the high affinity of Lba for oxygen and other ligands is determined primarily by enhanced accessibility and reactivity of the heme group.

Amino Acid Sequence↗
PubMed1997

Structure and dynamics of green fluorescent protein.

Many marine organisms are luminescent. The proteins that produce the light include a primary light producer (aequorin or luciferase) and often a secondary photoprotein that red shifts the light for better penetration in the ocean. Green fluorescent protein is one such secondary protein. It is remarkable in that it autocatalyzes the formation of its own fluorophore and thus can be expressed in a variety of organisms in its fluorescent form. The recent determination of its 3D structure and other physical characterizations are revealing its molecular mechanism of action.

Animals↗
PubMed1996

High resolution crystal structures of the deoxy, oxy, and aquomet forms of cobalt myoglobin.

The structures of the deoxy, oxy, and aquomet forms of native sperm whale myoglobin reconstituted with cobalt protoporphyrin IX have been determined by x-ray crystallography. As expected, cobalt myoglobin closely resembles native iron myoglobin in overall structure, especially in their respective aquomet forms. In the cobalt oxymyoglobin structure, the Nepsilon of distal histidine 64 lies within hydrogen bonding distance to both the oxygen atom directly bonded to the cobalt and the terminal oxygen atom, in agreement with previous EPR and resonance Raman studies. The metal atom in cobaltous myoglobin does show a small 0.06-A out-of-porphyrin plane displacement when moving from the oxy to deoxy state. In the case of the native iron-containing myoglobin, the oxy to deoxy transition results in a larger 0.16-A displacement of the metal farther out of the porphyrin plane, attributed to an increase in spin from S = 0 to S = 2. The small displacement in cobalt myoglobin is due to a change in coordination geometry, not spin state (S = 1/2 for both cobalt deoxy- and oxymyoglobin). The small out-of-porphyrin plane movement of cobalt which accompanies deoxygenation of myoglobin also occurs in cobalt hemoglobin and serves to explain why cooperativity, although reduced, is still preserved when iron is replaced by cobalt in human hemoglobin.

Animals↗
PubMed1996

Mechanism of NO-induced oxidation of myoglobin and hemoglobin.

Nitric oxide (NO) has been implicated as mediator in a variety of physiological functions, including neurotransmission, platelet aggregation, macrophage function, and vasodilation. The consumption of NO by extracellular hemoglobin and subsequent vasoconstriction have been suggested to be the cause of the mild hypertensive events reported during in vivo trials of hemoglobin-based O2 carriers. The depletion of NO from endothelial cells is most likely due to the oxidative reaction of NO with oxyhemoglobin in arterioles and surrounding tissue. In order to determine the mechanism of this key reaction, we have measured the kinetics of NO-induced oxidation of a variety of different recombinant sperm whale myoglobins (Mb) and human hemoglobins (Hb). The observed rates depend linearly on [NO] but show no dependence on [O2]. The bimolecular rate constants for NO-induced oxidation of MbO2 and HbO2 are large (k.ox,NO = 30-50 microM-1 s-1 for the wild-type proteins) and similar to those for simple nitric oxide binding to deoxygenated Mb and Hb. Both reversible NO binding and NO-induced oxidation occur in two steps: (1) bimolecular entry of nitric oxide into the distal portion of the heme pocket and (2) rapid reaction of noncovalently bound nitric oxide with the iron atom to produce Fe(2+)-N=O or with Fe(2+)-O-O delta- to produce Fe(3+)-OH2 and nitrate. Both the oxidation and binding rate constants for sperm whale Mb were increased when His(E7) was replaced by aliphatic residues. These mutants lack polar interactions in the distal pocket which normally hinder NO entry into the protein. Decreasing the volume of the distal pocket by replacing Leu(B10) and Val(E11) with aromatic amino acids markedly inhibits NO-induced oxidation of MbO2. The latter results provide a protein engineering strategy for reducing hypertensive events caused by extracellular hemoglobin-based O2 carriers. This approach has been explored by examining the effects of Phe(B10) and Phe(E11) substitutions on the rates of NO-induced oxidation of the alpha and beta subunits in recombinant human hemoglobin.

Animals↗
PubMed1996

Crystal structures of CO-, deoxy- and met-myoglobins at various pH values.

The distal histidine residue, His64(E7), and the proximal histidine residue, His93(F8), in myoglobin (Mb) are important for the function of the protein. For example, the increase in the association rate constant for CO binding at low pH has been suggested to be caused by the protonation of these histidine residues. In order to investigate the influence of protonation on the structure of myoglobin, we determined the crystal structures of sperm whale myoglobin to 2.0 A or better in different states of ligation (MbCO, deoxyMb and metMb) at pH values of 4, 5 and 6. The most dramatic change found at low pH is that His64 swings out of the distal pocket in the MbCO structure at pH 4, opening a direct channel from the solvent to the iron atom. This rotation seems to be facilitated by conformational changes in the CD corner. The benzyl side-chain of Phe46(CD4), which has been suggested to be a critical residue in controlling the rotation of His64, moves away from His64 at pH 4 in the deoxyMb structure, allowing more free rotation of His64. Arg45(CD3) is also important for the dynamics of myoglobin, since it influences the pK(a) of His64 and forms a hydrogen bond lattice that hinders the rotation of His64 at neutral pH. This hydrogen-bond lattice disappears at low pH. Although His64 rotates out of the distal pocket in the MbCO structure at pH 4, leaving more space for the CO ligand, the Fe-C-O angle refines to about 130 degrees, the same as those at pH 5 and 6. In the MbCO structure at pH 4, significant conformational changes appear in the EF corner. The peptide plane between Lys79(EF2) and Gly80(EF3) flips about 150 degrees. The occupancy of this conformation in the MbCO structures increases with decreases in pH. On the proximal side of the heme, the bond between the heme iron atom and N(epsilon) of His93 remains intact under the experimental conditions in the MbCO and deoxyMb structures, but appears elongated in the metMb structure at pH 4, representing either a weakened bond or the breakage of the bond in some fraction of the molecules in the crystal.

Animals↗
PubMed1996

Study of global motions in proteins by weighted masses molecular dynamics: adenylate kinase as a test case.

The weighted masses molecular dynamics (WMMD) technique is applied to the protein adenylate kinase. A novel set of restraints has been developed to allow the use of this technique with proteins. The WMMD simulation is successful in predicting the flexibility of the two mobile domains of the protein. The end product of the simulation is similar to the known open and AMP bound forms of the enzyme. The biological relevance of the restraints used and potential methods of improving the technique are discussed.

Adenosine Monophosphate↗
PubMed1996

Mechanical properties of tropomyosin and implications for muscle regulation.

Tropomyosin is a protein that controls the interactions of actin and myosin as a part of the regulation of muscle contraction. The 420 A long alpha-helical coiled-coil molecules form long filaments, both in muscle and in crystals. The x-ray diffraction data from tropomyosin crystals have indicated large scale motions of the filaments that can be related to the inherent mechanical properties of the molecule, and by extension, to the role of tropomyosin in the cooperative activation of the thin filaments of muscle. Diffuse scattering analysis has provided information about the amplitudes of the motions that has been used to calculate the intrinsic flexibility of the molecule. It can then be shown that each tropomyosin molecule by itself can only mediate interactions of the nearest-neighboring tropomyosin molecules along the filament. The repeating nature of the thin filament, however, allows the entire filament to activate cooperatively.

Actin Cytoskeleton↗
PubMed1996

Affinity and structure of complexes of tropomyosin and caldesmon domains.

The interaction of caldesmon domains with tropomyosin has been studied using x-ray crystallography and an optical biosensor. Only whole caldesmon and the carboxyl-terminal domain of caldesmon (CaD-4, chicken gizzard residues 597-756) bound to tropomyosin with greater than millimolar affinity at 100 and 150 microM salt. Under these conditions the affinities of whole caldesmon and CaD-4 were both in the micromolar range. Data from the x-ray studies showed that whole caldesmon bound to tropomyosin in several places, with the region of tightest interaction being at tropomyosin residues 70-100 and/or 230-260. Studies with CaD-4 revealed that this region corresponded to the strong binding site seen with whole caldesmon. Weaker association of other regions of caldesmon to tropomyosin residues 180-210 and 5-50 was also observed. The results suggest that the carboxyl-terminus of caldesmon binds tightly to tropomyosin and that other regions of caldesmon may interact with tropomyosin tightly only when they are held close to tropomyosin by the carboxyl-terminal domain. Four models are presented to show the possible interactions of caldesmon with tropomyosin.

Animals↗
PubMed1996

The molecular structure of green fluorescent protein.

The crystal structure of recombinant wild-type green fluorescent protein (GFP) has been solved to a resolution of 1.9 A by multiwavelength anomalous dispersion phasing methods. The protein is in the shape of a cylinder, comprising 11 strands of beta-sheet with an alpha-helix inside and short helical segments on the ends of the cylinder. This motif, with beta-structure on the outside and alpha-helix on the inside, represents a new protein fold, which we have named the beta-can. Two protomers pack closely together to form a dimer in the crystal. The fluorophores are protected inside the cylinders, and their structures are consistent with the formation of aromatic systems made up of Tyr66 with reduction of its C alpha-C beta bond coupled with cyclization of the neighboring glycine and serine residues. The environment inside the cylinder explains the effects of many existing mutants of GFP and suggests specific side chains that could be modified to change the spectral properties of GFP. Furthermore, the identification of the dimer contacts may allow mutagenic control of the state of assembly of the protein.

Animals↗
PubMed1995

A double mutant of sperm whale myoglobin mimics the structure and function of elephant myoglobin.

The functional, spectral, and structural properties of elephant myoglobin and the L29F/H64Q mutant of sperm whale myoglobin have been compared in detail by conventional kinetic techniques, infrared and resonance Raman spectroscopy, 1H NMR, and x-ray crystallography. There is a striking correspondence between the properties of the naturally occurring elephant protein and those of the sperm whale double mutant, both of which are quite distinct from those of native sperm whale myoglobin and the single H64Q mutant. These results and the recent crystal structure determination by Bisig et al. (Bisig, D. A., Di Iorio, E. E., Diederichs, K., Winterhalter, K. H., and Piontek, K. (1995) J. Biol. Chem. 270, 20754-20762) confirm that a Phe residue is present at position 29 (B10) in elephant myoglobin, and not a Leu residue as is reported in the published amino acid sequence. The single Gln64(E7) substitution lowers oxygen affinity approximately 5-fold and increases the rate of autooxidation 3-fold. These unfavorable effects are reversed by the Phe29(B10) replacement in both elephant myoglobin and the sperm whale double mutant. The latter, genetically engineered protein was originally constructed to be a blood substitute prototype with moderately low O2 affinity, large rate constants, and increased resistance to autooxidation. Thus, the same distal pocket combination that we designed rationally on the basis of proposed mechanisms for ligand binding and autooxidation is also found in nature.

Amino Acid Sequence↗
PubMed1995

The D-helix in myoglobin and in the beta subunit of hemoglobin is required for the retention of heme.

All globins consist of eight helices and interconnecting loops except alpha hemoglobin subunits which lack the D-helix due to deletion of five consecutive residues. Previous site-directed mutagenesis work suggested that this deletion is a neutral modification in hemoglobin with respect to equilibrium O2 binding [Komiyama, N. H., Shih, T.-B., Looker, D., Tame, J., & Nagai, K. (1991) Nature 352, 349-351]. To examine the role of the D-helix in myoglobin, we have measured the O2 and CO binding and hemin dissociation properties of recombinant sperm whale myoglobin mutants in which residues 52-56 have been deleted, Mb(-D), replaced by five alanines, Mb(Ala52-56), and substituted with four alanines and a methionine, Mb(Ala52-55Met56). Crystal structures of aquometMb(-D) and aquometMb(Ala52-55Met56) were determined to 2.0 A resolution and show that the conformation of the distal pocket is little affected by removal of the D-helix or mutations in this region. As a result, these mutations have little effect on O2 and CO binding. Diffuse electron density is observed in the region between the C- and E-helices of Mb(-D), indicating a highly mobile or heterogeneous conformation in this portion of the tertiary structure. This flexibility provides an explanation for the 50-fold higher rate of hemin loss from Mb(-D) as compared to that from wild-type myoglobin. Hemin loss from Mb(Ala52-56) is also rapid. In contrast, Mb(Ala52-55Met56) shows a well-defined D-helix and has a rate of hemin loss identical to that of wild-type holoprotein [corrected].(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

A sampling problem in molecular dynamics simulations of macromolecules.

Correlations in low-frequency atomic displacements predicted by molecular dynamics simulations on the order of 1 ns are undersampled for the time scales currently accessible by the technique. This is shown with three different representations of the fluctuations in a macromolecule: the reciprocal space of crystallography using diffuse x-ray scattering data, real three-dimensional Cartesian space using covariance matrices of the atomic displacements, and the 3N-dimensional configuration space of the protein using dimensionally reduced projections to visualize the extent to which phase space is sampled.

Computer Simulation↗
PubMed1995

Structural and functional effects of apolar mutations of the distal valine in myoglobin.

High-resolution structures of the aquomet, deoxy, and CO forms of Ala68, Ile68, Leu68, and Phe68 sperm whale myoglobins have been determined by X-ray crystallography. These 12 new structures, plus those of wild-type myoglobin, have been used to interpret the effects of mutations at position 68 and the effects of cobalt substitution on the kinetics of O2, CO, and NO binding. Molecular dynamics simulations based on crystal structures have provided information about the time-dependent behavior of photolyzed ligands for comparison with picosecond geminate recombination studies. The Val68-->Ala mutation has little effect on the structure and function of myoglobin. In Ala68 deoxymyoglobin, as in the wild-type protein, a water molecule hydrogen-bonded to the N epsilon atom of the distal histidine restricts ligand binding and appears to be more important in regulating the function of myoglobin than direct steric interactions between the ligand and the C gamma atoms of the native valine side-chain. This distal pocket water molecule is displaced by the larger side-chains at position 68 in the crystal structures of Leu68 and Ile68 deoxymyoglobins. The Leu68 side-chain can rotate about its C alpha-C beta and C beta-C gamma bonds to better accommodate bound ligands, resulting in net increases in overall association rate constants and affinities due to the absence of the distal pocket water molecule. However, the flexibility of Leu68 makes simulation of picosecond NO recombination difficult since multiple starting conformations are possible. In the case of Ile68, rotation of the substituted side-chain is restricted due to branching at the beta carbon, and as a result, the delta methyl group is located close to the iron atom in both the deoxy and liganded structures. The favorable effect of displacing the distal pocket water molecule is offset by direct steric hindrance between the bound ligand and the terminal carbon atom of the isoleucine side-chain, resulting in net decreases in affinity for all three ligands and inhibition of geminate recombination which is reproduced in the molecular dynamics simulations. In Phe68 myoglobin, the benzyl side-chain is pointed away from the ligand binding site, occupying a region in the back of the distal pocket. As in wild-type and Ala68 myoglobins, a well-defined water molecule is found hydrogen bonded to the distal histidine in Phe68 deoxymyoglobin. This water molecule, in combination with the large size of the benzyl side-chain, markedly reduces the speed and extent of ligand movement into the distal pocket. (ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Structure and dynamics of the water around myoglobin.

The interplay between simulations at various levels of hydration and experimental observables has led to a picture of the role of solvent in thermodynamics and dynamics of protein systems. One of the most studied protein-solvent systems is myoglobin, which serves as a paradigm for the development of structure-function relationships in many biophysical studies. We review here some aspects of the solvation of myoglobin and the resulting implications. In particular, recent theoretical and simulation studies unify much of the diverse set of experimental results on water near proteins.

Crystallography, X-Ray↗
PubMed1995

Automatic identification of discrete substates in proteins: singular value decomposition analysis of time-averaged crystallographic refinements.

The singular value decomposition (SVD) provides a method for decomposing a molecular dynamics trajectory into fundamental modes of atomic motion. The right singular vectors are projections of the protein conformations onto these modes showing the protein motion in a generalized low-dimensional basis. Statistical analysis of the right singular vectors can be used to classify discrete configurational substates in the protein. The configuration space portraits formed from the right singular vectors can also be used to visualize complex high-dimensional motion and to examine the extent of configuration space sampling by the simulation.

Computer Simulation↗
PubMed1995

Phe-46(CD4) orients the distal histidine for hydrogen bonding to bound ligands in sperm whale myoglobin.

The role of Phe-46(CD4) in modulating the functional properties of sperm whale myoglobin was investigated by replacing this residue with Leu, Ile, Val, Ala, Trp, Tyr, and Glu. This highly conserved amino acid almost makes direct contact with the distal histidine and has been postulated to affect ligand binding. The overall association rate constants for CO, O2, and NO binding were little affected by decreasing the size of residue 46 step-wise from Phe to Leu to Val to Ala. In contrast, the rates of CO, O2, and NO dissociation increased 4-, 10-, and 25-fold, respectively, for the same series of mutants, causing large decreases in the affinity of myoglobin for all three diatomic gases. The rates of autooxidation at 37 degrees C, pH 7.0 increased dramatically from approximately 0.1-0.3 h-1 for wild-type, Tyr-46, and Trp-46 myoglobins to 1.5, 5.2, 4.9, and 5.0 h-1 for the Leu-46, Ile-46, Val-46 and Ala-46 mutants, respectively. Rates of NO and O2 geminate recombination were measured using 35 ps and 9 ns laser excitation pulses. Decreasing the size of residue 46 causes significant decreases in the extent of both picosecond and nanosecond rebinding processes. High resolution structures of Leu-46 and Val-46 metmyoglobins, Val-46 CO-myoglobin, and Val-46 deoxymyoglobin were determined by X-ray crystallography. When Phe-46 is replaced by Val, the loss of internal packing volume is compensated by (1) contraction of the CD corner toward the core of the protein, (2) movement of the E-helix toward the mutation site, (3) greater exposure of the distal pocket to intruding solvent molecules, and (4) large disorder in the position of the side chain of the distal histidine (His-64). In wild-type myoglobin, the van der Waals contact between C zeta of Phe-46 and C beta of His-64 appears to restrict rotation of the imidazole side chain. Insertion of Val at position 46 relieves this steric restriction, allowing the imidazole side chain to rotate about the C alpha - C beta bond toward the surface of the globin and about the C beta - C gamma bond toward the space previously occupied by the native Phe-46 side chain. This movement disrupts hydrogen bonding with bound ligands, causing significant decreases in affinity, and opens the distal pocket to solvent water molecules, causing marked increases in the rate of autooxidation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗