bluuebunny/crossref_metadata_2025_split huggingface dataset:
Production of soluble αβ T‐cell receptor heterodimers suitable for biophysical analysis of ligand binding
Authors: Willcoj Benjamin E. , Gao George F. , Wyer Jessica R. , O'callaghan Christopher A. , Jakobsen Bent K. , Boulter Jonathan M. , Bell John I. , Jones E. Yvonne , Van Der Merwe P. Anton 
Published: February 2009

AbstractA method to produce αβ T‐cell receptors (TCRs) in a soluble form suitable for biophysical analysis was devised involving in vitro refolding of a TCR fusion protein. Polypeptides corresponding to the variable and constant domains of each chain of a human and a murine receptor, fused to a coiled coil heterodimerization motif from either c‐Jun (alpha) or v‐Fos (beta), were overexpressed separately in Escherichia coli. Following recovery from inclusion bodies, the two chains of each receptor were denatured, and then refolded together in the presence of denaturants. For the human receptor, which is specific for the immunodominant influenza A HLA‐A2‐restricted matrix epitope (M58‐66), a heterodimeric protein was purified in milligram yields and found to be homogeneous, monomeric, antibody‐reactive, and stable at concentrations lower than 1 μM. Using similar procedures, analogous results were obtained with a murine receptor specific for an influenza nucleoprotein epitope (366‐374) restricted by H2‐Db. Production of these receptors has facilitated a detailed analysis of viral peptide‐Major Histocompatibility Complex (peptide‐MHC) engagement by the TCR using both surface plasmon resonance (SPR) and, in the case of the human TCR, isothermal titration calorimetry (ITC) (Willcox et al., 1999). The recombinant methods described should enable a wide range of TCR‐peptide‐MHC interactions to be studied and may also have implications for the production of other heterodimeric receptor molecules.
Using genomic information to investigate the function of ThiI, an enzyme shared between thiamin and 4‐thiouridine biosynthesis
Authors: Mueller Eugene G. , Palenchar Peter M. 
Published: February 2009

AbstractThe gene thiI encodes a protein (ThiI) that plays a role in the transfer of sulfur from cysteine to both thiamin and 4‐thiouridine, but the reaction catalyzed by ThiI remains undetermined. Based upon sequence alignments, ThiI shares a unique “P‐loop” motif with the PPi synthetase family, four enzymes that catalyze adenylation and subsequent substitution of carbonyl oxygens. To test whether or not this motif is critical for ThiI function, the Asp in the motif was converted to Ala (D189A), and a screen for in vivo 4‐thiouridine production revealed the altered enzyme to be inactive. Further scrutiny of sequence data and the crystal structures of two members of the PPi synthetase family implicated Lys321 in the proposed adenylation function of ThiI, and the critical nature of Lys321 has been demonstrated by site‐directed mutagenesis and genetic screening. Our results, then, indicate that ThiI catalyzes the adenylation of a substrate at the expense of ATP, a narrowing of possible reactions that provides a strong new basis for deducing the early steps in the transfer of sulfur from cysteine to both thiamin and 4‐thiouridine.
Thermodynamics of replacing an α‐helical Pro residue in the P40S mutant of Escherichia coli thioredoxin
Authors: Chakrabarti Atis , Srivastava Sarika , Swaminathan Chittoor P. , Surolia Avadhesha , Varadarajan Raghavan 
Published: February 2009

AbstractEscherichia coli thioredoxin is a 108 amino acid oxidoreductase and contains a single Met residue at position 37. The protein contains a long α‐helical stretch between residues 32 and 49. The central residue of this helix, Pro40, has been replaced by Sen The stabilities of the oxidized states of two proteins, the single mutant M37L and the double mutant M37L, P40S, have been characterized by differential scanning calorimetry (DSC) and also by a series of isothermal guanidine hydrochloride (GuHCl) melts in the temperature range of 277 to 333 K. The P40S mutation was found to stabilize the protein at all temperatures upto 340 K though both proteins had similar Tm values of about 356 K. At 298 K, the M37L, P40S mutant was found to be more stable than M37L by 1.5 kcal/mol. A combined analysis of GuHCl and calorimetric data was carried out to determine the enthalpy, entropy, and heat capacity change upon unfolding. At 298 K there was a large, stabilizing enthalpic effect in P40S though significant enthalpy‐entropy compensation was observed and the two proteins had similar values of ΔCp. Thus, replacement of a Pro in the interior of an α helix can have substantial effects on protein stability.
Structures of yeast vesicle trafficking proteins
Authors: Tishgarten Tomer , Yin Fang Fang , Grant Tracy R. , Lipscomb Leigh Ann , Faucher Keith M. , Dluhy Richard A. , Stevens Tom H. , Mollard Gabriele Fischer Von 
Published: February 2009

AbstractIn protein transport between organelles, interactions of v‐ and t‐SNARE proteins are required for fusion of proteincontaining vesicles with appropriate target compartments. Mammalian SNARE proteins have been observed to interact with NSF and SNAP, and yeast SNAREs with yeast homologues of NSF and SNAP proteins. This observation led to the hypothesis that, despite low sequence homology, SNARE proteins are structurally similar among eukaryotes. SNARE proteins can be classified into two groups depending on whether they interact with SNARE binding partners via conserved glutamine (Q‐SNAREs) or arginine (R‐SNAREs). Much of the published structural data available is for SNAREs involved in exocytosis (either in yeast or synaptic vesicles). This paper describes circular dichroism, Fourier transform infrared spectroscopy, and dynamic light scattering data for a set of yeast v‐ and t‐SNARE proteins, Vti 1p and Pep12p, that are Q‐SNAREs involved in intracellular trafficking. Our results suggest that the secondary structure of Vti1p is highly α‐helical and that Vti 1p forms multimers under a variety of solution conditions. In these respects, Vti1p appears to be distinct from R‐SNARE proteins characterized previously. The α‐helicity of Vti1p is similar to that of Q‐SNARE proteins characterized previously. Pep12p, a Q‐SNARE, is highly α‐helical. It is distinct from other Q‐SNAREs in that it forms dimers under many of the solution conditions tested in our experiments. The results presented in this paper are among the first to suggest heterogeneity in the functioning of SNARE complexes.
A common mechanism for recombinant human NGF, BDNF, NT‐3, and murine NGF slow unfolding
Authors: De Young Linda R. , Schmelzer Charles H. , Burton Louis E. 
Published: February 2009

AbstractThe recombinant human nerve growth factor (hNGF), brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3), neurotrophin 4/5 (NT4/5), and murine NGF (mNGF) dimers all undergo rapid unfolding and dissociation to monomer in GdnHCl. Fluorescence spectroscopy, reversed‐phase high‐performance liquid chromatography, and sizeexclusion chromatography were used to show that this monomer M1 converts slowly to a more fully unfolded monomer, M2, by a first order process with half‐lives of 22, 2.5, 1.6, and 0.73 h for hNGF, mNGF, NT‐3, and BDNF, respectively, at 25 °C. Linear Arrhenius plots for the conversion of M1 to M2 yielded activation energies of 27, 22, 24, and 24 kcal/mol for hNGF, mNGF, NT‐3, and BDNF, respectively. The refolding of these neurotrophins from 5 M GdnHCl was also first order with NT‐3 the slowest to refold and BDNF the fastest. Threading of the N‐terminus out through the cystine‐knot loop present in each of these proteins is proposed as the slow step in unfolding. The number of amino acids in the cystine‐knot loop (14 for hNGF, mNGF, NT‐3, and BDNF; 21 for NT4/5), and the number and position of the proline residues in this loop (2 for hNGF; 1 for mNGF, NT‐3, BDNF, and NT4/5) correlate with the relative rates of unfolding. The smaller the loop and the greater the number of prolines, the more hindered and slower the unfolding.
The effects of alpha‐helix on the stability of Asn residues: Deamidation rates in peptides of varying helicity
Authors: Kosky Andrew A. , Razzaq Ursula O. , Treuheit Michael J. , Brems David N. 
Published: February 2009

AbstractAsn deamidation was monitored in Ala‐based octadecapeptides of varying α‐helicity. Gly was substituted for Ala residues at positions 6 and 16 to create a peptide with less helicity. Ala → Gly substitutions were made at three or more residues from the Asn to negate known primary sequence effects on deamidation rates. The extent of helicity and rate of Asn deamidation for alkaline aqueous solutions of each peptide was measured as a function of temperature by circular dichroism and reversed‐phase high‐performance liquid chromatography, respectively. The rate of deamidation in the peptides was inversely proportional to the extent of α‐helicity. The results support the conclusion that Asn deamidation only occurs in the nonhelical population of conformers.
Molecular basis for triclosan activity involves a flipping loop in the active site
Authors: Qiu Xiayang , Abdel‐Meguid Sherin S. , Janson Cheryl A. , Court Robert I. , Smyth Martin G. , Payne David J. 
Published: February 2009

AbstractThe crystal structure of the Escherichia coli enoyl reductase‐NAD +‐triclosan complex has been determined at 2.5 Å resolution. The Ile192‐Ser198 loop is either disordered or in an open conformation in the previously reported structures of the enzyme. This loop adopts a closed conformation in our structure, forming van der Waals interactions with the inhibitor and hydrogen bonds with the bound NAD+ cofactor. The opening and closing of this flipping loop is likely an important factor in substrate or ligand recognition. The closed conformation of the loop appears to be a critical feature for the enhanced binding potency of triclosan, and a key component in future structure‐based inhibitor design.
Thermolysin and mitochondrial processing peptidase: How far structure—functional convergence goes
Authors: Grishin Nick V. , Makarova Kira S. 
Published: February 2009

AbstractThe structure—functional convergence between two Zndependent proteases, namely thermolysin and mitochondrial processing peptidase (MPP), is described. These two families of nonhomologous enzymes show not only functional convergence of several active site residues as in chymotrypsin and subtilisin, but also structural convergence of overall molecular architectures including the β‐sheet arrangement and packing of the surrounding α‐helices. The major functionally important structural elements are present in both enzymes with different topological connections and often in reverse main‐chain orientation, but display similar packing. The structural comparison helps to rationalize sequence “inversion” of the HEXXH thermolysin consensus present as HXXEH in MPP. The described structural convergence may be due to a limited number of alternatives to build a Zn‐protease that utilizes hydrogen bonding between a substrate main chain and the enzyme β‐sheet for substrate binding.
Changing the surface of a virus shell fusion of an enzyme to polyoma VP1
Authors: Gleiter Stefan , Stubenrauch Kay , Lilie Hauke 
Published: February 2009

AbstractRecent developments on virus‐like particles have demonstrated their potential in transfecting eucaryotic cells. In the case of particles based on the major coat protein VP1 of polyoma virus, transfection occurs via binding of VP1 to sialic acids. Since sialic acid is present on almost every eucaryotic cell line, this results in an unspecific cell targeting. Generation of a cell‐type specificity of this system would imply the presentation of a new function on the surface of VP1. To analyze whether a new functional protein can be placed on VP1, we inserted dihydrofolate reductase from Escherichia coli as a model protein. The effect of such an insertion on both VP1 and the inserted protein was investigated, respectively. The function of VP1, like the formation of pentameric capsomers and its ability to assemble into capsids, was not influenced by the insertion. The inserted dihydrofolate reductase showed major changes when compared to the wild‐type form. The thermal stability of the enzyme was dramatically reduced in the fusion protein; nevertheless, the dihydrofolate reductase proved to be a fully active enzyme with only slightly increased KM values for its substrates. This model system provides the basis for further modifications of the VP1 protein to achieve an altered surface of VP1 with new properties.
Structure of the human glycogen‐associated protein phosphatase 1 regulatory subunit hGM: Homology modeling revealed an (α/β)8‐barrel‐like fold in the multidomain protein
Authors: Souchet Michel , Legave Marie‐Noelle , Bril Antoine , Berrebi‐Bertrand Isabelle , Jullian Nathalie , Bertrand Hugues‐Olivier 
Published: February 2009

AbstractProtein phosphatase 1 (PP1) is widely distributed among tissues and species and acts as a regulator of many important cellular processes. By targeting the catalytic part of PP1 (PP1C) toward particular loci and substrates, regulatory subunits constitute key elements conferring specificity to the holoenzyme. Here, we report the identification of an (α/β)8‐barrel‐like structure within the N‐ter stretch of the human PP1 regulatory subunit hGM, which is part of the family of diverse proteins associated with glycogen metabolism. Protein homology modeling gave rise to a three‐dimensional (3D) model for the 381 N‐ter residue stretch of hGM, based on sequence similarity with Streptomyces olivochromogenes xylose isomerase, identified by using FASTA. The alignment was subsequently extended by using hydrophobic cluster analysis. The homology‐derived model includes the putative glycogen binding area located within the 142–230 domain of hGM as well as a structural characterization of the PP1C interacting domain (segment 51–67). Refinement of the latter by molecular dynamics afforded a topology that is in agreement with previous X‐ray studies (Egloff et al., 1997). Finite difference Poisson‐Boltzmann calculations performed on the interacting domains of PP1C and hGM confirm the complementarity of the local electrostatic potentials of the two partners. This work highlights the presence of a conserved fold among distant species (mammalian, Caenorhabditis elegans, yeast) and, thus, emphasizes the involvement of PP1 in crucial basic cellular functions.
The structures of the neurotrophin 4 homodimer and the brain‐derived neurotrophic factor/neurotrophin 4 heterodimer reveal a common Trk‐binding site
Authors: Robinson Robert C. , Choe Senyon , Radziejewski Czeslaw , Spraggon Glen , Greenwald Jason , Kostura Mikey R. , Burtnick Leslie D. , Stuart David I. , Jones E. Yvonne 
Published: February 2009

AbstractThe neurotrophins are growth factors that are involved in the development and survival of neurons. Neurotrophin release by a target tissue results in neuron growth along the neurotrophin concentration gradient, culminating in the eventual innervation of the target tissue. These activities are mediated through trk cell surface receptors. We have determined the structures of the heterodimer formed between brain‐derived neurotrophic factor (BDNF) and neurotrophin 4 (NT4), as well as the structure of homodimer of NT4. We also present the structure of the Neurotrophin 3 homodimer, which is refined to higher resolution than previously published. These structures provide the first views of the architecture of the NT4 protomer. Comparison of the surface of a model of the BDNF homodimer with the structures of the neurotrophin homodimers reveals common features that may be important in the binding between the neurotrophins and their receptors. In particular, there exists an analogous region on the surface of each neurotrophin that is likely to be involved in trk receptor binding. Variations in sequence on the periphery of this common region serve to confer trk receptor specificity.
α1‐Microglobulin chromophores are located to three lysine residues semiburied in the lipocalin pocket and associated with a novel lipophilic compound
Authors: Berggård Tord , Lindqvist Annika , Cedervall Tommy , Åkerström BO , Thøgersen Ida B. , Enghild Jan J. , Cohen Arieh , Persson Per , Jönsson Jan‐Åke , Silow Maria 
Published: February 2009

Abstractα1‐Microglobulin (α1m) is an electrophoretically heterogeneous plasma protein. It belongs to the lipocalin superfamily, a group of proteins with a three‐dimensional (3D) structure that forms an internal hydrophobic ligand‐binding pocket, α1m carries a covalently linked unidentified chromophore that gives the protein a characteristic brown color and extremely heterogeneous optical properties. Twenty‐one different colored tryptic peptides corresponding to residues 88–94, 118–121, and 122–134 of human α1m were purified. In these peptides, the side chains of Lys92, Lys118, and Lys130 carried size heterogeneous, covalently attached, unidentified chromophores with molecular masses between 122 and 282 atomic mass units (amu). In addition, a previously unknown uncolored lipophilic 282 amu compound was found strongly, but noncovalently associated with the colored peptides. Uncolored tryptic peptides containing the same Lys residues were also purified. These peptides did not carry any additional mass (i.e., chromophore) suggesting that only a fraction of the Lys92, Lys118, and Lys130 are modified. The results can explain the size, charge, and optical heterogeneity of α1m. A 3D model of α1m, based on the structure of rat epididymal retinoic acid‐binding protein (ERABP), suggests that Lys92, Lys118, and Lys130 are semiburied near the entrance of the lipocalin pocket. This was supported by the fluorescence spectra of α1m under native and denatured conditions, which indicated that the chromophores are buried, or semiburied, in the interior of the protein. In human plasma, approximately 50% of α1m is complex bound to IgA. Only the free α1m carried colored groups, whereas α1m linked to IgA was uncolored.
Three‐dimensional solution structure of mouse [Cd7]‐metallothionein‐l by homonuclear and heteronuclear NMR spectroscopy
Authors: Zangger Klaus , ÖZ GÜLIN , Armitage Ian M. , Otvos James D. 
Published: February 2009

AbstractSequential 1H‐NMR assignments of mouse [Cd7]‐metallothionein‐l (MT1) have been carried out by standard homonuclear NMR methods and the use of an accordion‐heteronuclear multiple quantum correlation (HMQC) experiment for establishing the metal, 113Cd2+, to cysteine connectivities. The three‐dimensional structure was then calculated using the distance constraints from two‐dimensional nuclear Overhauser effect (NOE) spectroscopy spectra and the Cys‐Cd connectivities as input for a distance geometry‐dynamical simulated annealing protocol in X‐PLOR 3.851. Similar to the mammalian MT2 isoforms, the homologous primary structure of MT1 suggested two separate domains, each containing one metal cluster. Because there were no interdomain constraints, the structure calculation for the N‐terminal β‐ and the C‐terminal α‐domain were carried out separately. The structures are based on 409 NMR constraints, consisting of 381 NOEs and 28 cysteine‐metal connectivities. The only elements of regular secondary structure found were two short stretches of 310 helices along with some half‐turns in the α‐domain. Structural comparison with rat liver MT2 showed high similarity, with the β‐domain structure in mouse MT1 showing evidence of increased flexibility compared to the same domain in MT2. The latter was reflected by the presence of fewer interresidue NOEs, no slowly exchanging backbone amide protons, and enhanced cadmium‐cadmium exchange rates found in the β‐domain of MT1.
Methionine‐141 directly influences the binding of 4‐methylpyrazole in human σσ alcohol dehydrogenase
Authors: Xie Peiguang T. , Hurley Thomas D. 
Published: February 2009

AbstractPyrazole and its 4‐alkyl substituted derivatives are potent inhibitors for many alcohol dehydrogenases. However, the human σσ isoenzyme exhibits a 580‐fold lower affinity for 4‐methylpyrazole than does the human β1β1 isoenzyme, with which it shares 69% sequence identity. In this study, structural and kinetic studies were utilized in an effort to identify key structural features that affect the binding of 4‐methylpyrazole in human alcohol dehydrogenase isoenzymes. We have extended the resolution of the human σσ alcohol dehydrogenase (ADH) isoenzyme to 2.5 Å resolution. Comparison of this structure to the human β1β1 isoenzyme structure indicated that the side‐chain position for Met141 in σσ ADH might interfere with 4‐methylpyrazole binding. Mutation of Met141 in σσ ADH to Leu (σσ141L) lowers the Ki for 4‐methylpyrazole from 350 to 10 μM, while having a much smaller effect on the Ki for pyrazole. Thus, the mutagenesis results show that the residue at position 141, which lines the substrate‐binding pocket at a position close to the methyl group of 4‐methylpyrazole, directly affects the binding of the inhibitor. To rule out nonspecific structural changes due to the mutation, the X‐ray structure of the σ141L mutant enzyme was determined to 2.4 Å resolution. The three‐dimensional structure of the mutant enzyme is identical to the wild‐type enzyme, with the exception of the residue at position 141. Thus, the differences in 4‐methylpyrazole binding between the mutant and wild‐type σσ ADH isoenzymes can be completely ascribed to the local changes in the topology of the substrate binding site, and provides an explanation for the class‐specific differences in 4‐methylpyrazole binding to the human ADH isoenzymes.
Thermal stability of hydrophobic heme pocket variants of oxidized cytochrome c
Authors: Liggins John R. , Nall Barry T. , Lo Terence P. , Brayer Gary D. 
Published: February 2009

AbstractMicrocalorimetry has been used to measure the stabilities of mutational variants of yeast iso‐1 cytochrome c in which F82 and L85 have been replaced by other hydrophobic amino acids. Specifically, F82 has been replaced by Y and L85 by A. The double mutant F82Y, L85A iso‐1 has also been studied, and the mutational perturbations are compared to those for the two single mutants, F82Y iso‐1 and L85A iso‐1. Results are interpreted in terms of known crystallographic structures. The data show that (1) the destabilization of the mutant proteins is similar in magnitude to that which is theoretically predicted by the more obvious mutation‐induced structural effects; (2) the free energy of destabilization of the double mutant, F82Y, L85A iso‐1, is less than the sum of those of the two single mutants, almost certainly because, in the double mutant, the ‐OH group of Y82 is able to protrude into the cavity formed by the L85A substitution. The more favorable structural accommodation of the new ‐OH group in the double mutant leads to additional stability through (1) further decreases in the volumes of internal cavities and (2) formation of an extra protein‐protein hydrogen bond.
The mechanism of aconitase: 1.8 Å resolution crystal structure of the S642A:citrate complex
Authors: Lloyd S. J. , Lauble H. , Prasad G. S. , Stout C. D. 
Published: February 2009

AbstractThe crystal structure of the S642A mutant of mitochondrial aconitase (mAc) with citrate bound has been determined at 1.8 Å resolution and 100 K to capture this binding mode of substrates to the native enzyme. The 2.0 A resolution, 100 K crystal structure of the S642A mutant with isocitrate binding provides a control, showing that the Ser → Ala replacement does not alter the binding of substrates in the active site. The aconitase mechanism requires that the intermediate product, cis‐aconitate, flip over by 180° about the Cα‐Cβ double bond. Only one of these two alternative modes of binding, that of the isocitrate mode, has been previously visualized. Now, however, the structure revealing the citrate mode of binding provides direct support for the proposed enzyme mechanism.