bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structure and dynamics in solution of the complex of lactobacillus casei dihydrofolate reductase with the new lipophilic antifolate drug trimetrexate
Authors: Polshakov Vladimir I. , Birdsall Berry , Frenkiel Thomas A. , Gargaro Angelo R. , Feeney James 
Published: October 2010

AbstractWe have determined the three‐dimensional solution structure of the complex of Lactobacillus casei dihydrofolate reductase and the anticancer drug trimetrexate. Two thousand seventy distance, 345 dihedral angle, and 144 hydrogen bond restraints were obtained from analysis of multidimensional NMR spectra recorded for complexes containing 15N‐labeled protein. Simulated annealing calculations produced a family of 22 structures fully consistent with the constraints. Several intermolecular protein‐ligand NOEs were obtained by using a novel approach monitoring temperature effects of NOE signals resulting from dynamic processes in the bound ligand. At low temperature (5 °C) the trimethoxy ring of bound trimetrexate is flipping sufficiently slowly to give narrow signals in slow exchange, which give good NOE cross peaks. At higher temperature these broaden and their NOE cross peaks disappear thus allowing the signals in the lower‐temperature spectrum to be identified as NOEs involving ligand protons. The binding site for trimetrexate is well defined and this was compared with the binding sites in related complexes formed with methotrexate and trimethoprim. No major conformational differences were detected between the different complexes. The 2,4‐diaminopyrimidine‐containing moieties in the three drugs bind essentially in the same binding pocket and the remaining parts of their molecules adapt their conformations such that they can make effective van der Waals interactions with essentially the same set of hydrophobic amino acids, the side‐chain orientations and local conformations of which are not greatly changed in the different complexes (similar x1 and x2 values).
Solution assembly of the pseudo‐high affinity and intermediate affinity interleukin‐2 receptor complexes
Authors: Wu Zining , Goldstein Byron , Laue Thomas M. , Liparoto Stefano F. , Nemeth Michael J. , Ciardelli Thomas L. 
Published: October 2010

AbstractThe high affinity interleukin‐2 receptor is composed of three cell surface subunits, IL‐2Rα, IL‐2Rβ, and IL‐2Rγ. Functional forms of the IL‐2 receptor exist, however, that enlist only two of the three subunits. On activated T‐cells, the α‐ and β‐subunits combine as a preformed heterodimer (the pseudo‐high affinity receptor) that serves to capture IL‐2. On a subpopulation of natural killer cells, the β‐ and γ‐subunits interact in a ligand‐dependent manner to form the intermediate affinity receptor site. Previously, we have demonstrated the feasibility of employing coiled‐coil molecular recognition for the solution assembly of a heteromeric IL‐2 receptor complex. In that study, although the receptor was functional, the coiled‐coil complex was a trimer rather than the desired heterodimer. We have now redesigned the hydrophobic heptad sequences of the coiled‐coils to generate soluble forms of both the pseudo‐high affinity and the intermediate affinity heterodimeric IL‐2 receptors. The properties of these complexes were examined and their relevance to the physiological IL‐2 receptor mechanism is discussed.
Crystal structure of thymidylate synthase A from Bacillus subtilis
Authors: Fox Kristin M. , Maley Frank , Garibian Araik , Changchien Li‐Ming , Roey Patrick Van 
Published: October 2010

AbstractThymidylate synthase (TS) converts dUMP to dTMP by reductive methylation, where 5,10‐methylenetetrahydrofolate is the source of both the methylene group and reducing equivalents. The mechanism of this reaction has been extensively studied, mainly using the enzyme from Escherichia coli. Bacillus subtilis contains two genes for TSs, ThyA and ThyB. The ThyB enzyme is very similar to other bacterial TSs, but the ThyA enzyme is quite different, both in sequence and activity. In ThyA TS, the active site histidine is replaced by valine. In addition, the B. subtilis enzyme has a 2.4‐fold greater kcat than the E. coli enzyme. The structure of B. subtilis thymidylate synthase in a ternary complex with 5‐fluoro‐dUMP and 5,10‐methylenetetrahydrofolate has been determined to 2.5 Å resolution. Overall, the structure of B. subtilis TS (ThyA) is similar to that of the E. coli enzyme. However, there are significant differences in the structures of two loops, the dimer interface and the details of the active site. The effects of the replacement of histidine by valine and a serine to glutamine substitution in the active site area, and the addition of a loop over the carboxy terminus may account for the differences in kcat found between the two enzymes.
Two well‐defined motifs in the cAMP‐dependent protein kinase inhibitor (PKIα) correlate with inhibitory and nuclear export function
Authors: Hauer Jennifer A. , Barthe Philippe , Taylor Susan S. , Parello Joseph , Padilla André 
Published: October 2010

AbstractThe heat stable inhibitor of cAMP‐dependent protein kinase (PKIα) contains both a nuclear export signal (NES) and a high affinity inhibitory region that is essential for inhibition of the catalytic subunit of the kinase. These functions are sequentially independent. Two‐dimensional NMR spectroscopy was performed on uniformly [15N]‐labeled PKIα to examine its structure free in solution. Seventy out of 75 residues were identified, and examination of the CαH chemical shifts revealed two regions of upfield chemical shifts characteristic of α‐helices. When PKIα was fragmented into two functionally distinct peptides for study at higher concentrations, no significant alterations in chemical shifts or secondary structure were observed. The first ordered region, identified in PKIα (1–25), contains an α‐helix from residues 1–13. This helix extends by one turn the helix observed in the crystal structure of a PKIα (5–24) peptide bound to the catalytic subunit. The second region of well‐defined secondary structure, residues 35–47, overlaps with the nuclear export signal in the PKIα (26–75) fragment. This secondary structure consists of a helix with a hydrophobic face comprised of Leu37, Leu41, and Leu44, followed by a flexible turn containing Ile46. These four residues are critical for nuclear export function. The remainder of the protein in solution appears relatively unstructured, and this lack of structure surrounding a few essential and well‐defined signaling elements may be characteristic of a growing family of small regulatory proteins that interact with protein kinases.
Energetics of solvent and ligand‐induced conformational changes in α‐lactalbumin
Authors: Griko Yuri V. , Remeta David P. 
Published: October 2010

AbstractThe energetics of structural changes in the holo and apo forms of α‐lactalbumin and the transition between their native and denatured states induced by binding Ca2+ and Na+ have been studied by differential scanning and isothermal titration microcalorimetry and circular dichroism spectroscopy under various solvent conditions. Removal of Ca2+ from the protein enhances its sensitivity to pH and ionic conditions due to noncompensated negative charge‐charge interactions at the cation binding site, which significantly reduces its overall stability. At neutral pH and low ionic strength, the native structure of apo‐α‐lactalbumin is stable below 14 °C and undergoes a conformational change to a native‐like molten globule intermediate at temperatures above 25 °C. The denaturation of either holo‐ or apo‐α‐lactalbumin is a highly cooperative process that is characterized by an enthalpy of similar magnitude when calculated at the same temperature. Measured by direct calorimetric titration, the enthalpy of Ca2+‐binding to apo‐LA at pH 7.5 is –7.1 kJ mol–1 at 5.0 °C, which is essentially invariant to protonation effects. This small enthalpy effect infers that stabilization of α‐lactalbumin by Ca2+ is primarily an entropy driven process, presumably arising from electrostatic interactions and the hydration effect. In contrast to the binding of calcium, the interaction of sodium with apo‐LA does not produce a noticeable heat effect and is characterized by its ionic nature rather than specific binding to the metal‐binding site. Characterization of the conformational stability and ligand binding energetics of α‐lactalbumin as a function of solvent conditions furnishes significant insight regarding the molecular flexibility and regulatory mechanism mediated by this protein.
Analysis of interactive packing of secondary structural elements in α/β units in proteins
Authors: Reddy Boojala V.B. , Nagarajaram Hampapathalu A. , Blundell Tom L. 
Published: October 2010

AbstractAn α‐helix and a β‐strand are said to be interactively packed if at least one residue in each of the secondary structural elements loses 10% of its solvent accessible contact area on association with the other secondary structural element.An analysis of all such 5,975 nonidentical α/β units in protein structures, defined at 2.5Å resolution, shows that the interaxial distance between the α‐helix and theβ‐strand is linearly correlated with the residue‐dependent function, log[(V/nda)/n‐int], where V is the volume of amino acid residues in the packing interface,nda is the normalized difference in solventaccessible contact area of the residues in packed and unpacked secondary structural elements, and n‐int is the number of residues in the packing interface. The β‐sheet unit (βu), defined as a pair of adjacent parallel or antiparallel hydrogen‐bonded β‐strands, packing with an α‐helix shows a better correlation between the interaxial distance and log (V/nda) for the residues in the packing interface.This packing relationship is shown to be useful in the prediction of interaxial distances inα/β units using the interacting residue information of equivalentα/β units of homologous proteins.It is, therefore, of value in comparative modeling of protein structures.
Functional insights from structural predictions: Analysis of the Escherichia coli genome
Authors: Rychlewski Leszek , Zhang Baohong , Godzik Adam 
Published: October 2010

AbstractFold assignments for proteins from the Escherichia coli genome are carried out using BASIC, a profile–profile alignment algorithm, recently tested on fold recognition benchmarks and on the Mycoplasma genitalium genome and PSI BLAST, the newest generation of the de facto standard in homology search algorithms. The fold assignments are followed by automated modeling and the resulting three‐dimensional models are analyzed for possible function prediction.Close to 30% of the proteins encoded in the E. coli genome can be recognized as homologous to a protein family with known structure. Most of these homologies (23% of the entire genome) can be recognized both by PSI BLAST and BASIC algorithms, but the latter recognizes an additional 260 homologies. Previous estimates suggested that only 10–15% of E. coli proteins can be characterized this way. This dramatic increase in the number of recognized homologies between E. coli proteins and structurally characterized protein families is partly due to the rapid increase of the database of known protein structures, but mostly it is due to the significant improvement in prediction algorithms.Knowing protein structure adds a new dimension to our understanding of its function and the predictions presented here can be used to predict function for uncharacterized proteins. Several examples, analyzed in more detail in this paper, include the DPS protein protecting DNA from oxidative damage (predicted to be homologous to ferritin with iron ion acting as a reducing agent) and the ahpC/tsa family of proteins, which provides resistance to various oxidating agents (predicted to be homologous to glutathione peroxidase).
Trifluoroethanol‐induced conformational transitions of proteins: Insights gained from the differences between α‐lactalbumin and ribonuclease A
Authors: Gast Klaus , Zirwer Dietrich , Müller‐Frohne Marlies , Damaschun Gregor 
Published: October 2010

AbstractThe trifluoroethanol (TFE)‐induced structural changes of two proteins widely used in folding experiments, bovine α‐lactalbumin, and bovine pancreatic ribonuclease A, have been investigated. The experiments were performed using circular dichroism spectroscopy in the far‐and near‐UV region to monitor changes in the secondary and tertiary structures, respectively, and dynamic light scattering to measure the hydrodynamic dimensions and the intermolecular interactions of the proteins in different conformational states. Both proteins behave rather differently under the influence of TFE: α‐lactalbumin exhibits a molten globule state at low TFE concentrations before it reaches the so‐called TFE state, whereas ribonuclease A is directly transformed into the TFE state at TFE concentrations above 40% (v/v). The properties of the TFE‐induced states are compared with those of equilibrium and kinetic intermediate states known from previous work to rationalize the use of TFE in yielding information about the folding of proteins. Additionally, we report on the properties of TFE/water and TFE/buffer mixtures derived from dynamic light scattering investigations under conditions used in our experiments.
Combinatorial codons: A computer program to approximate amino acid probabilities with biased nucleotide usage
Authors: Wolf Ethan , Kim Peter S. 
Published: October 2010

AbstractUsing techniques from optimization theory, we have developed a computer program that approximates a desired probability distribution for amino acids by imposing a probability distribution on the four nucleotides in each of the three codon positions. These base probabilities allow for the generation of biased codons for use in mutational studies and in the design of biologically encoded libraries. The dependencies between codons in the genetic code often makes the exact generation of the desired probability distribution for amino acids impossible. Compromises are often necessary. The program, therefore, not only solves for the “optimal” approximation to the desired distribution (where the definition of “optimal” is influenced by several types of parameters entered by the user), but also solves for a number of “sub‐optimal” solutions that are classified into families ofsimilar solutions. A representative of each family is presented to the program user, who can then choose the type of approximation that is best for the intended application. The Combinatorial Codons program is available for use over the web from http://www.wi.mit.edu/kim/computing.html.
Dissection of the structural and functional role of a conserved hydration site in RNase T1
Authors: Langhorst Ulrike , Loris Remy , Denisov Vladimir P. , Doumen Jan , Roose Patrice , Maes Dominique , Halle Bertil , Steyaert Jan 
Published: October 2010

AbstractThe reoccurrence of water molecules in crystal structures of RNase T1 was investigated. Five waters were found to be invariant in RNase T1 as well as in six other related fungal RNases. The structural, dynamical, and functional characteristics of one of these conserved hydration sites (WAT1) were analyzed by protein engineering, X‐ray crystallography, and 17O and 2H nuclear magnetic relaxation dispersion (NMRD). The position of WAT1 and its surrounding hydrogen bond network are unaffected by deletions of two neighboring side chains. In the mutant Thr93Gln, the Gln93Ne2 nitrogen replaces WAT1 and participates in a similar hydrogen bond network involving Cys6, Asn9, Asp76, and Thr91. The ability of WAT1 to form four hydrogen bonds may explain why evolution has preserved a water molecule, rather than a side‐chain atom, at the center of this intricate hydrogen bond network. Comparison of the 17O NMRD profiles from wild‐type and Thr93Gln RNase T1 yield a mean residence time of 7 ns at 27°C and an orientational order parameter of 0.45. The effects of mutations around WAT1 on the kinetic parameters of RNase T1 are small but significant and probably relate to the dynamics of the active site.
Structural basis of neurophysin hormone specificity: Geometry, polarity, and polarizability in aromatic ring interactions
Authors: Breslow Esther , Mombouyran Valerie , Deeb Ruba , Zheng Changsheng , Rose John P. , Wang Bi‐Cheng , Haschemeyer Rudy H. 
Published: October 2010

AbstractThe structural origins of the specificity of the neurophysin hormone‐binding site for an aromatic residue in peptide position 2 were explored by analyzing the binding of a series of peptides in the context of the crystal structure of liganded neurophysin. A new modeling method for describing the van der Waals surface of binding sites assisted in the analysis. Particular attention was paid to the unusually large (5 kcal/mol) difference in binding free energy between Phe and Leu in position 2, a value representing more than three times the maximum expected based on hydrophobicity alone, and additionally remarkable since modeling indicated that the Leu side chain was readily accommodated by the binding pocket. Although evidence was obtained of a weak thermodynamic linkage between the binding interactions of the residue 2 side chain and of the peptide α‐amino group, two factors are considered central. (1) The bound Leu side chain can establish only one‐third of the van der Waals contacts available to a Phe side chain. (2) The bound Phe side chain appears to be additionally stabilized relative to Leu by more favorable dipole and induced dipole interactions with nonaromatic polar and sulfur ligands in the binding pocket, as evidenced by examination of its interactions in the pocket, analysis of the detailed energetics of transfer of Phe and Leu side chains from water to other phases, and comparison with thermodynamic and structural data for the binding of residue 1 side chains in this system. While such polar interactions of aromatic rings have been previously observed, the present results suggest their potential for significant thermodynamic contributions to protein structure and ligand recognition.
WW: An isolated three‐stranded antiparallel β‐sheet domain that unfolds and refolds reversibly; evidence for a structured hydrophobic cluster in urea and GdnHCl and a disordered thermal unfolded state
Authors: Koepf Edward K. , Petrassi1 H. Michael , Sudol Marius , Kelly Jeffery W. 
Published: October 2010

AbstractThe objective of this study was to evaluate the suitability of the WW domain as a desirable model system to understand the folding and stability of an isolated three‐stranded antiparallel β‐sheet structure. The WW domain was subjected to thermal and chaotropic denaturation/reconstitution utilizing a variety of biophysical methods. This three‐stranded sheet folds reversibly and cooperatively utilizing both urea and GdnHCl as denaturants; however, the denatured state retains structure in the form of a hydrophobic cluster involving at least one aromatic side chain. In contrast to chaotropic denaturation, thermal denaturation appears to be more complete and may be a two state process. The suitability of the WW domain for future studies aimed at understanding the kinetics and thermodynamics of antiparallel β‐sheet folding clearly emerges from this initial study. The most exciting and significant result in this manuscript is the finding that the chaotropic denatured state of WW has a hydrophobic cluster as discerned by near‐UV CD evidence. The role that the denatured state plays in the folding and stability of a three‐stranded β‐sheets, and its capacity for preventing aggregation may be particularly important and is the subject of ongoing studies.
Specificity of coenzyme analogues and fragments in promoting or impeding the refolding of clostridial glutamate dehydrogenase
Authors: Aghajanian Suren , Walsh Thomas P. , Engel Paul C. 
Published: October 2010

AbstractNAD+facilitates high‐yield reactivation of clostridial glutamate dehydrogenase (GDH) after unfolding in urea. The specificity of this effect has been explored by using analogues and fragments of NAD+.The adenine portion, unlike the nicotinamide portion, is important for reactivation. Alteration in the nicotinamide portion, in acetylpyridine adenine dinucleotide, has little effect, whereas loss of the 6‐NH2substitution on the adenine ring, in 6‐deamino NAD, diminishes the effectiveness of the nucleotide in promoting refolding. Also ADP‐ribose, lacking nicotinamide, promotes reactivation whereas NMN‐phosphoribose, lacking the adenine, does not.Of the smaller fragments, those containing an adenosine moiety, and especially those with one or more phosphate groups, impede the refolding ability of NAD+, and are able to bind to the folding intermediate though unable to facilitate refolding.These results are interpreted in terms of the known 3D structure for clostridial glutamate dehydrogenase. It is assumed that the refolding intermediate has a more or less fully formed NAD+‐binding domain but a partially disordered substrate‐binding domain and linking region. Binding of NAD+or ADP‐ribose appears to impose new structural constraints that result in completion of the correct folding of the second domain, allowing association of enzyme molecules to form the native hexamer.
The loss in hydrophobic surface area resulting from a Leu to Val mutation at the N‐terminus of the aldehyde dehydrogenase presequence prevents import of the protein into mitochondria
Authors: Hammen Philip K. , Heard Thomas S. , Waltner Mary , Weiner Henry 
Published: October 2010

AbstractAn apparent conservative mutation, Leu to Val, at the second residue of the rat liver mitochondrial aldehyde dehydrogenase (ALDH) presequence resulted in a precursor protein that was not imported into mitochondria. Additional mutants were made to substitute various amino acids with nonpolar side chains for Leu2. The Ile, Phe, and Trp mutants were imported to an extent similar to that of the native precursor, but the Ala mutant was imported only about one‐fourth as well. It was shown that the N‐terminal methionine was removed from the L2V mutant in a reaction catalyzed by methionine aminopeptidase. The N‐terminal methionine of native pALDH and the other mutant presequences was blocked, presumably by acetylation. Because of the difference in co‐translational modification, the L2V mutant sustained a significant loss in the available hydrophobic surface of the presequence. Import competence was restored to the L2V mutant when it was translated using a system that did not remove Met1. The removal of anArg‐Gly‐Pro helix linker segment (residues 11–14) from the L2V mutant, which shifted three leucine residues toward the N‐terminus, also restored import competence. These results lead to the conclusion that a minimum amount of hydrophobic surface area near the N‐termini of mitochondrial presequences is an essential property to determine their ability to be imported. As a result, both electrostatic and hydrophobic components must be considered when trying to understand the interactions between precursor proteins and proteins of the mitochondrial import apparatus.
Protein structural topology: Automated analysis and diagrammatic representation
Authors: Westhead David R. , Slidel Timothy W.F. , Flores Tomas P.J. , Thornton Janet M. 
Published: October 2010

AbstractThe topology of a protein structure is a highly simplified description of its fold including only the sequence of secondary structure elements, and their relative spatial positions and approximate orientations. This information can be embodied in a two‐dimensional diagram of protein topology, called a TOPS cartoon. These cartoons are useful for the understanding of particular folds and making comparisons between folds. Here we describe a new algorithm for the production of TOPS cartoons, which is more robust than those previously available, and has a much higher success rate. This algorithm has been used to produce a database of protein topology cartoons that covers most of the data bank of known protein structures.