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George N Phillips

Publications and source records attributed to George N Phillips.

At least 19 recordsLinked to original sources
PubMed2005

Identifying and engineering ion pairs in adenylate kinases. Insights from molecular dynamics simulations of thermophilic and mesophilic homologues.

Molecular dynamics simulations were performed to study thermal stabilization of proteins via electrostatic interactions of ion pairs. Dynamic motions of four ion pairs previously proposed to be important in thermal stability of adenylate kinase from the thermophile Bacillus stearothermophilus were monitored during the simulation. One of the four ion pairs identified in the crystal structure, Lys180-Asp114, was not maintained in close contact suggesting that the ion pair does not contribute to thermal stability. Among the other three ion pairs, the ion pair Arg116-Glu198 was proposed to be the most important for stability. To predict behaviors of the ion pairs when engineered into a mesophilic homologue to increase stability, in silico mutants of adenylate kinase from the mesophile Bacillus subtilis were generated, and their molecular dynamics simulations were carried out. The ion pairs in the mutant simulations displayed similar behaviors to those in the simulation of the thermophilic protein. To validate the results of the simulations experimentally, the same mutants were produced in vitro and their thermal stabilities were measured using differential scanning calorimetry. In agreement with the simulations, the Lys180-Asp114 did not result in any increase in stability by itself or additive effect with other ion pairs, whereas a mutant with the Arg116-Glu198 exhibited the highest stability among the mutants having one of the four ion pairs. These results provide specific knowledge about stability in adenylate kinases and more generally suggest that molecular dynamics simulations can provide valuable information for identifying and engineering ion pairs.

Adenylate Kinase↗
PubMed2005

Comparison of cell-based and cell-free protocols for producing target proteins from the Arabidopsis thaliana genome for structural studies.

We describe a comparative study of protein production from 96 Arabidopsis thaliana open reading frames (ORFs) by cell-based and cell-free protocols. Each target was carried through four pipeline protocols used by the Center for Eukaryotic Structural Genomics (CESG), one for the production of unlabeled protein to be used in crystallization trials and three for the production of 15N-labeled proteins to be analyzed by 1H-15N NMR correlation spectroscopy. Two of the protocols involved Escherichia coli cell-based and two involved wheat germ cell-free technology. The progress of each target through each of the protocols was followed with all failures and successes noted. Failures were of the following types: ORF not cloned, protein not expressed, low protein yield, no cleavage of fusion protein, insoluble protein, protein not purified, NMR sample too dilute. Those targets that reached the goal of analysis by 1H-15N NMR correlation spectroscopy were scored as HSQC+ (protein folded and suitable for NMR structural analysis), HSQC+/- (protein partially disordered or not in a single stable conformational state), HSQC- (protein unfolded, misfolded, or aggregated and thus unsuitable for NMR structural analysis). Targets were also scored as X- for failing to crystallize and X+ for successful crystallization. The results constitute a rich database for understanding differences between targets and protocols. In general, the wheat germ cell-free platform offers the advantage of greater genome coverage for NMR-based structural proteomics whereas the E. coli platform when successful yields more protein, as currently needed for crystallization trials for X-ray structure determination.

Arabidopsis↗
PubMed2005

Identification of transcribed sequences in Arabidopsis thaliana by using high-resolution genome tiling arrays.

Using a maskless photolithography method, we produced DNA oligonucleotide microarrays with probe sequences tiled throughout the genome of the plant Arabidopsis thaliana. RNA expression was determined for the complete nuclear, mitochondrial, and chloroplast genomes by tiling 5 million 36-mer probes. These probes were hybridized to labeled mRNA isolated from liquid grown T87 cells, an undifferentiated Arabidopsis cell culture line. Transcripts were detected from at least 60% of the nearly 26,330 annotated genes, which included 151 predicted genes that were not identified previously by a similar genome-wide hybridization study on four different cell lines. In comparison with previously published results with 25-mer tiling arrays produced by chromium masking-based photolithography technique, 36-mer oligonucleotide probes were found to be more useful in identifying intron-exon boundaries. Using two-dimensional HPLC tandem mass spectrometry, a small-scale proteomic analysis was performed with the same cells. A large amount of strongly hybridizing RNA was found in regions "antisense" to known genes. Similarity of antisense activities between the 25-mer and 36-mer data sets suggests that it is a reproducible and inherent property of the experiments. Transcription activities were also detected for many of the intergenic regions and the small RNAs, including tRNA, small nuclear RNA, small nucleolar RNA, and microRNA. Expression of tRNAs correlates with genome-wide amino acid usage.

Arabidopsis↗
PubMed2005

Biophysical and kinetic characterization of HemAT, an aerotaxis receptor from Bacillus subtilis.

HemAT from Bacillus subtilis is a new type of heme protein responsible for sensing oxygen. The structural and functional properties of the full-length HemAT protein, the sensor domain (1-178), and Tyr-70 mutants have been characterized. Kinetic and equilibrium measurements reveal that both full-length HemAT and the sensor domain show two distinct O(2) binding components. The high-affinity component has a K(dissociation) approximately 1-2 microM and a normal O(2) dissociation rate constant, k(O2) = 50-80 s(-1). The low-affinity component has a K(dissociation) approximately 50-100 microM and a large O(2) dissociation rate constant equal to approximately 2000 s(-1). The low n-value and biphasic character of the equilibrium curve indicate that O(2) binding to HemAT involves either independent binding to high- and low-affinity subunits in the dimer or negative cooperativity. Replacement of Tyr-70(B10) with Phe, Leu, or Trp in the sensor domain causes dramatic increases in k(O2) for both the high- and low-affinity components. In contrast, the rates and affinity for CO binding are little affected by loss of the Tyr-70 hydroxyl group. These results suggest highly dynamic behavior for the Tyr-70 side chain and the fraction of the "up" versus "down" conformation is strongly influenced by the nature of the iron-ligand complex. As a result of having both high- and low-affinity components, HemAT can respond to oxygen concentration gradients under both hypoxic (0-10 microM) and aerobic (50-250 microM) conditions, a property which could, in principle, be important for a robust sensing system. The unusual ligand-binding properties of HemAT suggest that asymmetry and apparent negative cooperativity play an important role in the signal transduction pathway.

Bacillus subtilis↗
PubMed2005

High-throughput purification and quality assurance of Arabidopsis thaliana proteins for eukaryotic structural genomics.

The Center for Eukaryotic Structural Genomics (CESG) has established procedures for the purification of Arabidopsis proteins in a high-throughput mode. Recombinant proteins were fused with (His)(6)-MBP tags at their N-terminus and expressed in Escherichia coli. Using an automated AKTApurifier system, fusion proteins were initially purified by immobilized metal affinity chromatography (IMAC). After cleavage of (His)(6)-MBP tags by TEV protease, (His)(6)-MBP tags were separated from target proteins by a subtractive 2nd IMAC. As a part of quality assurance, all purified proteins were subjected to MALDI-TOF and ESI mass spectrometry to confirm target identity and integrity, and determine incorporation of seleno-methionine (SeMet) and (15)N and (13)C isotopes. The protocols have been used successfully to provide high quality proteins that are suitable for structural studies by X-ray crystallography and NMR.

Arabidopsis↗
PubMed2005

Protocols for production of selenomethionine-labeled proteins in 2-L polyethylene terephthalate bottles using auto-induction medium.

Protocols have been developed and applied in the high-throughput production of selenomethionine labeled fusion proteins using the conditional Met auxotroph Escherichia coli B834. The large-scale growth and expression uses a chemically defined auto-induction medium containing 125 mg L(-1) selenomethionine, salts and trace metals, other amino acids including 10 mg L(-1) of methionine, vitamins except vitamin B12, and glucose, glycerol, and alpha-lactose. A schematic for a shaker rack that can hold up to twenty-four 2-L polyethylene terephthalate beverage bottles in a standard laboratory refrigerated floor shaker is provided. The growth cycle from inoculation of the culture bottle through the growth, induction, and expression was timed to take approximately 24 h. Culture growth in the auto-induction medium gave an average final optical density at 600 nm of approximately 6 and an average wet cell mass yield of approximately 14 g from 2 L of culture in greater than 150 expression trials. A simple method for visual scoring of denaturing electrophoresis gels for total protein expression, solubility, and effectiveness of fusion protein proteolysis was developed and applied. For the favorably scored expression trials, the average yield of purified, selenomethionine-labeled target protein obtained after proteolysis of the fusion protein was approximately 30 mg. Analysis by mass spectrometry showed greater than 90% incorporation of selenomethionine over a approximately 8-fold range of selenomethionine concentrations in the growth medium, with higher growth rates observed at the lower selenomethionine concentrations. These protein preparations have been utilized to solve X-ray crystal structures by multiwavelength anomalous diffraction phasing.

Cell Culture Techniques↗
PubMed2004

Automatic domain decomposition of proteins by a Gaussian Network Model.

Proteins are often comprised of domains of apparently independent folding units. These domains can be defined in various ways, but one useful definition divides the protein into substructures that seem to move more or less independently. The same methods that allow fairly accurate calculation of motion can be used to help classify these substructures. We show how the Gaussian Network Model (GNM), commonly used for determining motion, can also be adapted to automatically classify domains in proteins. Parallels between this physical network model and graph theory implementation are apparent. The method is applied to a nonredundant set of 55 proteins, and the results are compared to the visual assignments by crystallographers. Apart from decomposing proteins into structural domains, the algorithm can generally be applied to any large macromolecular system to decompose it into motionally decoupled sub-systems.

Algorithms↗
PubMed2004

On the equivalence between a commonly used correlation coefficient and a least-squares function.

Many objective functions have been proposed in X-ray crystallography to solve the molecular replacement (MR) problem and other optimization problems. This paper establishes the equivalence of optimizing two of these target functions, a commonly used correlation coefficient and a least-squares function. This equivalence may exist only in the neighborhoods about the global optima or the entire MR variable space depending on whether the mean values of the observed and calculated data are subtracted from the data. In addition, an argument is presented that the correlation coefficient between structure-factor magnitudes is likely to perform better than the correlation coefficient between intensities, especially when low-resolution data are used. This prediction was tested during coarse grid searches at low resolution using the MR program SOMoRe.

Journal Article↗
PubMed2004

Structures and analysis of highly homologous psychrophilic, mesophilic, and thermophilic adenylate kinases.

The crystal structures of adenylate kinases from the psychrophile Bacillus globisporus and the mesophile Bacillus subtilis have been solved and compared with that from the thermophile Bacillus stearothermophilus. This is the first example we know of where a trio of protein structures has been solved that have the same number of amino acids and a high level of identity (66-74%) and yet come from organisms with different operating temperatures. The enzymes were characterized for their own thermal denaturation and inactivation, and they exhibited the same temperature preferences as their source organisms. The structures of the three highly homologous, dynamic proteins with different temperature-activity profiles provide an opportunity to explore a molecular mechanism of cold and heat adaptation. Their analysis suggests that the maintenance of the balance between stability and flexibility is crucial for proteins to function at their environmental temperatures, and it is achieved by the modification of intramolecular interactions in the process of temperature adaptation.

Adenylate Kinase↗
PubMed2004

Kinetic proofreading by the cavity system of myoglobin: protection from poisoning.

Throughout its matrix of atoms, myoglobin has a network of cavities that are inhabited for short lengths of time by ligands released by photolysis from the myoglobin heme. The purpose or effect of this cavity network is not clear. A recently published kinetic scheme that fits data from many native and mutant myoglobin oxygen photolysis experiments can be modified easily into a kinetic scheme that includes kinetic proofreading. Proofreading would provide protection against contaminants and, specifically, might help protect the cell from carbon monoxide poisoning. Here we present a two-part model: (1) myoglobin represented by a kinetic description, which includes proofreading reactions associated with the cavities, and (2) a reaction-diffusion description of a myocyte model in which the part 1 myoglobin acts as a mobile buffer in simultaneous carbon monoxide and oxygen gradients. The non-equilibrium nature of part 2 should promote the proofreading function of part 1. A simulation using the model demonstrates that the cavity system can in principle proofread, reducing mitochondrial enzyme contamination.

Animals↗
PubMed2003

Molecular engineering of myoglobin: influence of residue 68 on the rate and the enantioselectivity of oxidation reactions catalyzed by H64D/V68X myoglobin.

In the elucidation of structural requirements of heme vicinity for hydrogen peroxide activation, we found that the replacement of His-64 of myoglobin (Mb) with a negatively charged aspartate residue enhanced peroxidase and peroxygenase activities by 78- and 580-fold, respectively. Since residue 68 is known to influence the ligation of small molecules to the heme iron, we constructed H64D/V68X Mb bearing Ala, Ser, Leu, Ile, and Phe at position 68 to improve the oxidation activity. The Val-68 to Leu mutation of H64D Mb accelerates the reaction with H(2)O(2) to form a catalytic species, called compound I, and improves the one-electron oxidation of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (i.e., peroxidase activity) approximately 2-fold. On the other hand, H64D/V68I Mb oxygenates thioanisole 2.7- and 1600-fold faster than H64D and wild-type Mb, respectively. In terms of the enantioselectivity, H64D/V68A and H64D/V68S Mb were good chiral catalysts for thioanisole oxidation and produced the (R)-sulfoxide dominantly with 84% and 88% ee, respectively [Kato, S., et al. (2002) J. Am. Chem. Soc. 124, 8506-8507]. On the contrary, the substitution of Val-68 in H64D Mb with an isoleucine residue alters the dominant sulfoxide product from the (R)- to the (S)-isomer. The crystal structures of H64D/V68A and H64D/V68S Mb elucidated in this study do not clearly indicate residues interacting with thioanisole. However, comparison of the active site structures provides the basis to interpret the changes in oxidation activity: (1) direct steric interactions between residue 68 and substrates (i.e., H(2)O(2), ABTS, thioanisole) and (2) the polar interactions between tightly hydrogen-bonded water molecules and substrates.

Amino Acid Substitution↗