bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pseudodihedrals: Simplified protein backbone representation with knowledge‐based energy
Authors: Dewitte R. S. , Shakhnovich E. I. 
Published: February 2009

AbstractPairwise contact energies do not explicitly take protein secondary structure into account, and so provide an incomplete description of conformational energy. In order to construct a Hamiltonian that specifically relates to protein backbone conformations, a simplified backbone angle is used. The pseudodihedral angle (the torsion angle between planes defined by 4 consecutive α‐carbon atoms) provides a simplified backbone representation and continues to manifest information about secondary‐structure elements: the pseudo‐Ramachandran plot contains helical and sheetlike regions. The distribution of pseudodihedral angles is highly sensitive to the identity of the central pair of amino acids. Therefore, a sequence‐dependent, knowledge‐based potential energy was found according to a quasichemical approximation. These functions form complementary additions to the contact potentials currently in use. This pseudodihedral potential greatly enhances the ability to design sequences that are specific to a given conformation and also improves the ability to discriminate a native conformation from many other conformations.
The three‐dimensional structures of mutants of porphobilinogen deaminase: Toward an understanding of the structural basis of acute intermittent porphyria
Authors: Brownlie Paul D. , Cooper Jon B. , Wood Steve P. , Lambert Richard , Blundell Tom L. , Louie Gordon V. , Jordan Peter M. , Warren Martin J. 
Published: February 2009

AbstractMutations in the human gene for the enzyme porphobilinogen deaminase give rise to an inherited disease of heme biosynthesis, acute intermittent porphyria. Knowledge of the 3‐dimensionai structure of human porphobilinogen deaminase, based on the structure of the bacterial enzyme, allows correlation of structure with gene organization and leads to an understanding of the relationship between mutations in the gene, structural and functional changes of the enzyme, and the symptoms of the disease. Most mutations occur in exons 10 and 12, often changing amino acids in the active site. Several of these are shown to be involved in binding the primer or substrate; none modifies Asp 84, which is essential for catalytic activity.
Role and mechanism of the maturation cleavage of VP0 in poliovirus assembly: Structure of the empty capsid assembly intermediate at 2.9 Å resolution
Authors: Basavappa R. , Filman D.J. , Syed R. , Flore O. , Icenogle J.P. , Hogle J.M. 
Published: February 2009

AbstractThe crystal structure of the P1/Mahoney poliovirus empty capsid has been determined at 2.9 Å resolution. The empty capsids differ from mature virions in that they lack the viral RNA and have yet to undergo a stabilizing maturation cleavage of VPO to yield the mature capsid proteins VP4 and VP2. The outer surface and the bulk of the protein shell are very similar to those of the mature virion. The major differences between the 2 structures are focused in a network formed by the N‐terminal extensions of the capsid proteins on the inner surface of the shell. In the empty capsids, the entire N‐terminal extension of VP1, as well as portions corresponding to VP4 and the N‐terminal extension of VP2, are disordered, and many stabilizing interactions that are present in the mature virion are missing. In the empty capsid, the VP1 scissile bond is located some 20 Å away from the positions in the mature virion of the termini generated by VP0 cleavage. The scissile bond is located on the rim of a trefoilshaped depression in the inner surface of the shell that is highly reminiscent of an RNA binding site in bean pod mottle virus. The structure suggests plausible (and ultimately testable) models for the initiation of encapsidation, for the RNA‐dependent autocatalytic cleavage of VP0, and for the role of the cleavage in establishing the ordered N‐terminal network and in generating stable virions.
Probing the catalytic roles of n2‐site glutamate residues in Escherichia coli glutamine synthetase by mutagenesis
Authors: Witmer Mark R. , Villafranca Joseph J. , Palmieri‐Young Donna 
Published: February 2009

AbstractThe contribution of metal ion ligand type and charge to catalysis and regulation at the lower affinity metal ion site (n2 site) of Escherichia coli glutamine synthetase (GS) was tested by mutagenesis and kinetic analysis. The 2 glutamate residues at the n2 site, E129 and E357, were changed to E129D, E129H, E357H, E357Q, and E357D, representing conservative and nonconservative alterations. Unadenylylated and fully adenylylated enzyme forms were studied. The Mn2+‐KD values, UV‐vis, and fluorescence emission properties were similar for all mutants versus WTGS, except E129H. For kinetic determinations with both Mn2+ and Mg2+, nonconservative mutants (E357H, E129H, E357Q) showed lower biosynthetic activities than conservative mutants (E129D, E357D). Relative to WTGS, all the unadenylylated Mn2+‐activated enzymes showed reduced kcar/Km values for ATP (>7‐fold) and for glutamate (> 10‐fold). Of the unadenylylated Mg2+‐activated enzymes, only E129D showed kinetic parameters competitive with WTGS, and adenylylated E129D was a 20‐fold better catalyst than WTGS. We propose the n2‐site metal ion activates ADP for departure in the phosphorylation of glutamate by ATP to generate γ‐glutamyl phosphate. Alteration of the charge density at this metal ion alters the transition‐state energy for phosphoryl group transfer and may affect ATP binding and/or ADP release. Thus, the steady‐state kinetic data suggest that modifying the charge density increases the transition‐state energies for chemical steps. Importantly, the data demonstrate that each ligand position has a specialized spatial environment and the charge of the ligand modulates the catalytic steps occurring at the metal ion. The data are discussed in the context of the known X‐ray structures of GS.
Identification of amino acid residues involved in the binding of Huperzine A to cholinesterases
Authors: Saxena Ashima , Doctor Bhupendra P. , Qian Naifeng , Kovach Ildiko M. , Kozikowski A.P. , Pang Y.P. , Vellom Daniel C. , Radic Zoran , Quinn Daniel , Taylor Palmer 
Published: February 2009

AbstractHuperzine A, a potential agent for therapy in Alzheimer's disease and for prophylaxis of organophosphate toxicity, has recently been characterized as a reversible inhibitor of cholinesterases. To examine the specificity of this novel compound in more detail, we have examined the interaction of the 2 stereoisomers of Huperzine A with cholinesterases and site‐specific mutants that detail the involvement of specific amino acid residues. Inhibition of fetal bovine serum acetylcholinesterase by (—)‐Huperzine A was 35‐fold more potent than (+)‐Huperzine A, with K1 values of 6.2 nM and 210 nM, respectively. In addition, (—)‐Huperzine A was 88‐fold more potent in inhibiting Torpedo acetylcholinesterase than (+)‐Huperzine A, with K1 values of 0.25 μM and 22 μM, respectively. Far larger K1 values that did not differ between the 2 stereoisomers were observed with horse and human serum butyrylcholinesterases. Mammalian acetylcholinesterase, Torpedo acetylcholinesterase, and mammalian butyrylcholinesterase can be distinguished by the amino acid Tyr, Phe, or Ala in the 330 position, respectively. Studies with mouse acetylcholinesterase mutants, Tyr 337(330) Phe and Tyr 337(330) Ala yielded a difference in reactivity that closely mimicked the native enzymes. In contrast, mutation of the conserved Glu 199 residue to Gln in Torpedo acetylcholinesterase produced only a 3‐fold increase in K1 value for the binding of Huperzine A. Molecular mechanics energy minimization of the complexes formed between each of the 2 stereoisomers of Huperzine A and fetal bovine serum acetylcholinesterase, Torpedo acetylcholinesterase, or human butyrylcholinesterase also revealed that (—)‐Huperzine A gave a better fit than (+)‐Huperzine A and implicated Tyr 337(330) in the stereoselectivity of Huperzine A.
Solution structure of the DNA‐binding domain of the heat shock transcription factor determined by multidimensional heteronuclear magnetic resonance spectroscopy
Authors: Damberger Fred F. , Pelton Jeffrey G. , Harrison Celia J. , Nelson Hillary C.M. , Wemmer David E. 
Published: February 2009

AbstractThe solution structure of the 92‐residue DNA‐binding domain of the heat shock transcription factor from Kluyveromyces lactis has been determined using multidimensional NMR methods. Three‐dimensional (3D) triple resonance, 1H‐13C‐13C‐1H total correlation spectroscopy, and 15N‐separated total correlation spectroscopy‐heteronuclear multiple quantum correlation experiments were used along with various 2D spectra to make nearly complete assignments for the backbone and side‐chain 1H, 15N, and 13C resonances. Five‐hundred eighty‐three NOE constraints identified in 3D 13C‐ and l5N‐separated NOE spectroscopy (NOESY)‐heteronuclear multiple quantum correlation spectra and a 4‐dimensional 13C/13C‐edited NOESY spectrum, along with 35 ø, 9 χ1, and 30 hydrogen bond constraints, were used to calculate 30 structures by a hybrid distance geometry/simulated annealing protocol, of which 24 were used for structural comparison. The calculations revealed that a 3‐helix bundle packs against a small 4‐stranded antiparallel β‐sheet. The backbone RMS deviation (RMSD) for the family of structures was 1.03 ± 0.19 Å with respect to the average structure. The topology is analogous to that of the C‐terminal domain of the catabolite gene activator protein and appears to be in the helix‐turn‐helix family of DNA‐binding proteins. The overall fold determined by the NMR data is consistent with recent crystallographic work on this domain (Harrison CJ, Bohm AA, Nelson HCM, 1994, Science 263:224) as evidenced by RMSD between backbone atoms in the NMR and X‐ray structures of 1.77 ± 0.20 Å. Several differences were identified some of which may be due to protein‐protein interactions in the crystal.
Discovering structural correlations in α‐helices
Authors: Klingler Tod M. , Brutlag Douglas L. 
Published: February 2009

AbstractWe have developed a new representation for structural and functional motifs in protein sequences based on correlations between pairs of amino acids and applied it to α‐helical and β‐sheet sequences. Existing probabilistic methods for representing and analyzing protein sequences have traditionally assumed conditional independence of evidence. In other words, amino acids are assumed to have no effect on each other. However, analyses of protein structures have repeatedly demonstrated the importance of interactions between amino acids in conferring both structure and function. Using Bayesian networks, we are able to model the relationships between amino acids at distinct positions in a protein sequence in addition to the amino acid distributions at each position. We have also developed an automated program for discovering sequence correlations using standard statistical tests and validation techniques. In this paper, we test this program on sequences from secondary structure motifs, namely α‐helices and β‐sheets. In each case, the correlations our program discovers correspond well with known physical and chemical interactions between amino acids in structures. Furthermore, we show that, using different chemical alphabets for the amino acids, we discover structural relationships based on the same chemical principle used in constructing the alphabet. This new representation of 3‐dimensional features in protein motifs, such as those arising from structural or functional constraints on the sequence, can be used to improve sequence analysis tools including pattern analysis and database search.
Nerve growth factor: Structure/function relationships
Authors: Bradshaw Ralph A. , Murray‐Rust Judith , Blundell Tom L. , Mcdonald Neil Q. , Lapatto Risto , Ibáñez Carlos F. 
Published: February 2009

AbstractNerve growth factor (NGF), which has a tertiary structure based on a cluster of 3 cystine disulfides and 2 very extended, but distorted β‐hairpins, is the prototype of a larger family of neurotrophins. Prior to the availability of cloning techniques, the mouse submandibular gland was the richest source of NGF and provided sufficient material to enable its biochemical characterization. It binds as a dimer to at least 2 cell‐surface receptor types expressed in a variety of neuronal and non‐neuronal cells. Residues involved in these interactions and in the maintenance of tertiary and quaternary structure have been identified by chemical modification and site‐directed mutagenesis, and this information can be related to their location in the 3‐dimensional structure. For example, interactions between aromatic residues contribute to the stability of the NGF dimer, and specific surface lysine residues participate in receptor contacts. The conclusion from these studies is that receptor interactions involve broad surface regions, which may be composed of residues from both protomers in the dimer.
Common structural features of the luxF protein and the subunits of bacterial luciferase: Evidence for a (βα)8 fold in luciferase
Authors: Moore Stanley A. , James Michael N. G. 
Published: February 2009

AbstractThe amino acid sequence identity and potential structural similarity between the subunits of bacterial luciferase and the recently determined structure of the luxF molecule are examined. The unique β/α barrel fold found in luxF appears to be conserved in part in the luciferase subunits. From secondary structural predictions of both luciferase subunits, and from structural comparisons between the protein product of the luxF gene, NFP, and glycolate oxidase, we propose that it is feasible for both luciferase subunits to adopt a (βα)8 barrel fold with at least 2 excursions from the (βα)8 topology. Amino acids conserved between NFP and the luciferase subunits cluster together in 3 distinct “pockets” of NFP, which are located at hydrophobic interfaces between the β‐strands and α‐helices. Several tight turns joining the C‐termini of β‐strands and the N‐termini of α‐helices are found as key components of these conserved regions. Helix start and end points are easily demarcated in the luciferase subunit protein sequences; the N‐cap residues are the most strongly conserved structural features. A partial model of the luciferase β subunit from Photobacterium leiognathi has been built based on our crystallographically determined structure of luxF at 1.6 Å resolution.
Fast folding of a prototypic polypeptide: The immunoglobulin binding domain of streptococcal protein G
Authors: Kuszewski John , Clore G. Marius , Gronenborn Angela M. 
Published: February 2009

AbstractThe folding of the small (56 residues) highly stable B1 immunoglobulin binding domain (GB1) of streptococcal protein G has been investigated by quenched‐flow deuterium‐hydrogen exchange. This system represents a paradigm for the study of protein folding because it exhibits no complicating features superimposed upon the intrinsic properties of the polypeptide chain. Collapse to a semicompact state exhibiting partial order, reflected in protection factors for ND‐NH exchange up to 10‐fold higher than that expected for a random coil, occurs within the dead time (≤1 ms) of the quenched flow apparatus. This is followed by the formation of the fully native state, as monitored by the fractional proton occupancy of 26 backbone amide groups spread throughout the protein, in a single rapid concerted step with a half‐life of 5.2 ms at 5 °C.
Surfactant effects on protein structure examined by electrospray ionization mass spectrometry
Authors: Loo R. R. Ogorzalek , Dales N. , Andrews P. C. 
Published: February 2009

AbstractElectrospray ionization mass spectrometry (ESI‐MS) has proven to be a useful tool for examining noncovalent complexes between proteins and a variety of ligands. It has also been used to distinguish between denatured and refolded forms of proteins. Surfactants are frequently employed to enhance solubilization or to modify the tertiary or quaternary structure of proteins, but are usually considered incompatible with mass spectrometry. A broad range of ionic, nonionic, and zwitterionic surfactants was examined to characterize their effects on ESI‐MS and on protein structure under ESI‐MS conditions. Solution conditions studied include 4% acetic acid/50% acetonitrile/46% H2O and 100% aqueous. Of the surfactants examined, the nonionic saccharides, such as n‐dodecyl‐β‐D‐gluco‐pyranoside, at 0.1% to 0.01% (w/v) concentrations, performed best, with limited interference from chemical background and adduct formation. Under the experimental conditions used, ESI‐MS performance in the presence of surfactants was found to be unrelated to critical micelle concentration. It is demonstrated that surfactants can affect both the tertiary and quaternary structures of proteins under conditions used for ESI‐MS. However, several of the surfactants caused significant shifts in the charge‐state distributions, which appeared to be independent of conformational effects. These observations suggest that surfactants, used in conjunction with ESI‐MS, can be useful for protein structure studies, if care is used in the interpretation of the results.
Glu‐50 in the catalytic chain of Escherichia coli aspartate transcarbamoylase plays a crucial role in the stability of the R quaternary structure
Authors: Tauc Patrick , Keiser Richard T. , Kantrowitz Evan R. , Vachette Patrice 
Published: February 2009

AbstractGlu‐50 of aspartate transcarbamoylase from Escherichia coli forms a set of interdomain bridging interactions between the 2 domains of the catalytic chain; these interactions are critical for stabilization of the high‐activity high‐affinity form of the enzyme. The mutant enzyme with an alanine substituted for Glu‐50 (Glu‐50 → Ala) exhibits significantly reduced activity, little cooperativity, and altered regulatory behavior (Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:1444–1451). A study of the structural consequences of replacing Glu‐50 by alanine using solution X‐ray scattering is reported here. Correspondingly, in the absence of substrates, the mutant enzyme is in the same, so‐called T quaternary conformation as is the wild‐type enzyme. In the presence of a saturating concentration of the bisubstrate analog N‐phosphonacetyl‐L‐aspartate (PALA), the mutant enzyme is in the same, so‐called R quaternary conformation as the wild‐type enzyme. However, the Glu‐50 → Ala enzyme differs from the wild‐type enzyme, in that its scattering pattern is hardly altered by a combination of carbamoyl phosphate and succinate. Addition of ATP under these conditions does result in a slight shift toward the R structure. Steady‐state kinetic studies indicate that, in contrast to the wild‐type enzyme, the Glu‐50 → Ala enzyme is activated by PALA at saturating concentrations of carbamoyl phosphate and aspartate, and that PALA increases the affinity of the mutant enzyme for aspartate. These data suggest that the enzyme does not undergo the normal T to R transition upon binding of the physiological substrates and verifies the previous suggestion that the interdomain bridging interactions involving Glu‐50 are critical for the creation of the high‐activity, high‐affinity R state of the enzyme.
Analysis of hydrophobicity in the α and β chemokine families and its relevance to dimerization
Authors: Covell David G. , Smythers Gary W. , Gronenborn Angela M. , Clore G. Marius 
Published: February 2009

AbstractThe chemokine family of chemotactic cytokines plays a key role in orchestrating the immune response. The family has been divided into 2 subfamilies, α and β, based on the spacing of the first 2 cysteine residues, function, and chromosomal location. Members within each subfamily have 25–70% sequence identity, whereas the amino acid identity between members of the 2 subfamilies ranges from 20 to 40%. A quantitative analysis of the hydrophobic properties of 11 α and 9 β chemokine sequences, based on the coordinates of the prototypic α and β chemokines, interleukin‐8 (IL‐8), and human macrophage inflammatory protein‐10 (hMIP‐10), respectively, is presented. The monomers of the α and β chemokines have their strongest core hydrophobic cluster at equivalent positions, consistent with their similar tertiary structures. In contrast, the pattern of monomer surface hydrophobicity between the α and β chemokines differs in a manner that is fully consistent with the observed differences in quaternary structure. The most hydrophobic surface clusters on the monomer subunits are located in very different regions of the α and β chemokines and comprise in each case the amino acids that are buried at the interface of their respective dimers. The theoretical analysis of hydrophobicity strongly supports the hypothesis that the distinct dimers observed for IL‐8 and hMIP‐1β are preserved for all the α and β chemokines, respectively. This provides a rational explanation for the lack of receptor crossbinding and reactivity between the α and β chemokine subfamilies.
Docking of nitrogenase iron‐and molybdenum‐iron proteins for electron transfer and MgATP hydrolysis: The role of arginine 140 and lysine 143 of the Azotobacter vinelandii iron protein
Authors: Seefeldt Lance C. 
Published: February 2009

AbstractDocking of the nitrogenase component proteins, the iron protein (FeP) and the molybdenum‐iron protein (MoFeP), is required for MgATP hydrolysis, electron transfer between the component proteins, and substrate reductions catalyzed by nitrogenase. The present work examines the function of 3 charged amino acids, Arg 140, Glu 141, and Lys 143, of the Azotobacter vinelandii FeP in nitrogenase component protein docking. The function of these amino acids was probed by changing each to the neutral amino acid glutamine using site‐directed mutagenesis. The altered FePs were expressed in A. vinelandii in place of the wild‐type FeP. Changing Glu 141 to Gln (E141Q) had no adverse effects on the function of nitrogenase in whole cells, indicating that this charged residue is not essential to nitrogenase function. In contrast, changing Arg 140 or Lys 143 to Gln (R140Q and K143Q) resulted in a significant decrease in nitrogenase activity, suggesting that these charged amino acid residues play an important role in some function of the FeP. The function of each amino acid was deduced by analysis of the properties of the purified R140Q and K143Q FePs. Both altered proteins were found to support reduced substrate reduction rates when coupled to wild‐type MoFeP. Detailed analysis revealed that changing these residues to Gln resulted in a dramatic reduction in the affinity of the altered FeP for binding to the MoFeP. This was deduced in FeP titration, NaCl inhibition, and MoFeP protection from Fe2+ chelation experiments. In addition, it was found that changing Arg 140 and Lys 143 to Gln resulted in a significant uncoupling of MgATP hydrolysis from intercomponent electron transfer rates between FeP and MoFeP. The wild‐type complex was found to hydrolyze 2.5 MgATP per electron transferred, whereas the R140Q and K143Q proteins, when coupled to wild‐type MoFeP, required 6 and 31 MgATP for each electron transferred, respectively. It is concluded that both Arg 140 and Lys 143 of the A. vinelandii nitrogenase FeP are important in the docking interaction with the MoFeP and that these residues probably function in aligning the FeP with the MoFeP for intercomponent electron transfer. A model is discussed in which these positively charged amino acids of the FeP might interact with negatively charged amino acids (Asp and Glu) on the MoFeP near its 8Fe cluster.