bluuebunny/crossref_metadata_2025_split huggingface dataset:
Crystal structure of checkpoint kinase 2 in complex with NSC 109555, a potent and selective inhibitor
Authors: Lountos George T. , Tropea Joseph E. , Zhang Di , Jobson Andrew G. , Pommier Yves , Shoemaker Robert H. , Waugh David S. 
Published: February 2009

AbstractCheckpoint kinase 2 (Chk2), a ser/thr kinase involved in the ATM‐Chk2 checkpoint pathway, is activated by genomic instability and DNA damage and results in either arrest of the cell cycle to allow DNA repair to occur or apoptosis if the DNA damage is severe. Drugs that specifically target Chk2 could be beneficial when administered in combination with current DNA‐damaging agents used in cancer therapy. Recently, a novel inhibitor of Chk2, NSC 109555, was identified that exhibited high potency (IC50 = 240 nM) and selectivity. This compound represents a new chemotype and lead for the development of novel Chk2 inhibitors that could be used as therapeutic agents for the treatment of cancer. To facilitate the discovery of new analogs of NSC 109555 with even greater potency and selectivity, we have solved the crystal structure of this inhibitor in complex with the catalytic domain of Chk2. The structure confirms that the compound is an ATP‐competitive inhibitor, as the electron density clearly reveals that it occupies the ATP‐binding pocket. However, the mode of inhibition differs from that of the previously studied structure of Chk2 in complex with debromohymenialdisine, a compound that inhibits both Chk1 and Chk2. A unique hydrophobic pocket in Chk2, located very close to the bound inhibitor, presents an opportunity for the rational design of compounds with higher binding affinity and greater selectivity.
Backbone structure of a small helical integral membrane protein: A unique structural characterization
Authors: Page Richard C. , Lee Sangwon , Moore Jacob D. , Opella Stanley J. , Cross Timothy A. 
Published: February 2009

AbstractThe structural characterization of small integral membrane proteins pose a significant challenge for structural biology because of the multitude of molecular interactions between the protein and its heterogeneous environment. Here, the three‐dimensional backbone structure of Rv1761c from Mycobacterium tuberculosis has been characterized using solution NMR spectroscopy and dodecylphosphocholine (DPC) micelles as a membrane mimetic environment. This 127 residue single transmembrane helix protein has a significant (10 kDa) C‐terminal extramembranous domain. Five hundred and ninety distance, backbone dihedral, and orientational restraints were employed resulting in a 1.16 Å rmsd backbone structure with a transmembrane domain defined at 0.40 Å. The structure determination approach utilized residual dipolar coupling orientation data from partially aligned samples, long‐range paramagnetic relaxation enhancement derived distances, and dihedral restraints from chemical shift indices to determine the global fold. This structural model of Rv1761c displays some influences by the membrane mimetic illustrating that the structure of these membrane proteins is dictated by a combination of the amino acid sequence and the protein's environment. These results demonstrate both the efficacy of the structural approach and the necessity to consider the biophysical properties of membrane mimetics when interpreting structural data of integral membrane proteins and, in particular, small integral membrane proteins.
Effect of trehalose on protein structure
Authors: Jain Nishant Kumar , Roy Ipsita 
Published: February 2009

AbstractTrehalose is a ubiquitous molecule that occurs in lower and higher life forms but not in mammals. Till about 40 years ago, trehalose was visualized as a storage molecule, aiding the release of glucose for carrying out cellular functions. This perception has now changed dramatically. The role of trehalose has expanded, and this molecule has now been implicated in a variety of situations. Trehalose is synthesized as a stress‐responsive factor when cells are exposed to environmental stresses like heat, cold, oxidation, desiccation, and so forth. When unicellular organisms are exposed to stress, they adapt by synthesizing huge amounts of trehalose, which helps them in retaining cellular integrity. This is thought to occur by prevention of denaturation of proteins by trehalose, which would otherwise degrade under stress. This explanation may be rational, since recently, trehalose has been shown to slow down the rate of polyglutamine‐mediated protein aggregation and the resultant pathogenesis by stabilizing an aggregation‐prone model protein. In recent years, trehalose has also proved useful in the cryopreservation of sperm and stem cells and in the development of a highly reliable organ preservation solution. This review aims to highlight the changing perception of the role of trehalose over the last 10 years and to propose common mechanisms that may be involved in all the myriad ways in which trehalose stabilizes protein structures. These will take into account the structure of trehalose molecule and its interactions with its environment, and the explanations will focus on the role of trehalose in preventing protein denaturation.
Parallel screening and optimization of protein constructs for structural studies
Authors: Rasia Rodolfo M. , Noirclerc‐Savoye Marjolaine , Bologna Nicolás G. , Gallet Benoit , Plevin Michael J. , Blanchard Laurence , Palatnik Javier F. , Brutscher Bernhard , Vernet Thierry , Boisbouvier Jérôme 
Published: February 2009

AbstractA major challenge in structural biology remains the identification of protein constructs amenable to structural characterization. Here, we present a simple method for parallel expression, labeling, and purification of protein constructs (up to 80 kDa) combined with rapid evaluation by NMR spectroscopy. Our approach, which is equally applicable for manual or automated implementation, offers an efficient way to identify and optimize protein constructs for NMR or X‐ray crystallographic investigations.
Amino acid sequence of fibrolase, a direct‐acting fibrinolytic enzyme from agkistrodon contortrix contortrix venom
Authors: Randolph Anne , Chamberlain Scott H. , Chu H.‐L. Cathy , Masiarz Frank R. , Retzios Anastassios D. , Markland Francis S. 
Published: February 2009

AbstractThe complete amino acid sequence of fibrolase, a fibrinolytic enzyme from southern copperhead (Agkistrodon contortrix contortrix) venom, has been determined. This is the first report of the sequence of a direct‐acting, non‐hemorrhagic fibrinolytic enzyme found in snake venom. The majority of the sequence was established by automated Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. The amino‐terminus is blocked by a cyclized glutamine (pyroglutamic acid) residue, and the sequence of this region of the molecule was determined by mass spectrometry. Fibrolase is composed of 203 residues in a single polypeptide chain with a molecular weight of 22,891, as determined by the sequence. Its sequence is homologous to the sequence of the hemorrhagic toxin Ht‐d of Crotalus atrox venom and with the sequences of two metalloproteinases from Trimeresurus flavoviridis venom. Microheterogeneity in the sequence was found at both the amino‐terminus and at residues 189 and 192. All six cysteine residues in fibrolase are involved in disulfide bonds. A disulfide bond between cysteine‐118 and cysteine‐198 has been established and bonds between cysteines‐158/165 and between cysteines‐160/192 are inferred from the homology to Ht‐d. Secondary structure prediction reveals a very low percentage of α‐helix (4%), but much greater β‐structure (39.5%). Analysis of the sequence reveals the absence of asparagine‐linked glycosylation sites defined by the consensus sequence: asparagine‐X‐serine/threonine.
Mutation of conserved residues in Escherichia coli thioredoxin: Effects on stability and function
Authors: Gleason Florence K. 
Published: February 2009

AbstractMutations were made in three highly conserved residues in Escherichia coli thioredoxin. An internal charged residue, Asp‐26, was changed to an alanine. The mutant protein was more stable than the wild type. It can function as a substrate for thioredoxin reductase with a 10‐fold increase in the Km over the wild type. Although the redox potential was not substantially changed from that of the wild type, thioredoxin D26A was a poor reducing agent for ribonucleotide reductase. Asp‐26 apparently serves to maintain an optimal charge distribution in the active site region for interaction with other proteins. Mutation of a surface Pro‐34 in the active site disulfide ring to a serine had little effect on protein stability. A slight decrease in the redox potential (9 mV) made thioredoxin P34S a better reducing agent for ribonucleotide reductase. In contrast, mutation of the internal cis Pro‐76 to an alanine destabilized the protein. The data indicate a change had also occurred in the charge distribution in the active site region. Thioredoxin P76A had a higher redox potential than the wild type protein and was not an effective reducing agent for ribonucleotide reductase. It was concluded that this residue is essential for maintaining the conformation of the active site and the redox potential of thioredoxin.
Optical activity associated with the sulfur to metal charge transfer bands of Zn and Cd GAL4
Authors: Basile Lena A. , Coleman Joseph E. 
Published: February 2009

AbstractThe N‐terminal DNA‐binding domain of the GAL4 transcription factor, isolated as a proteolytic fragment of 63 amino acid residues, GAL4(63), or cloned as a 62‐residue subdomain, GAL4(62*), binds 2 Cd(II) ions. Both Cd derivatives show intense ellipticity bands associated with ‐S− → Cd charge transfer bands at 250 nm (+), 233 nm (‐), and 218 nm (+). Molar ellipticity values are (5–7) x 104 deg · cm2 · dmol−1. The energies and pattern (+, ‐, +) of these ellipticity bands are similar to those observed for the Cd(II) clusters of metallothionein. The latter are believed to be due to the presence of bridging cysteine ligands (Willner, H.W., Vasak, M., & Kägi, J.H.R., 1987, Biochemistry 26, 6287–6292). Circular dichroism provides further evidence that the GAL4 DNA‐binding domain forms a binuclear Cd cluster with the amino acid sequence ‐C11‐X2‐C14‐X6‐C21‐X6‐C28‐X2‐C31‐X6‐C38‐found in GAL4(62*). Zn2GAL4(62*) has much weaker ellipticity associated with the higher energy ‐S− → Zn charge transfer bands. Removal of the metal ions from GAL4(62*) results in substantial changes in the near UV circular dichroism due to both loss of the charge transfer bands and changes in the conformation of the peptide backbone. Binding of the UASG enhancer DNA element to either Zn or Cd GAL4(62*) alters the ellipticity of the ‐S− → metal charge transfer bands suggesting that DNA binding is associated with some changes in conformation around the metal cluster.
Assembly of polypeptide and protein backbone conformations from low energy ensembles of short fragments: Development of strategies and construction of models for myoglobin, lysozyme, and thymosin β4
Authors: Sippl Manfred J. , Hendlich Manfred , Lackner Peter 
Published: February 2009

AbstractRecently we developed methods for the construction of knowledge‐based mean fields from a data base of known protein structures. As shown previously, this approach can be used to calculate ensembles of probable conformations for short fragments of polypeptide chains. Here we develop procedures for the assembly of short fragments to complete three‐dimensional models of polypeptide chains.The amino acid sequence of a given protein is decomposed into all possible overlapping fragments of a given length, and an ensemble of probable conformations is calculated for each fragment. The fragments are assembled to a complete model by choosing appropriate conformations from the individual ensembles and by averaging over equivalent angles. Finally a consistent model is obtained by rebuilding the conformation from the average angles. From the average angles the local variability of the structure can be calculated, which is a useful criterion for the reliability of the model.The procedure is applied to the calculation of the local backbone conformations of myoglobin and lysozyme whose structures have been solved by X‐ray analysis and thymosin β4, a polypeptide of 43 amino acid residues whose structure was recently investigated by NMR spectroscopy. We demonstrate that substantial fractions of the calculated local backbone conformations are similar to the experimentally determined structures.
Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding
Authors: Wilcox William , Eisenberg David 
Published: February 2009

AbstractThe tetramerization of melittin, a 26‐amino acid peptide from Apis mellifera bee venom, has been studied as a model for protein folding. Melittin converts from a monomeric random coil to an α‐helical tetramer as the pH is raised from 4.0 to 9.5, as ionic strength is increased, as temperature is raised or lowered from about 37 °C, or as phosphate is added. The thermodynamics of this tetramerization (termed “folding”) are explored using circular dichroism. The melittin tetramer has two pKa values of 7.5 and 8.5 corresponding to protonation of the N‐terminus and Lys 23, respectively. pKa values calculated with the program DelPhi (Gilson, M.K., Sharp, K.A., & Honig, B.H., 1987, J. Comp. Chem. 9, 327–335; Gilson, M.K. & Honig, B.H., 1988a, Proteins3, 32–52; Gilson, M.K. & Honig, B.H., 1988b, Proteins 4, 7–18) agree with experimental titration data. Greater electrostatic repulsion of these protonated groups destabilizes the tetramer by 3.6 kcal/mol at pH 4.0 compared to pH 9.5. Increasing the concentration of NaCl in the solution from 0 to 0.5 M stabilizes the tetramer by 5–6 kcal/mol at pH 4.0. The effect of NaCl is modeled with a ligand‐binding approach. The melittin tetramer is found to have a temperature of maximum stability ranging from 35.5 to 43 °C depending on the pH, unfolding above and below that temperature. ΔCp0 for folding ranges from –0.085 to –0.102 cal g−1 K−1 comparable to that of other small globular proteins (Privalov, P.L., 1979, Adv. Protein Chem. 33, 167–241). ΔH0 and ΔS0 are found to decrease with temperature, presumably due to the hydrophobic effect (Kauzmann, W., 1959, Adv. Protein Chem. 14, 1–63). Phosphate is found to perturb the equilibrium substantially with a maximal effect at 150 mM, stabilizing the tetramer at pH 7.4 and 25 °C by 4.6 kcal/mol. The enthalpy change due to addition of phosphate (–7.5 kcal/mol at 25 °C) can be accounted for by simple dielectric screening. Both circular dichroism and crystallographic results suggest that phosphate may bind Lys 23 at the ends of the elongated tetramer. These detailed measurements give insight into the relative importance of various forces for the stability of melittin in the folded form and may provide an experimental standard for future tests of computational energetics on this simple protein system.
A proposed link between nitrogen and carbon metabolism involving protein phosphorylation in bacteria
Authors: Reizer Jonathan , Reizer Aiala , Saier Milton H. , Jacobson Gary R. 
Published: February 2009

AbstractWe demonstrate that certain phosphoryl transfer proteins of the bacterial phosphotransferase system (PTS), the fructose‐ and mannitol‐specific IIA proteins or domains, are homologous to a class of proteins, one of which is known to affect transcription of some of the nitrogen‐regulatory σ54‐dependent operons in Klebsiella pneumoniae. The phosphorylatable histidyl residue in the homologous PTS proteins and the consensus sequence in the vicinity of the active‐site histidine are fully conserved in all members that comprise this family of proteins. A phylogenetic tree of the eight protein members of this family was constructed, and a “signature” sequence that can serve for the identification of new protein members of this family is proposed. These observations suggest that PTS‐catalyzed protein phosphorylation may provide a regulatory link between carbon and nitrogen assimilation in bacteria.
Electrostatic interactions in the association of proteins: An analysis of the thrombin–hirudin complex
Authors: Karshikov Andrej , Bode Wolfram , Tulinsky Alexander , Stone Stuart R. 
Published: February 2009

AbstractThe role of electrostatic interactions in stabilization of the thrombin–hirudin complex has been investigated by means of two macroscopic approaches: the modified Tanford–Kirkwood model and the finite‐difference method for numerical solution of the Poisson–Boltzmann equations. The electrostatic potentials around the thrombin and hirudin molecules were asymmetric and complementary, and it is suggested that these fields influence the initial orientation in the process of the complex formation. The change of the electrostatic binding energy due to mutation of acidic residues in hirudin has been calculated and compared with experimentally determined changes in binding energy. In general, the change in electrostatic binding energy for a particular mutation calculated by the modified Tanford–Kirkwood approach agreed well with the experimentally observed change. The finite‐difference approach tended to overestimate changes in binding energy when the mutated residues were involved in short‐range electrostatic interactions. Decreases in binding energy caused by mutations of amino acids that do not make any direct ionic interactions (e.g., Glu 61 and Glu 62 of hirudin) can be explained in terms of the interaction of these charges with the positive electrostatic potential of thrombin. Differences between the calculated and observed changes in binding energy are discussed in terms of the crystal structure of the thrombin–hirudin complex.
The solution structure of eglin c based on measurements of many NOEs and coupling constants and its comparison with X‐ray structures
Authors: Hyberts Sven G. , Goldberg Matthew S. , Havel Timothy F. , Wagner Gerhard 
Published: February 2009

AbstractA high‐precision solution structure of the elastase inhibitor eglin c was determined by NMR and distance geometry calculations. A large set of 947 nuclear Overhauser (NOE) distance constraints was identified, 417 of which were quantified from two‐dimensional NOE spectra at short mixing times. In addition, a large number of homonuclear 1H‐1H and heteronuclear 1H‐15N vicinal coupling constants were used, and constraints on 42 χ1 and 38 ϕ angles were obtained. Structure calculations were carried out using the distance geometry program DG‐II. These calculations had a high convergence rate, in that 66 out of 75 calculations converged with maximum residual NOE violations ranging from 0.17 Å to 0.47 Å. The spread of the structures was characterized with average root mean square deviations (<rmsd>) between the structures and a mean structure. To calculate the <rmsd> unbiased toward any single structure, a new procedure was used for structure alignment. A canonical structure was calculated from the mean distances, and all structures were aligned relative to that. Furthermore, an angular order parameter S was defined and used to characterize the spread of structures in torsion angle space. To obtain an accurate estimate of the precision of the structure, the number of calculations was increased until the <rmsd> and the angular order parameters stabilized. This was achieved after approximately 40 calculations. The structure consists of a well‐defined core whose backbone deviates from the canonical structure ca. 0.4 Å, a disordered N‐terminal heptapeptide whose backbone deviates by 0.8–12 Å, and a proteinase‐binding loop whose backbone deviates up to 3.0 Å. Analysis of the angular order parameters and inspection of the structures indicates that a hinge‐bending motion of the binding loop may occur in solution. Secondary structures were analyzed by comparison of dihedral angle patterns. The high precision of the structure allows one to identify subtle differences with four crystal structures of eglin c determined in complexes with proteinases.
Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho. I. Binding of ATP
Authors: Geiselmann Johannes , Von Hippel Peter H. 
Published: February 2009

AbstractEscherichia coli transcription termination factor rho is an RNA‐dependent ATPase, and ATPase activity is required for all its functions. We have characterized the binding of ATP to the physiologically relevant hexameric association state of rho in the absence of RNA and have shown that there are six ATP binding sites per rho hexamer. This stoichiometry has been verified by a number of different techniques, including ultracentrifugation, ultrafiltration, and fluorescence titration studies. We have also shown that ATP can bind to isolated monomers of rho when the hexamer is dissociated with the mild denaturant myristyltrimethylammonium bromide, demonstrating that each protomer of rho carries an ATP binding site. The six binding sites that we observe in the rho hexamer are not equivalent; the hexamer contains three strong (Ka ≈︁ 3 × 106 M−1) and three weak (Ka ≈︁ 105 M−1) binding sites for ATP. The binding constant of the weak binding sites is just the reciprocal of the enzymatic Km for ATP as a substrate; thus these weak sites, as well as the strong sites, can, in principle, take part in the catalytic cycle. The asymmetry induced (or manifested) by ATP binding reduces the symmetry of the rho hexamer from a D3 to a pseudo‐D3 state. This “breakage” of symmetry has implications for the molecular mechanism of rho, because an asymmetric structure can lead to directional helicase activity by invoking directionally distinct RNA binding and release reactions (see Geiselmann, J., Yager, T.D., & von Hippel, P.H., 1992c, Protein Sci. 1, 861–873).
Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho. II. Binding of RNA
Authors: Geiselmann Johannes , Yager Thomas D. , Von Hippel Peter H. 
Published: February 2009

AbstractThe rho protein of Escherichia coli interacts with the nascent RNA transcript while RNA polymerase is paused at specific rho‐dependent termination sites on the DNA template, and (in a series of steps that are still largely undefined) brings about transcript termination at these sites. In this paper we characterize the interactions of rho with RNA and relate these interactions to the quaternary structure of the functional form of rho. We use CD spectroscopy and analytical ultracentrifugation to determine the binding interactions of rho with RNA ligands of defined length ([rC]n where n ≥ 6). Rho binds to long RNA chains as a hexamer characterized by D3 symmetry. Each hexamer binds ∼70 residues of RNA. We show by ultracentrifugation and dynamic laser light scattering that, in the presence of RNA ligands less than 22 nucleotide residues in length, rho changes its quaternary structure and becomes a homogeneous dodecamer. The dodecamer contains six strong binding sites for short RNA ligands: i.e., one site for every two rho protomers. The measured association constant of these short RNAs to rho increases with increasing (rC)n length, up to n = 9, suggesting that the binding site of each rho protomer interacts with 9 RNA nucleotide residues. Oligo(rC) ligands bound to the strong RNA binding sites on the rho dodecamer do not significantly stimulate the RNA‐dependent ATPase activity of rho. Based on these features of the rho‐RNA interaction and other experimental data we propose a molecular model of the interaction of rho with its cofactors.