Crystal structure of gene locus At3g16990 from Arabidopsis thaliana.
Explore the source record for details and available documents.
Arabidopsis Proteins↗
Biomedical subjects
George N Phillips
Publications and source records attributed to George N Phillips.
Crystal structure of the protein from gene At3g17210 of Arabidopsis thaliana.
Explore the source record for details and available documents.
Amino Acid Sequence↗
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↗
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↗
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↗
Structure of the hypothetical protein At3g17210 from Arabidopsis thaliana.
Explore the source record for details and available documents.
Amino Acid Sequence↗
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↗
Structures of thermophilic and mesophilic adenylate kinases from the genus Methanococcus.
The crystal structures of adenylate kinases from the thermophile Methanococcus thermolithotrophicus and the mesophile Methanococcus voltae have been solved to resolutions of 2.8A and 2.5A, respectively. The structures of the enzymes are similar to that of the adenylate kinase from archaeal Sulfolobus acidocaldarius in many respects such as the extended central beta-sheets, the short LID domain, and the trimeric state. The analysis of unligated and AMP-bound subunits of M.voltae suggests that movements of two mobile domains are not independent of each other. The methanococcal structures are examined with respect to their lack of the "invariant" Lys residue within the phosphate-binding loop, and two Arg residues in the LID domain are proposed as substituting residues based on their conservation among archaeal adenylate kinases and mobility within the structures. Since S.acidocaldarius adenylate kinase has the invariant Lys residue as well as the two Arg residues, its phosphate-binding loop is examined and compared with those of other adenylate kinases. On the basis of the comparison and other available biochemical data, the unusual conformation of the Lys residue in S.acidocaldarius adenylate kinase is explained. Despite possessing 78% sequence identity, the methanococcal enzymes exhibit significantly different thermal stabilities. To study the determinants of thermostability, several structural features including salt-links, hydrogen bonds, packing density, surface to volume ratio and buried surface area are compared between the enzymes. From their difference in apolar buried surface area, hydrophobic interaction is proposed to be a basis for the disparate thermostabilities, and the corresponding free energy difference is also estimated. Results of previous mutational studies are interpreted in terms of the crystal structures, and support the importance of hydrophobic interactions in thermostability.
Adenosine Monophosphate↗
Watching a protein as it functions with 150-ps time-resolved x-ray crystallography.
We report picosecond time-resolved x-ray diffraction from the myoglobin (Mb) mutant in which Leu29 is replaced by Phe (L29Fmutant). The frame-by-frame structural evolution, resolved to 1.8 angstroms, allows one to literally "watch" the protein as it executes its function. Time-resolved mid-infrared spectroscopy of flash-photolyzed L29F MbCO revealed a short-lived CO intermediate whose 140-ps lifetime is shorter than that found in wild-type protein by a factor of 1000. The electron density maps of the protein unveil transient conformational changes far more dramatic than the structural differences between the carboxy and deoxy states and depict the correlated side-chain motion responsible for rapidly sweeping CO away from its primary docking site.
Amino Acid Substitution↗
Crystallization and X-ray diffraction analysis of the sensor domain of the HemAT aerotactic receptor.
HemAT is a 432-amino-acid protein with two structural domains identified from Bacillus subtilis. It is responsible for sensing oxygen and delivering the signal to the downstream signal transduction cascade through a two-component system [Hou et al. (2000), Nature (London), 403, 540-544]. The consequence of such events is to change the flagellar movement and alter the swimming behavior of bacteria. To elucidate the molecular mechanism of oxygen sensing, the sensor domain of HemAT from B. subtilis was cloned, expressed and crystallized. Multiple-wavelength anomalous dispersion (MAD) data were collected from the intrinsic anomalous scatterer, iron, using synchrotron radiation. Three-wavelength iron MAD data sets were collected to 2.8 A resolution. The native data set was collected to 2.15 A resolution. Initial crystallographic analysis revealed the crystals to belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.00, b = 80.12, c = 85.95 A. There is one dimer in the asymmetric unit, with 40% solvent content. Structure determination using MAD methods and model building are currently under way.
Bacillus subtilis↗
SOMoRe: a multi-dimensional search and optimization approach to molecular replacement.
Commonly used traditional molecular-replacement (MR) methods, though often successful, have difficulty solving certain classes of MR problems. In addition, MR problems are generally very difficult global optimization problems because of the enormous number of local minima in traditionally computed target functions. As a result, a new MR program called SOMoRe is introduced that implements a new global optimization strategy that has two major components: (i) a six-dimensional global search of a target function computed from low-resolution data and (ii) multi-start local optimization. Because the target function computed from low-resolution data is relatively smooth, the global search can coarsely sample the MR variable space to identify good starting points for extensive multi-start local optimization. Consequently, SOMoRe was able to straightforwardly solve four realistic test problems, including two that could not be directly solved by traditional MR programs, and SOMoRe solved a problem using a less complete model than those required by two traditional programs and a stochastic six-dimensional program. Based on these results, this new strategy promises to extend the applicability and robustness of MR.
Algorithms↗
Structure of the oxygen sensor in Bacillus subtilis: signal transduction of chemotaxis by control of symmetry.
Much is now known about chemotaxis signaling transduction for Escherichia coli and Salmonella typhimurium. The mechanism of chemotaxis of Bacillus subtilis is, in a sense, reversed. Attractant binding strengthens the activity of histidine kinase in B. subtilis, instead of an inhibition reaction. The HemAT from B. subtilis can detect oxygen and transmit the signal to regulatory proteins that control the direction of flagella rotation. We have determined the crystal structures of the HemAT sensor domain in liganded and unliganded forms at 2.15 A and 2.7 A resolution, respectively. The liganded structure reveals a highly symmetrical organization. Tyrosine70 shows distinct conformational changes on one subunit when ligands are removed. Our study suggests that disruption of the symmetry of HemAT plays an important role in initiating the chemotaxis signaling transduction cascade.
Amino Acid Sequence↗
Project management system for structural and functional proteomics: Sesame.
A computing infrastructure (Sesame) has been designed to manage and link individual steps in complex projects. Sesame is being developed to support a large-scale structural proteomics pilot project. When complete, the system is expected to manage all steps from target selection to data-bank deposition and report writing. We report here on the design criteria of the Sesame system and on results demonstrating successful achievement of the basic goals of its architecture. The Sesame software package, which follows the client/server paradigm, consists of a framework, which supports secure interactions among the three tiers of the system (the client, server, and database tiers), and application modules that carry out specific tasks. The framework utilizes industry standards. The client tier is written in Java2 and can be accessed anywhere through the Internet. All the development on the server tier is also carried out in Java2 so as to accommodate a wide variety of computer platforms. The database tier employs a commercial database management system. Each Sesame application module consists of a simple user interface in the client tier, corresponding objects in the server tier, and relevant data stored in the centralized database. For security, access to stored data is controlled by access privileges. The system facilitates both local and remote collaborations. Because users interact with the system using Java Web Start or through a web browser, access is limited only by the availability of an Internet connection. We describe several Sesame modules that have been developed to the point where they are being utilized routinely to support steps involved in structural and functional proteomics. This software is available to parties interested in using it and assisting to guide its further development.
Database Management Systems↗
Understanding protein flexibility through dimensionality reduction.
This work shows how to decrease the complexity of modeling flexibility in proteins by reducing the number of dimensions necessary to model important macromolecular motions such as the induced-fit process. Induced fit occurs during the binding of a protein to other proteins, nucleic acids, or small molecules (ligands) and is a critical part of protein function. It is now widely accepted that conformational changes of proteins can affect their ability to bind other molecules and that any progress in modeling protein motion and flexibility will contribute to the understanding of key biological functions. However, modeling protein flexibility has proven a very difficult task. Experimental laboratory methods, such as x-ray crystallography, produce rather limited information, while computational methods such as molecular dynamics are too slow for routine use with large systems. In this work, we show how to use the principal component analysis method, a dimensionality reduction technique, to transform the original high-dimensional representation of protein motion into a lower dimensional representation that captures the dominant modes of motions of proteins. For a medium-sized protein, this corresponds to reducing a problem with a few thousand degrees of freedom to one with less than fifty. Although there is inevitably some loss in accuracy, we show that we can obtain conformations that have been observed in laboratory experiments, starting from different initial conformations and working in a drastically reduced search space.
Aldehyde Reductase↗
Asymmetric sulfoxidation and amine binding by H64D/V68A and H64D/V68S Mb: mechanistic insight into the chiral discrimination step.
The H64D/V68A and H64D/V68S mutants of Myoglobin are found to oxidize thioanisole with high enantioselectivity and reactivity. These mutants are also capable of enantioselective binding of alpha-methylbenzylamine, which mimics an expected sulfoxidation intermediate. The kinetic study of the amine binding shows that the Fe-O bond cleavage in the intermediate may be the chiral discrimination step of the sulfoxidation.
Kinetics↗
Dynamics of proteins in crystals: comparison of experiment with simple models.
The dynamic behavior of proteins in crystals is examined by comparing theory and experiments. The Gaussian network model (GNM) and a simplified version of the crystallographic translation libration screw (TLS) model are used to calculate mean square fluctuations of C(alpha) atoms for a set of 113 proteins whose structures have been determined by x-ray crystallography. Correlation coefficients between the theoretical estimations and experiment are calculated and compared. The GNM method gives better correlation with experimental data than the rigid-body libration model and has the added benefit of being able to calculate correlations between the fluctuations of pairs of atoms. By incorporating the effect of neighboring molecules in the crystal the correlation is further improved.
Biophysical Phenomena↗
Molecular mechanisms of calcium and magnesium binding to parvalbumin.
Molecular dynamics simulations have been used to investigate the relationship between the coordinating residues of the EF-hand calcium binding loop of parvalbumin and the overall plasticity and flexibility of the protein. The first simulation modeled the transition from Ca(2+) to Mg(2+) coordination by varying the van der Waals parameters for the bound metal ions. The glutamate at position 12 could be accurately and reversibly seen to be a source of selective bidentate ligation of Ca(2+) in the simulations. A second simulation correlated well with the experimental observation that an E101D substitution at EF loop position 12 results in a dramatically less tightly bound monodentate Ca(2+) coordination by aspartate. A final set of simulations investigated Ca(2+) binding in the E101D mutant loop in the presence of applied external forces designed to impose bidentate coordination. The results of these simulations illustrate that the aspartate is capable of attaining a suitable orientation for bidentate coordination, thus implying that it is the inherent rigidity of the loop that prevents bidentate coordination in the parvalbumin E101D mutant.
Amino Acid Sequence↗
Probing substates in sperm whale myoglobin using high-pressure crystallography.
Pressures in the 100 MPa range are known to have an enormous number of effects on the action of proteins, but straightforward means for determining the structural basis of these effects have been lacking. Here, crystallography has been used to probe effects of pressure on sperm whale myoglobin structure. A comparison of pressure effects with those seen at low pH suggests that structural changes under pressure are interpretable as a shift in the populations of conformational substates. Furthermore, a novel high-pressure protein crystal-cooling method has been used to show low-temperature metastability, providing an alternative to room temperature, beryllium pressure cell-based techniques. The change in protein structure due to pressure is not purely compressive and involves conformational changes important to protein activity. Correlation with low-pH structures suggests observed structural changes are associated with global conformational substates. Methods developed here open up a direct avenue for exploration of the effects of pressure on proteins.
Animals↗