bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use of electrospray ionization mass spectrometry to study binding interactions between a replication terminator protein and DNA
Authors: Kapur Amit , Beck Jennifer L. , Brown Susan E. , Dixon Nicholas E. , Sheil Margaret M. 
Published: October 2010

AbstractTus protein binds tightly to specific DNA sequences (Ter) on the Escherichia coli chromosome halting replication. We report here conditions for detecting the 1 : 1 Tus–Ter complex by electrospray ionization mass spectrometry (ESI‐MS). ESI mass spectra of a mixture of Tus and nonspecific DNA showed ions predominantly from uncomplexed Tus protein, indicating that the Tus–Ter complex observed in the gas phase was the result of a specific interaction rather than nonspecific associations in the ionization source. The Tus–Ter complex was very stable using a spray solvent of 10 mM ammonium acetate at pH 8.0, and initial attempts to distinguish binding affinities of Tus and mutant Tus proteins for Ter DNA were unsuccessful. Increasing the ammonium acetate concentration in the electrospray solvent (800 mM at pH 8.0) increased the dissociation constants sufficiently such that relative orders of binding affinity for Tus and various mutant Tus proteins for various DNA sequences could be determined. These were in agreement with the dissociation constants determined in solution studies. A dissociation constant of 700 × 10−9 M for the binding of the mutant Tus protein A173T (where residue 173 is changed from alanine to threonine) to Ter DNA was estimated, compared with a value of ≤2 × 10−9 M for Tus where A173 was unchanged. This is the first example in which ESI‐MS has been used to compare binding affinities of a DNA‐binding protein with mutant proteins for specific DNA recognition sequences. It was also possible to estimate the strength of the interaction between Tus and a DNA sequence (TerH) that had been identified by database searching.
Hemoglobin Porto Alegre forms a tetramer of tetramers superstructure
Authors: Baudin‐Creuza Véronique , Fablet Christophe , Zal Franck , Green Brian N. , Promé Danielle , Marden Michael C. , Pagnier Josée , Wajcman Henri 
Published: October 2010

AbstractThe effects of the mutation β9(A6)Ser → Cys on the interactions between the human hemoglobin molecules were investigated, and comparisons were made with other variants having an additional cysteine residue. In hemoglobin Porto Alegre (PA), the β9 mutation induces polymerization by forming interchain disulfide bonds via the extra cysteine. The hemolysate from a heterozygote was separated by gel filtration into a tetrameric fraction and a higher‐molecular‐weight oligomeric fraction (30%). Reversed‐phase high‐performance liquid chromatography and electrospray ionization mass spectrometry (ESI‐MS) under denaturing conditions showed that the tetrameric fraction contained only normal α‐ and β‐chains, whereas the oligomeric fraction contained only normal α‐chain and disulfide‐linked βPA dimer. Under native conditions, ESI‐MS of the oligomeric fraction revealed a principal complex of mass 258,400 Da corresponding to a tetramer of tetramers, and 10% of minor components. Transmission electron microscopy corroborated this structure by showing four spheres of 140 Å diameter surrounding a central cavity. Equilibrium experiments on the oligomer at different concentrations, using gel filtration and dimer exchange experiments with metHbA‐CN, showed that the tetramer of tetramers dissociates into smaller species, probably by breaking the dimer–dimer allosteric interface. None of the other variants investigated formed such a large oligomer.
Identification of Tyr438 as the major in vitro c‐Src phosphorylation site in human gelsolin: A mass spectrometric approach
Authors: Corte Veerle De , Demol Hans , Goethals Mark , Damme Jozef Van , Gettemans Jan , Vandekerckhove Joël 
Published: October 2010

AbstractGelsolin is an actin‐binding protein (82 kDa) consisting of six repeated segments (S1—S6), each approximately 120 residues long. It interacts with phospholipids and we previously showed that phosphatidylinositol 4,5‐bisphosphate promotes phosphorylation of gelsolin by the tyrosine kinase c‐Src. We used a combination of different methods, such as thin‐layer chromatography and anti‐phosphotyrosine‐agarose immunoprecipitation of phosphopeptides combined with matrix assisted laser desorption ionization‐mass spectrometry (MALDI‐MS) and post source decay (PSD) analysis, to identify the phosphorylation sites in gelsolin. The major phosphorylation site (Tyr438) was located in subdomain 4 (S4). Phosphorylation of gelsolin in the gelsolin‐actin2 complex was inhibited by 90%. Gelsolin phosphorylation by c‐Src in the presence of lysophosphatidic acid also revealed Tyr438 as the most prominent site. Additional minor sites were found using the anti‐phosphotyrosine bead immunoprecipitation method followed by MALDI‐MS and PSD analysis. These sites, representing ∼5% of the total phosphate incorporation, were identified as Tyr59, Tyr382, Tyr576, and Tyr624. Based on these results we generated antibodies which specifically recognize Tyr438 phosphorylated gelsolin.
Comparison of anionic and cationic trypsinogens: The anionic activation domain is more flexible in solution and differs in its mode of BPTI binding in the crystal structure
Authors: Pasternak Annette , Ringe Dagmar , Hedstrom Lizbeth 
Published: October 2010

AbstractUnlike bovine cationic trypsin, rat anionic trypsin retains activity at high pH. This alkaline stability has been attributed to stabilization of the salt bridge between the N‐terminal Ile16 and Asp194 by the surface negative charge (Soman K, Yang A‐S, Honig B, Fletterick R., 1989, Biochemistry 28:9918—9926). The formation of this salt bridge controls the conformation of the activation domain in trypsin. In this work we probe the structure of rat trypsinogen to determine the effects of the surface negative charge on the activation domain in the absence of the Ile16—Asp194 salt bridge. We determined the crystal structures of the rat trypsin‐BPTI complex and the rat trypsinogen‐BPTI complex at 1.8 and 2.2 å, respectively. The BPTI complex of rat trypsinogen resembles that of rat trypsin. Surprisingly, the side chain of Ile16 is found in a similar position in both the rat trypsin and trypsinogen complexes, although it is not the N‐terminal residue and cannot form the salt bridge in trypsinogen. The resulting position of the activation peptide alters the conformation of the adjacent autolysis loop (residues 142—153). While bovine trypsinogen and trypsin have similar CD spectra, the CD spectrum of rat trypsinogen has only 60% of the intensity of rat trypsin. This lower intensity most likely results from increased flexibility around two conserved tryptophans, which are adjacent to the activation domain. The NMR spectrum of rat trypsinogen contains high field methyl signals as observed in bovine trypsinogen. It is concluded that the activation domain of rat trypsinogen is more flexible than that of bovine trypsinogen, but does not extend further into the protein core.
On the role of the cis‐proline residue in the active site of DsbA
Authors: Charbonnier Jean‐Baptiste , Belin Pascal , Moutiez Mireille , Stura Enrico A. , Quéméneur Eric 
Published: October 2010

AbstractIn addition to the Cys‐Xaa‐Xaa‐Cys motif at position 30—33, DsbA, the essential catalyst for disulfide bond formation in the bacterial periplasm shares with other oxidoreductases of the thioredoxin family a cis‐proline in proximity of the active site residues. In the variant DsbAP151A, this residue has been changed to an alanine, an almost isosteric residue which is not disposed to adopt the cis conformation. The substitution strongly destabilized the structure of DsbA, as determined by the decrease in the free energy of folding. The pKa of the thiol of Cys30 was only marginally decreased. Although in vivo the variant appeared to be correctly oxidized, it exhibited an activity less than half that of the wild‐type enzyme with respect to the folding of alkaline phosphatase, used as a reporter of the disulfide bond formation in the periplasm. DsbAP151A crystallized in a different crystal form from the wild‐type protein, in space group P21 with six molecules in the asymmetric unit. Its X‐ray structure was determined to 2.8 Å resolution. The most significant conformational changes occurred at the active site. The loop 149—152 adopted a new backbone conformation with Ala151 in a trans conformation. This rearrangement resulted in the loss of van der Waals interactions between this loop and the disulfide bond. His32 from the Cys‐Xaa‐Xaa‐Cys sequence presented in four out of six molecules in the asymmetric unit a gauche—‐conformation not observed in the wild‐type protein. The X‐ray structure and folding studies on DsbAP151A were consistent with the cis‐proline playing a major role in the stabilization of the protein. A role for the positioning of the substrate is discussed. These important properties for the enzyme function might explain the conservation of this residue in DsbA and related proteins possessing the thioredoxin fold.
Crystal structure of human muscle aldolase complexed with fructose 1,6‐bisphosphate: Mechanistic implications
Authors: Dalby Andrew , Dauter Zbigniev , Littlechild Jennifer A. 
Published: October 2010

AbstractFructose 1,6‐bisphosphate aldolase catalyzes the reversible cleavage of fructose 1,6‐bisphosphate and fructose 1‐phosphate to dihydroxyacetone phosphate and either glyceraldehyde 3‐phosphate or glyceraldehyde, respectively. Catalysis involves the formation of a Schiff's base intermediate formed at the ϵ‐amino group of Lys229. The existing apo‐enzyme structure was refined using the crystallographic free‐R‐factor and maximum likelihood methods that have been shown to give improved structural results that are less subject to model bias. Crystals were also soaked with the natural substrate (fructose 1,6‐bisphosphate), and the crystal structure of this complex has been determined to 2.8 å. The apo structure differs from the previous Brookhaven‐deposited structure (1ald) in the flexible C‐terminal region. This is also the region where the native and complex structures exhibit differences. The conformational changes between native and complex structure are not large, but the observed complex does not involve the full formation of the Schiff's base intermediate, and suggests a preliminary hydrogen‐bonded Michaelis complex before the formation of the covalent complex.
Thermodynamic analysis of halide binding to haloalkane dehalogenase suggests the occurrence of large conformational changes
Authors: Krooshof Geja H. , Floris René , Tepper Armand W.J.W. , Janssen Dick B. 
Published: October 2010

AbstractHaloalkane dehalogenase (DhlA) hydrolyzes short‐chain haloalkanes to produce the corresponding alcohols and halide ions. Release of the halide ion from the active‐site cavity can proceed via a two‐step and a three‐step route, which both contain slow enzyme isomerization steps. Thermodynamic analysis of bromide binding and release showed that the slow unimolecular isomerization steps in the three‐step bromide export route have considerably larger transition state enthalpies and entropies than those in the other route. This suggests that the three‐step route involves different and perhaps larger conformational changes than the two‐step export route. We propose that the three‐step halide export route starts with conformational changes that result in a more open configuration of the active site from which the halide ion can readily escape. In addition, we suggest that the two‐step route for halide release involves the transfer of the halide ion from the halide‐binding site in the cavity to a binding site somewhere at the protein surface, where a so‐called collision complex is formed in which the halide ion is only weakly bound. No large structural rearrangements are necessary for this latter process.
Identification of a novel domain shared by putative components of the endocytic and cytoskeletal machinery
Authors: Kay Brian K. , Yamabhai Montarop , Wendland Beverly , Emr Scott D. 
Published: October 2010

AbstractWe have identified a ∼140 amino acid domain that is shared by a variety of proteins in budding and fission yeast, nematode, rat, mouse, frog, oat, and man. Typically, this domain is located within 20 residues of the N‐terminus of the various proteins. The percent identity among the domains in the 12 proteins ranges from 42 to 93%, with 16 absolutely conserved residues: N‐x11–13‐V‐x2‐A‐T‐x34–36‐R‐x7–8‐W‐R‐x3‐K‐x12‐G‐x‐E‐x15‐L‐x11–12‐D‐x‐G‐R‐x11‐D‐x7‐R. Even though these proteins share little beyond their segment of homology, data are emerging that several of the proteins are involved in endocytosis and or regulation of cytoskeletal organization. We have named this protein segment the ENTH domain, for Epsin N‐terminal Homology domain, and hypothesize that it is a candidate for binding specific ligands and/or enzymatic activity in the cell.
The solution structure of a superpotent b‐chain‐shortened single‐replacement insulin analogue
Authors: Kurapkat Günther , Siedentop Michael , Gattner Hans‐Gregor , Hagelstein Michael , Brandenburg Dietrich , Grötzinger Joachim , Wollmer Axel 
Published: October 2010

AbstractThis paper reports on an insulin analogue with 12.5‐fold receptor affinity, the highest increase observed for a single replacement, and on its solution structure, determined by NMR spectroscopy.The analogue is [D‐AlaB26]des‐(B27‐B30)‐tetrapeptide‐insulin‐B26‐amide. C‐terminal truncation of the B‐chain by four (or five) residues is known not to affect the functional properties of insulin, provided the new carboxylate charge is neutralized. As opposed to the dramatic increase in receptor affinity caused by the substitution of D‐Ala for the wild‐type residue TyrB26 in the truncated molecule, this very substitution reduces it to only 18% of that of the wild‐type hormone when the B‐chain is present in full length.The insulin molecule in solution is visualized as an ensemble of conformers interrelated by a dynamic equilibrium. The question is whether the “active” conformation of the hormone, sought after in innumerable structure/function studies, is or is not included in the accessible conformational space, so that it could be adopted also in the absence of the receptor. If there were any chance for the active conformation, or at least a predisposed state to be populated to a detectable extent, this chance should be best in the case of a superpotent analogue. This was the motivation for the determination of the three‐dimensional structure of [D‐AlaB26]des‐(B27‐B30)‐tetrapeptide‐insulin‐B26‐amide. However, neither the NMR data nor CD spectroscopic comparison of a number of related analogues provided a clue concerning structural features predisposing insulin to high receptor affinity. After the present study it seems more likely than before that insulin will adopt its active conformation only when exposed to the force field of the receptor surface.
Domain exchange experiments in duck d‐crystallins: Functional and evolutionary implications
Authors: Sampaleanu Liliana M. , Davidson Alan R. , Graham Caroline , Wistow Graeme J. , Howell P. Lynne 
Published: October 2010

Abstractδ‐Crystallin, the major soluble protein component of the avian and reptilian eye lens, is homologous to the urea cycle enzyme argininosuccinate lyase (ASL). In duck lenses there are two δ crystallins, denoted δ1 and δ2. Duck δ2 is both a major structural protein of the lens and also the duck orthologue of ASL, an example of gene recruitment. Although 94% identical to δ2/ASL in the amino acid sequence, δ1 is enzymatically inactive. A series of hybrid proteins have been constructed to assess the role of each structural domain in the enzymatic mechanism. Five chimeras—221, 122, 121, 211, and 112, where the three numbers correspond to the three structural domains and the value of 1 or 2 represents the protein of origin, δ1 or δ2, respectively—were constructed and thermodynamically and kinetically analyzed. The kinetic analysis indicates that only domain 1 is crucial for restoring ASL activity to d 1 crystallin, and that amino acid substitutions in domain 2 may play a role in substrate binding. These results confirm the hypothesis that only one domain, domain 1, is responsible for the loss of catalytic activity in d 1. The thermodynamic characterization of human ASL (hASL) and duck δ1 and δ2 indicate that δ crystallins are slightly less stable than hASL, with the δ1 being the least stable. The DGs of unfolding are 57.25, 63.13, and 70.71 kcal mol–1 for δ1, δ2, and hASL, respectively. This result was unexpected, and we speculate that δ crystallins have adapted to their structural role by adopting a slightly less stable conformation that might allow for enhanced protein‐protein and protein‐solvent interactions.
A survey of left‐handed polyproline II helices
Authors: Stapley Benjamin J. , Creamer Trevor P. 
Published: October 2010

AbstractLeft‐handed polyproline II helices (PPII) are contiguous elements of protein secondary structure in which the ø and Ψ angles of constituent residues are restricted to around –75° and 145°, respectively. They are important in structural proteins, in unfolded states and as ligands for signaling proteins. Here, we present a survey of 274 nonhomologous polypeptide chains from proteins of known structure for regions that form these structures. Such regions are rare, but the majority of proteins contain at least one PPII helix. Most PPII helices are shorter than five residues, although the longest found contained 12 amino acids. Proline predominates in PPII, but Gln and positively charged residues are also favored. The basis of Gln's prevalence is its ability to form an i, i + 1 side‐chain to main‐chain hydrogen bond with the backbone carbonyl oxygen of the proceeding residue; this helps to fix the Ψ angle of the Gln and the ø and Ψ of the proceeding residue in PPII conformations and explains why Gln is favored at the first position in a PPII helix. PPII helices are highly solvent exposed, which explains why apolar amino acids are disfavored despite preferring this region of ø/Ψ space when in isolation. PPII helices have perfect threefold rotational symmetry and within these structures we find significant correlation between the hydrophobicity of residues at i and i + 3; thus, PPII helices in globular proteins can be considered to be amphipathic.
The mechanism of sugar phosphate isomerization by glucosamine 6‐phosphate synthase
Authors: Teplyakov Alexei , Obmolova Galya , Badet‐Denisot Marie‐Ange , Badet Bernard 
Published: October 2010

AbstractGlucosamine 6‐phosphate synthase converts fructose‐6P into glucosamine‐6P or glucose‐6P depending on the presence or absence of glutamine. The isomerase activity is associated with a 40‐kDa C‐terminal domain, which has already been characterized crystallographically. Now the three‐dimensional structures of the complexes with the reaction product glucose‐6P and with the transition state analog 2‐amino‐2‐deoxyglucitol‐6P have been determined. Glucose‐6P binds in a cyclic form whereas 2‐amino‐2‐deoxyglucitol‐6P is in an extended conformation. The information on ligand‐protein interactions observed in the crystal structures together with the isotope exchange and site‐directed mutagenesis data allow us to propose a mechanism of the isomerase activity of glucosamine‐6P synthase. The sugar phosphate isomerization involves a ring opening step catalyzed by His504 and an enolization step with Glu488 catalyzing the hydrogen transfer from C1 to C2 of the substrate. The enediol intermediate is stabilized by a helix dipole and the ‐amino group of Lys603. Lys485 may play a role in deprotonating the hydroxyl O1 of the intermediate.
Covalent modification of lys19 in the ctp binding site of cytidine 5′‐monophosphate n‐acetylneuraminic acid synthetase
Authors: Tullius Michael V. , Vann Willie F. , Gibson Bradford W. 
Published: October 2010

AbstractPeriodate oxidized CTP (oCTP) was used to investigate the importance of lysine residues in the CTP binding site of the cytidine 5′‐monophosphate N‐acetylneuraminic acid (CMP‐NeuAc) synthetase (EC 2.7.7.43) from Haemophilus ducreyi. The reaction of oCTP with the enzyme follows pseudo‐first‐order saturation kinetics, giving a maximum rate of inactivation of 0.6 min–1 and a KI of 6.0 mM at pH 7.1. Mass spectrometric analysis of the modified enzyme provided data that was consistent with β‐elimination of triphosphate after the reaction of oCTP with the enzyme. A fully reduced enzyme‐oCTP conjugate, retaining the triphosphate moiety, was obtained by inclusion of NaBH3CN in the reaction solution. The β‐elimination product of oCTP reacted several times more rapidly with the enzyme compared to equivalent concentrations of oCTP This compound also formed a stable reduced morpholino adduct with CMP‐NeuAc synthetase when the reaction was conducted in the presence of NaBH3CN, and was found to be a useful lysine modifying reagent. The substrate CTP was capable of protecting the enzyme to a large degree from inactivation by oCTP and its β‐elimination product. Lys19, a residue conserved in CMP‐NeuAc synthetases, was identified as being labeled with the β‐elimination product of oCTP
Common structural features of mapeg—a widespread superfamily of membrane associated proteins with highly divergent functions in eicosanoid and glutathione metabolism
Authors: Jakobsson Per‐Johan , Morgenstern Ralf , Mancini Joseph , Ford‐Hutchinson Anthony , Persson Bengt 
Published: October 2010

AbstractA novel superfamily designated MAPEG (Membrane Associated Proteins in Eicosanoid and Glutathione metabolism), including members of widespread origin with diversified biological functions is defined according to enzymatic activities, sequence motifs, and structural properties. Two of the members are crucial for leukotriene biosynthesis, and three are cytoprotective exhibiting glutathione S‐transferase and peroxidase activities. Expression of the most recently recognized member is strongly induced by p53, and may therefore play a role in apoptosis or cancer development. In spite of the different biological functions, all six proteins demonstrate common structural characteristics typical of membrane proteins. In addition, homologues are identified in plants, fungi, and bacteria, demonstrating this superfamily to be generally occurring.
In vitro evolution of thermostable p53 variants
Authors: Matsumura Ichiro , Ellington Andrew D. 
Published: October 2010

AbstractThe tumor suppressor p53 is conformationally unstable at physiological temperature. Even the activated p53Δ30 variant, which lacks the self‐inhibiting carboxy terminal domain, has a half‐life of only 8 min at 37°C in vitro. We have developed a genetic approach to identify p53 variants that stabilize the active conformation. The human p53Δ30 gene was randomly mutated, and the resulting library was expressed in Escherichia coli under conditions that apparently denatured the parental protein. Stable p53 variants were identified based on their ability to specifically bind a p53 consensus site. The initial thermostable variants were randomly recombined by DNA shuffling, and substitutions that were functionally additive or synergistic were identified in a second more stringent round of screening. The DNA binding activity of N239Y/N268D/E336V p53Δ30 variant has a half‐life of 100 min at 37 °C, 12 times longer than that of the parental protein. The thermostable variants should be more amenable to crystallographic studies and more effective in gene therapies than the wild‐type protein.