bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mammalian integral membrane receptors are homologous to facilitators and antiporters of yeast, fungi, and eubacteria
Authors: Reizer Jonathan , Reizer Aiala , Saier Milton H. , Finley Kim , Kakuda Donald , Macleod Carol L. 
Published: July 2010

AbstractWe demonstrate that three integral membrane receptors of mammals — the ecotropic retroviral leukemia receptor (ERR), the human retroviral receptor (HRR), and the T‐cell early activator (Tea) — are homologous to a family of transporters specific for amino acids, polyamines, and choline (APC), which catalyze solute uniport, solute:cation symport, or solute:solute antiport in yeast, fungi, and eubacteria. Interestingly, the ERR membrane protein was recently shown to function as a cation:amino acid cotransporter. A binary sequence similarity matrix and an evolutionary tree of the 14 members of this family, illustrating their sequence similarities and divergences, were constructed. Other proteins, including the developmentally controlled GerAII spore germination protein of Bacillus subtilis and the acetylcholine receptor of Drosophila melanogaster gave sequence comparison scores of a sufficiently large magnitude to suggest (but not to establish) a common evolutionary origin with members of the APC family.We report an extended and corrected Tea cDNA sequence and show that the mammalian Tea and ERR encoding genes are differentially expressed in tissues and cell lines. Furthermore, the two mammalian cDNA sequences hybridize with other vertebrate and yeast genomic DNAs under stringent conditions. These observations support the notion that cell surface receptor proteins in mammals are transport proteins that share a common origin with transport proteins of single‐celled organisms. Thus, permeases of essential metabolites may function pathologically as viral receptors.
An automated method for modeling proteins on known templates using distance geometry
Authors: Srinivasan Subhashini , March Carl J. , Sudarsanam Sucha 
Published: July 2010

AbstractWe present an automated method incorporated into a software package, FOLDER, to fold a protein sequence on a given three‐dimensional (3D) template. Starting with the sequence alignment of a family of homologous proteins, tertiary structures are modeled using the known 3D structure of one member of the family as a template. Homologous interatomic distances from the template are used as constraints. For nonhomologous regions in the model protein, the lower and the upper bounds for the interatomic distances are imposed by steric constraints and the globular dimensions of the template, respectively. Distance geometry is used to embed an ensemble of structures consistent with these distance bounds. Structures are selected from this ensemble based on minimal distance error criteria, after a penalty function optimization step. These structures are then refined using energy optimization methods.The method is tested by simulating the α‐chain of horse hemoglobin using the α‐chain of human hemoglobin as the template and by comparing the generated models with the crystal structure of the α‐chain of horse hemoglobin. We also test the packing efficiency of this method by reconstructing the atomic positions of the interior side chains beyond Cβ atoms of a protein domain from a known 3D structure. In both test cases, models retain the template constraints and any additionally imposed constraints while the packing of the interior residues is optimized with no short contacts or bond deformations. To demonstrate the use of this method in simulating structures of proteins with nonhomologous disulfides, we construct a model of murine interleukin (IL)‐4 using the NMR structure of human IL‐4 as the template. The resulting geometry of the nonhomologous disulfide in the model structure for murine IL‐4 is consistent with standard disulfide geometry.
Controlling the regiospecificity and coupling of cytochrome P450cam: T185F mutant increases coupling and abolishes 3‐hydroxynorcamphor product
Authors: Paulsen Mark D. , Ornstein Rick L. , Filipovic Djordje , Sligar Stephen G. 
Published: July 2010

AbstractCytochrome P450cam (P450CIA1) catalyzes the hydroxylation of camphor and several substrate analogues such as norcamphor and 1‐methyl‐norcamphor. Hydroxylation was found experimentally at the 3, 5, and 6 positions of norcamphor, but only at the 5 and 6 positions of 1‐methyl‐norcamphor. In the catalytic cycle, the hydroxylation of substrate is coupled to the consumption of NADH. For camphor, the degree of coupling is 100%, but for both norcamphor and 1‐methyl‐norcamphor, the efficiency is dramatically lowered to 12% and 50%, respectively. Based on an examination of the active site of P450cam, it appeared that mutating position 185 might dramatically alter the product specificity and coupling of hydroxylation of norcamphor by P450cam. Analysis of molecular dynamics trajectories of norcamphor bound to the T185F mutant of cytochrome P450cam predicted that hydroxylation at the 3 position should be abolished and that the coupling should be dramatically increased. This mutant was constructed and the product profile and coupling experimentally determined. The coupling was doubled, and hydroxylation at the 3 position was essentially abolished. Both of these results are in agreement with the prediction.
Orientational constraints as three‐dimensional structural constraints from chemical shift anisotropy: The polypeptide backbone of gramicidin A in a lipid bilayer
Authors: Mai W. , Hu W. , Wang C. , Cross T.A. 
Published: July 2010

AbstractChemical shifts observed from samples that are uniformly aligned with respect to the magnetic field can be used as very high‐resolution structural constraints. This constraint takes the form of an orientational constraint rather than the more familiar distance constraint. The accuracy of these constraints is dependent upon the quality of the tensor characterization. Both tensor element magnitudes and tensor orientations with respect to the molecular frame need to be considered. Here these constraints have been used to evaluate models for the channel conformation of gramicidin A. Of the three models used, the one experimentally derived model of gramicidin in sodium dodecyl sulfate micelles fits the data least well.
G proteins: Critical control points for transmembrane signals
Authors: Neer Eva J. 
Published: July 2010

AbstractHeterotrimeric GTP‐binding proteins (G proteins) that are made up of α and βγy subunits couple many kinds of cell‐surface receptors to intracellular effector enzymes or ion channels. Every cell contains several types of receptors, G proteins, and effectors. The specificity with which G protein subunits interact with receptors and effectors defines the range of responses a cell is able to make to an external signal. Thus, the G proteins act as a critical control point that determines whether a signal spreads through several pathways or is focused to a single pathway. In this review, I will summarize some features of the structure and function of mammalian G protein subunits, discuss the role of both a and by subunits in regulation of effectors, the role of the βγ subunit in macromolecular assembly, and the mechanisms that might make some responses extremely specific and others rather diffuse.
A two‐domain structure for the two subunits of NAD(P)H:quinone acceptor oxidoreductase
Authors: Chen Shiuan , Deng Paulis S.K. , Bailey Jerome M. , Swiderek Kristine M. 
Published: July 2010

AbstractNAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) (DT‐diaphorase) is a FAD‐containing reductase that catalyzes a unique 2‐electron reduction of quinones. It consists of 2 identical subunits. In this study, it was found that the carboxyl‐terminal portion of the 2 subunits can be cleaved by various proteases, whereas the amino‐terminal portion cannot. It was also found that proteolytic digestion of the enzyme can be blocked by the prosthetic group FAD, substrates NAD(P)H and menadione, and inhibitors dicoumarol and phenindione. Interestingly, chrysin and Cibacron blue, 2 additional inhibitors, cannot protect the enzyme from proteolytic digestion. The results obtained from this study indicate that the subunit of the quinone reductase has a 2‐domain structure, i.e., an aminoterminal compact domain and a carboxyl‐terminal flexible domain. A structural model of the quinone reductase is generated based on results obtained from amino‐terminal and carboxyl‐terminal protein sequence analyses and electrospray mass spectral analyses of hydrolytic products of the enzyme generated by trypsin, chymotrypsin, andStaphylococcus aureusprotease. Furthermore, based on the data, it is suggested that the binding of substrates involves an interaction between 2 structural domains.
Kinetics and thermodynamics of thermal denaturation in acyl carrier protein
Authors: Horvath L.A. , Sturtevant J.M. , Prestegard J.H. 
Published: July 2010

AbstractThe denaturation of Escherichia coli acyl carrier protein (ACP) in buffers containing both monovalent and divalent cations was followed by variable‐temperature NMR and differential scanning calorimetry. Both high concentrations of monovalent salts (Na+) and moderate concentrations of divalent salts (Ca2+) raise the denaturation temperature, but calorimetry indicates that a significant increase in the enthalpy of denaturation is obtained only with the addition of a divalent salt. NMR experiments in both low ionic strength monovalent buffers and low ionic strength monovalent buffers containing calcium ions show exchange between native and denatured forms to be slow on the NMR time scale. However, in high ionic strength monovalent buffers, where the temperature of denaturation is elevated as it is in the presence of Ca2+, the transition is fast on the NMR time scale. These results suggest that monovalent and divalent cations may act to stabilize ACP in different ways. Monovalent ions may nonspecifically balance the intrinsic negative charge of this protein in a way that is similar for native, denatured, and intermediate forms. Divalent cations provide stability by binding to specific sites present only in the native state.
On the rate of proton exchange with solvent of the catalytic histidine in flavocytochrome b2 (yeast L‐lactate dehydrogenase)
Authors: Balme Alexis , Lederer Florence 
Published: July 2010

AbstractThe family of FMN‐dependent, α‐hydroxy acid‐oxidizing enzymes catalyzes substrate dehydrogenation by a mechanism the first step of which is abstraction of the substrate α‐proton (so‐called carbanion mechanism). For flavocytochrome b2 and lactate oxidase, it was shown that once on the enzyme this proton is lost only slowly to the solvent (Lederer F, 1984, In: Bray RC, Engel PC, Mayhew SG, eds, Flavins & flavoproteins, Berlin: Walter de Gruyter & Co., pp 513‐526; Urban P, Lederer F, 1985, J Biol Chem 260:11115‐11122). This suggested the occurrence of a pKa increase of the catalytic histidine upon enzyme reduction by substrate. For flavocytochrome b2, the crystal structure indicated 2 possible origins for the stabilization of the imidazolium form of His 373: either a network of hydrogen bonds involving His 373, Tyr 254, flavin N5 and O4, a heme propionate, and solvent molecules, and/or electrostatic interactions with Asp 282 and with the reduced cofactor N1 anion. In this work, we probe the effect of the hydrogen bond network at the active site by studying proton exchange with solvent for 2 mutants: Y254F and the recombinant flavodehydrogenase domain, in which this network should be disrupted. The rate of proton exchange, as determined by intermolecular hydrogen transfer experiments, appears identical in the flavodehydrogenase domain and the wild‐type enzyme, whereas it is about 3‐fold faster in the Y254F mutant. It thus appears that specific hydrogen bonds to the solvent do not play a major role in stabilizing the acid form of His 373 in reduced flavocytochrome b2. Removal of the Y254 phenol group induces a pKa drop of about half a pH unit for His 373 in the reduced enzyme. Even then, the rate of exchange of the imidazolium proton with solvent is still lower by several orders of magnitude than that of a normally ionizing histidine. Other factors must then also contribute to the pKa increase, such as the electrostatic interactions with D282 and the anionic reduced cofactor, as suggested by the crystal structure.
Improving the sensitivity of the sequence profile method
Authors: Lüthy Roland , Xenarios Ioannis , Bucher Philipp 
Published: July 2010

AbstractThe sequence profile method (Gribskov M, McLachlan AD, Eisenberg D, 1987, Proc Natl Acad Sci USA 84:4355‐4358) is a powerful tool to detect distant relationships between amino acid sequences. A profile is a table of posi‐tion‐specific scores and gap penalties, providing a generalized description of a protein motif, which can be used for sequence alignments and database searches instead of an individual sequence. A sequence profile is derived from a multiple sequence alignment. We have found 2 ways to improve the sensitivity of sequence profiles: (1) Sequence weights: Usage of individual weights for each sequence avoids bias toward closely related sequences. These weights are automatically assigned based on the distance of the sequences using a published procedure (Sib‐bald PR, Argos P, 1990, J Mol Biol 216:813‐818). (2) Amino acid substitution table: In addition to the alignment, the construction of a profile also needs an amino acid substitution table. We have found that in some cases a new table, the BLOSUM45 table (Henikoff S, Henikoff JG, 1992, Proc Natl Acud Sci USA 89:10915‐10919), is more sensitive than the original Dayhoff table or the modified Dayhoff table used in the current implementation. Profiles derived by the improved method are more sensitive and selective in a number of cases where previous methods have failed to completely separate true members from false positives.
Identification of succinimide sites in proteins by N‐terminal sequence analysis after alkaline hydroxylamine cleavage
Authors: Kwong May Y. , Harris Reed J. 
Published: July 2010

AbstractUnder favorable conditions, Asp or Asn residues can undergo rearrangement to a succinimide (cyclic imide), which may also serve as an intermediate for deamidation and/or isoaspartate formation. Direct identification of such succinimides by peptide mapping is hampered by their lability at neutral and alkaline pH. We determined that incubation in 2 M hydroxylamine, 0.2 M Tris buffer, pH 9, for 2 h at 45 °C will specifically cleave on the C‐terminal side of succinimides without cleavage at Asn‐Gly bonds; yields are typically ∼ 50%. N‐terminal sequence analysis can then be used to identify an internal sequence generated by cleavage of the succinimide, hence identifying the succinimide site.
Structure and dynamics of the active site gorge of acetylcholinesterase: Synergistic use of molecular dynamics simulation and X‐ray crystallography
Authors: Axelsen Paul H. , Harel Michal , Silman Israel , Sussman Joel L. 
Published: July 2010

AbstractThe active site of acetylcholinesterase (AChE) fromTorpedo californicais located 20 Å from the enzyme surface at the bottom of a narrow gorge. To understand the role of this gorge in the function of AChE, we have studied simulations of its molecular dynamics. When simulations were conducted with pure water filling the gorge, residues in the vicinity of the active site deviated quickly and markedly from the crystal structure. Further study of the original crystallographic data suggests that a bis‐quaternary decamethonium (DECA) ion, acquired during enzyme purification, resides in the gorge. There is additional electron density within the gorge that may represent small bound cations. When DECA and 2 cations are placed within the gorge, the simulation and the crystal structure are dramatically reconciled. The small cations, more so than DECA, appear to stabilize part of the gorge wall through electrostatic interactions. This part of the gorge wall is relatively thin and may regulate substrate, product, and water movement through the active site.
Interaction of semisynthetic variants of RNase A with ribonuclease inhibitor
Authors: Neumann Ulf , Hofsteenge Jan 
Published: July 2010

AbstractDerivatives of ribonuclease A (RNase A) with modifications in positions 1 and/or 7 were prepared by subtilisincatalyzed semisynthesis starting from synthetic RNase 1‐20 peptides and S‐protein (RNase 21‐124). The lysyl residue at position 1 was replaced by alanine, whereas Lys‐7 was replaced by cysteine that was specifically modified prior to semisynthesis. The enzymes obtained were characterized by protein chemical methods and were active toward uridylyl‐3′,5′‐adenosine and yeast RNA. When Lys‐7 was replaced by S‐methyl‐cysteine or S‐carboxamido‐methyl‐cysteine, binding of recombinant ribonuclease inhibitor (RI) from porcine liver was strongly affected. In contrast, the catalytic properties were only slightly altered. The dissociation constant for the RNase A‐RI complex increased from 74 fM (RNase A) to 4.5 pM (Lys‐1, Cys‐7‐methyl RNase), corresponding to a decrease in binding energy of 10 kJ mol−1. Modifications that introduced a positive charge in position 7 (S‐aminoethyl‐ or S‐ethylpyridyl‐cysteine) led to much smaller losses. The replacement of Lys‐1 resulted in a 4‐kJ mol−1 loss in binding energy. S‐protein bound to R1 with K1 = 63.4 pM, 800‐fold weaker than RNase A. This corresponded to a 16‐kJ mol−1 difference in binding energy. The results show that the N‐terminal portion of RNase A contributes significantly to binding of ribonuclease inhibitor and that ionic interactions of Lys‐7 and to a smaller extent of Lys‐1 provide most of the binding energy.
The primary structure and properties of thioltransferase (glutaredoxin) from human red blood cells
Authors: Papov Vladimir V. , Gravina Stephen A. , Mieyal John J. , Biemann Klaus 
Published: July 2010

AbstractThioltransferase (glutaredoxin) was purified from human red blood cells essentially as described previously (Mieyal JJ et al., 1991a, Biochemistry 30:6088–6097). The primary sequence of the HPLC‐pure enzyme was determined by tandem mass spectrometry and found to represent a 105‐amino acid protein of molecular weight 11,688 Da. The physicochemical and catalytic properties of this enzyme are common to the group of proteins called glutaredoxins among the family of thiol: disulfide oxidoreductases that also includes thioredoxin and protein disulfide isomerase. Although this human red blood Cell glutaredoxin (hRBC Grx) is highly homologous to the 3 other mammalian Grx proteins whose sequences are known (calf thymus, rabbit bone marrow, and pig liver), there are a number of significant differences. Most notably an additional cysteine residue (Cys‐7) occurs near the N‐terminus of the human enzyme in place of a serine residue in the other proteins. In addition, residue 51 of hRBC Grx displayed a mixture of Asp and Asn. This result is consistent with isoelectric focusing analysis, which revealed 2 distinct bands for either the oxidized or reduced forms of the protein. Because the enzyme was prepared from blood combined from a number of individual donors, it is not clear whether this Asp/Asn ambiguity represents inter‐individual variation, gene duplication, or a deamidation artifact of purification.
Novel phosphotransferase system genes revealed by bacterial genome analysis: Unique, putative fructose‐ and glucoside‐specific systems
Authors: Reizer Jonathan , Michotey Valerie , Reizer Aiala , Saier Milton H. 
Published: July 2010

AbstractAnalyses of sequences made available through the Escherichia coli genome project in the 87.2‐89.2‐min and 81.5‐84.5‐min regions have revealed 2 putative operons encoding proteins of the phosphoenolpyruvate: sugar phosphotransferase system (PTS). The first putative operon, designated frv, includes 4 open reading frames (ORFs), ORFf147, ORFf485, ORFf356, and ORFf582. ORFf147 and ORFf485 comprise an Enzyme IIA‐Enzyme IIBC pair of the PTS. The sequence similarity of ORFf485 to previously characterized fructose‐specific Enzymes IIBC suggests that ORFf485 may be specific for fructose. ORFf 147 encodes a protein with comparable degrees of sequence similarity to fructose and mannitol‐specific Enzymes IIA as well as homologous proteins implicated in σ54‐dependent transcriptional regulation. Unique features of this system include a detached IIA protein and the absence of a IIB domain duplication. ORFf356 and ORFf582 are functionally unidentified and nonhomologous to other ORFs in the current protein databases, but ORFf582 contains 2 N‐terminal helix‐turn‐helix motifs, suggestive of a role in frv operon transcriptional regulation.The second putative operon, designated glv, includes 3 ORFs, ORFf455, ORFf 161, and ORFf212. We suggest that ORFf455 was incorrectly assigned and should be designated ORFf368. ORFf368 and ORFfl61 encode an Enzyme IIC and IIB pair of the PTS showing greatest sequence similarity to Enzymes II specific for sugars of the gluco configuration. ORFf212 encodes a protein with sequence similarity to a phospho‐β‐glucosidase and an α‐galactosidase. No putative transcriptional regulator of the glv operon was found. This operon is the first one encoding a putative PTS permease with detached Enzymes IIB and IIC and lacking an Enzyme IIA. It is suggested that both the frv and glv operons are cryptic in E. coli and that additional genes encoding novel PTS‐related proteins will be revealed by bacterial genome sequence analyses.
Multiple copy sampling in protein loop modeling: Computational efficiency and sensitivity to dihedral angle perturbations
Authors: Zheng Qiang , Rosenfeld Rakefet , Delisi Charles , Kyle Donald J. 
Published: July 2010

AbstractMultiple copy sampling and the bond scaling‐relaxation technique are combined to generate 3‐dimensional conformations of protein loop segments. The computational efficiency and sensitivity to initial loop copy dispersion are analyzed. The multicopy loop modeling method requires approximately 20‐50% of the computational time required by the single‐copy method for the various protein segments tested. An analytical formula is proposed to estimate the computational gain prior to carrying out a multicopy simulation. When 7‐residue loops within flexible proteins are modeled, each multicopy simulation can sample a set of loop conformations with initial dispersions up to ±15° for backbone and ±3° for side‐chain rotatable dihedral angles. The dispersions are larger for shorter and smaller for longer and/or surface loops. The degree of convergence of loop copies during a simulation can be used to complement commonly used target functions (such as potential energy) for distinguishing between native and misfolded conformations. Furthermore, this convergence also reflects the conformational flexibility of the modeled protein segment. Application to simultaneously building all 6 hypervariable loops of an antibody is discussed.
Protein folding dynamics: The diffusion‐collision model and experimental data
Authors: Karplus Martin , Weaver David L. 
Published: July 2010

AbstractThe diffusion‐collision model of protein folding is assessed. A description is given of the qualitative aspects and quantitative results of the diffusion‐collision model and their relation to available experimental data. We consider alternative mechanisms for folding and point out their relationship to the diffusion‐collision model. We show that the diffusion‐collision model is supported by a growing body of experimental and theoretical evidence, and we outline future directions for developing the model and its applications.