bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cluster analysis of consensus water sites in thrombin and trypsin shows conservation between serine proteases and contributions to ligand specificity
Authors: Sanschagrin Paul C. , Kuhn Leslie A. 
Published: February 2009

AbstractCluster analysis is presented as a technique for analyzing the conservation and chemistry of water sites from independent protein structures, and applied to thrombin, trypsin, and bovine pancreatic trypsin inhibitor (BPTI) to locate shared water sites, as well as those contributing to specificity. When several protein structures are superimposed, complete linkage cluster analysis provides an objective technique for resolving the continuum of overlaps between water sites into a set of maximally dense microclusters of overlapping water molecules, and also avoids reliance on any one structure as a reference. Water sites were clustered for ten superimposed thrombin structures, three trypsin structures, and four BPTI structures. For thrombin, 19% of the 708 microclusters, representing unique water sites, contained water molecules from at least half of the structures, and 4% contained waters from all 10. For trypsin, 77% of the 106 microclusters contained water sites from at least half of the structures, and 57% contained waters from all three. Water site conservation correlated with several environmental features: highly conserved microclusters generally had more protein atom neighbors, were in a more hydrophilic environment, made more hydrogen bonds to the protein, and were less mobile. There were significant overlaps between thrombin and trypsin conserved water sites, which did not localize to their similar active sites, but were concentrated in buried regions including the solvent channel surrounding the Na+ site in thrombin, which is associated with ligand selectivity. Cluster analysis also identified water sites conserved in thrombin but not trypsin, and vice versa, providing a list of water sites that may contribute to ligand discrimination. Thus, in addition to facilitating the analysis of water sites from multiple structures, cluster analysis provides a useful tool for distinguishing between conserved features within a protein family and those conferring specificity.
Differences between the electronic environments of reduced and oxidized Escherichia coli DsbA inferred from heteronuclear magnetic resonance spectroscopy
Authors: Couprie Joel , Remerowski M. Lyndsay , Bailleul Arnaud , Courçon Marie , Gilles Nicolas , Quéméneur Eric , Jamin Nadege 
Published: February 2009

AbstractDsbA is the strongest protein disulfide oxidant yet known and is involved in catalyzing protein folding in the bacterial periplasm. Its strong oxidizing power has been attributed to the lowered pKa of its reactive active site cysteine and to the difference in thermodynamic stability between the oxidized and the reduced form. However, no structural data are available for the reduced state. Therefore, an NMR study of DsbA in its two redox states was undertaken. We report here the backbone 1HN, 15N, 13Cα, 13CO, 1Hα, and 13Cß NMR assignments for both oxidized and reduced Escherichia coli DsbA (189 residues). Ninety‐nine percent of the frequencies were assigned using a combination of triple (1H‐13C‐15N) and double resonance (1H‐15N or 1H‐13C) experiments. Secondary structures were established using the CSI (Chemical Shift Index) method, NOE connectivity patterns, 3JHNHα and amide proton exchange data. Comparison of chemical shifts for both forms reveals four regions of the protein, which undergo some changes in the electronic environment. These regions are around the active site (residues 26 to 43), around His60 and Pro151, and also around Gln97. Both the number and the amplitude of observed chemical shift variations are more substantial in DsbA than in E. coli thioredoxin. Large 13Cα chemical shift variations for residues of the active site and residues Phe28, Tyr34, Phe36, Ile42, Ser43, and Lys98 suggest that the backbone conformation of these residues is affected upon reduction.
Structure of the xylanase from Penicillium simplicissimum
Authors: Schmidt Andrea , Kratky Christoph , Schlacher Anton , Schwab Helmut , Steiner Walter 
Published: February 2009

AbstractDespite its relatively low pH and temperature optimum, the xylanase from Penicillium simplicissimum performs exceedingly well under conditions of paper bleaching. We have purified and characterized this enzyme, which belongs to family 10 of glycosyl hydrolases. Its gene was cloned, and the sequence of the protein was deduced from the nucleotide sequence. The xylanase was crystallized from ammonium sulfate at pH 8.4, and X‐ray data were collected at cryo‐temperature to a Crystallographic resolution of 1.75 Å. The crystal structure was solved by molecular replacement using the catalytic domain of the Clostridium thermocellum xylanase as a search model, and refined to a residual of R = 20% (Rfree = 23%) for data between 10 and 1.75 Å. The xylanase folds in an (α/β)8 barrel (TIM‐barrel), with additional helices and loops arranged at the “top” forming the active site cleft. In its overall shape, the P simplicissimum xylanase structure is similar to other family 10 xylanases, but its active site cleft is much shallower and wider. This probably accounts for the differences in catalysis and in the mode of action of this enzyme. Three glycerol molecules were observed to bind within the active site groove, one of which interacts directly with the catalytic glutamate residues. It appears that they occupy putative xylose binding subsites.
Tertiary structure‐dependence of misfolding substitutions in loops of the maltose‐binding protein
Authors: Raffy Sébastien , Sassoon Nathalie , Hofnung Maurice , Betton Jean‐Michel 
Published: February 2009

AbstractWe previously identified and characterized amino acid substitutions in a loop connecting helix I to strand B, the αI/βB loop, of the N‐domain that are critical for in vivo folding of the maltose‐binding protein (MalE31). The tertiary context‐dependence of this mutation in MalE folding was assessed by probing the tolerance of an equivalent αβ loop of the C‐domain to the same amino acid substitutions (MaIE219). Moving the loop mutation from the N‐ to the C‐domain eliminated the in vivo misfolding step that led to the formation of inclusion bodies. In vitro, both loop variants exhibited an important decrease of stability, but their intrinsic tendency to aggregate was well correlated with their periplasmic fates inEscherichia coli. Furthermore, the noncoincidence of the unfolding and refolding transition curves and increase of light scattering during the refolding of MalE31 indicate that a competing off‐pathway reaction could occurs on the folding pathway of this variant. These results strongly support the notion that the formation of super‐secondary structures of the N‐domain is a rate‐limiting step in the folding pathway of MalE.
Calcium‐binding and structural stability of echidna and canine milk lysozymes
Authors: Kikuchi Masayuki , Kawano Keiichi , Nitta Katsutoshi 
Published: February 2009

AbstractFor echidna and canine milk lysozymes, which were presumed to be the calcium‐binding lysozymes by their amino acid sequences, we have quantitated their calcium‐binding strength and examined their guanidine unfolding profiles.The calcium‐binding constants of echidna and canine lysozymes were determined to be 8.6 × 106 M−1 and 8.9 X 106 M−1 in 0.1 M KCl at pH 7.1 and 20 °C, respectively. The unfolding of decalcified canine lysozyme proceeds in the same manner as that of α‐lactalbumin, through a stable molten globule intermediate. However, neither calcium‐bound nor decalcified echidna lysozyme shows a stable molten globule intermediate. This unfolding profile of echidna lysozyme is identical to that of conventional lysozymes and pigeon egg‐white lysozyme, avian calcium‐binding lysozyme. This result supports the suggestion of Prager and Jolles (Prager EM, Jolles P. 1996. Animal lysozymes c and g: An overview. In: Jolles P, ed. Lysozymes: Model enzymes in biochemistry and biology. Basel‐Boston‐Berlin: Birkhauzer Verlag. pp 9‐31) that the lineage of avian and echidna calcium‐binding lysozymes and that of eutherian calcium‐binding lysozymes diverged separately from that of conventional lysozymes.
The functional importance of structural differences between the mannitol‐specific IIAmannitol and the regulatory IIAnitrogen
Authors: Van Montfort Rob L. M. , Dijkstra Bauke W. 
Published: February 2009

AbstractThe three‐dimensional structures of the IIA domain of the mannitol‐specific phosphoenol‐pyruvate dependent phos‐photransferase system (PTS) of Escherichia coli and the regulatory IIAntr enzyme have been compared. The enzymes are 20% identical in sequence and contain the sequence motif specific for IIA proteins belonging to the mannitol‐fructose family of the PTS. The fold of the enzymes is nearly identical, which confirms their evolution from a common ancestor. Moreover, the phosphorylation site of IIAmtl (His65) and one of the observed conformations of the active site Arg49 are extremely similar to their equivalents (His73 and Arg57) in IIAntr. In contrast, His l20, the equivalent of the second active site His111 of IIAmtl, is not located in the active site of IIAntr but points into the solvent on the other side of the molecule. The different position of His120 makes it unlikely that this residue assists in phosphoryl transfer in the regulatory IIAntrs. As His120 is conserved in all IIAntr enzymes, it could have an essential role in the recognition of the target protein of IIAntr.
Mapping the lifetimes of local opening events in a native state protein
Authors: Kragelund Birthe B. , Heinemann Bo , Knudsen Jens , Poulsen Flemming M. 
Published: February 2009

AbstractThe rate constants for the processes that lead to local opening and closing of the structures around hydrogen bonds in native proteins have been determined for most of the secondary structure hydrogen bonds in the four‐helix protein acyl coenzyme A binding protein. In an analysis that combines these results with the energies of activation of the opening processes and the stability of the local structures, three groups of residues in the protein structure have been identified. In one group, the structures around the hydrogen bonds have frequent openings, every 600 to 1,500 s, and long lifetimes in the open state, around 1 s.In another group of local structures, the local opening is a very rare event that takes place only every 15 to 60 h.For these the lifetime in the open state is also around 1 s.The majority of local structures have lifetimes between 2,000 and 20,000 s and relatively short lifetimes of the open state in the range between 30 and 400 ms. Mapping of these groups of amides to the tertiary structure shows that the openings of the local structures are not cooperative at native conditions, and they rarely if ever lead to global unfolding.The results suggest a mechanism of hydrogen exchange by progressive local openings.
Semisynthesis of cytotoxic proteins using a modified protein splicing element
Authors: Evans Thomas C. , Benner Jack , Xu Ming‐Qun 
Published: February 2009

AbstractTwo cytotoxic proteins, bovine pancreatic ribonuclease A (RNase A), and a restriction endonuclease from Haemophilus parainfluenzae (HpaI), were produced using a novel semisynthetic approach that utilizes a protein splicing element, an intein, to generate a reactive thioester at the C‐terminus of a recombinant protein. Nucleophilic attack on this thioester by the N‐terminal cysteine of a synthetic peptide ultimately leads to the ligation of the two reactants through a native peptide bond. This strategy was used to produce RNase A and HpaI by isolating inactive truncated forms of these proteins, the first 109 and 223 amino acids of RNase A and HpaI, respectively, as fusion proteins consisting of the target protein, an intein, and a chitin binding domain. Thiol‐induced cleavage of the precursor led to the liberation of the target protein with a C‐terminal thioester‐tag. Addition of synthetic peptides representing the amino acids missing from the truncated forms led to the generation of full‐length products that displayed catalytic activity indicative of the wild‐type enzymes. The turnover numbers and Km for ligated and renatured RNase A were 8. 2 s‐1 and 1. 5 mM, in good agreement with reported values of 8. 3 s‐1 and 1. 2 mM (Hodges&Merrifield, 1975). Ligated HpaI had a specific activity of 0. 5–1. 5 × 106 U/mg, which compared favorably with the expected value of 1–2 × 106 U/mg (J. Benner, unpubl. obs. ). Besides assisting in the production of cytotoxic proteins, this technique could allow the easy insertion of unnatural amino acids into a protein sequence.
Solution structures of stromelysin complexed to thiadiazole inhibitors
Authors: Stockman Brian J. , Waldon Daniel J. , Gates Jo A. , Scahill Terrence A. , Kloosterman David A. , Mizsak Stephen A. , Jacobsen E. Jon , Belonga Kenneth L. , Mitchell Mark A. , Mao Boryeu , Petke James D. , Goodman Linda , Powers Elaine A. , Ledbetter Steven R. , Kaytes Paul S. , Vogeli Gabriel , Marshall Vincent P. , Petzold Gary L. , Poorman Roger A. 
Published: February 2009

AbstractUnregulated or overexpressed matrix metalloproteinases (MMPs), including stromelysin, collagenase, and gelatinase, have been implicated in several pathological conditions including arthritis and cancer. Small‐molecule MMP inhibitors may have therapeutic value in the treatment of these diseases. In this regard, the solution structures of two stromelysin/inhibitor complexes have been investigated using H, 13C, and 15N NMR spectroscopy. Both inhibitors are members of a novel class of matrix metalloproteinase inhibitor that contain a thiadiazole group and that interact with stromelysin in a manner distinct from other classes of inhibitors. The inhibitors coordinate the catalytic zinc atom through their exocyclic sulfur atom, with the remainder of the ligand extending into the S1‐S3 side of the active site. The binding of inhibitor containing a protonated or fluorinated aromatic ring was investigated using H and 19F NMR spectroscopy. The fluorinated ring was found to have a reduced ring‐flip rate compared to the protonated version. A strong, coplanar interaction between the fluorinated ring of the inhibitor and the aromatic ring of Tyr155 is proposed to account for the reduced ring‐flip rate and for the increase in binding affinity observed for the fluorinated inhibitor compared to the protonated inhibitor. Binding interactions observed for the thiadiazole class of ligands have implications for the design of matrix metalloproteinase inhibitors.
Singular points of protein β‐sheets
Authors: Liu Wei‐Min , Chou Kuo‐Chen 
Published: February 2009

AbstractProtein ß‐sheets can be regarded as surfaces. Two surfaces can be connected along a common edge to form a larger surface, or two edges of a surface can coalesce to form a closed sheet such as a ß‐barrel. Singular points are locations where these connections are not perfect. In protein ß‐sheets, a singular point is characterized by a residue separating two ß‐ladders. In this paper, we study the singular points of protein ß‐sheets from the surface topologic viewpoint, summarize our search results from the protein structural data in the Protein Data Bank, and present examples where singular points are near the active sites and may contribute to forming the proper relative positions of catalytic residues.
Role of the electrostatic loop charged residues in Cu, Zn superoxide dismutase
Authors: Polticelli Fabio , Battistoni Andrea , O'Neill Peter , Rotilio Giuseppe , Desideri Alessandro 
Published: February 2009

AbstractWe have expressed and characterized a mutant of Xenopus laevis Cu, Zn superoxide dismutase in which four highly conserved charged residues belonging to the electrostatic loop have been replaced by neutral side chains: Lys120 → Leu, Asp130 → Gln, Glu131 → Gln, and Lys134 → Thr. At low ionic strength, the mutant enzyme is one of the fastest superoxide dismutases ever assayed (k = 6. 7 × 109 M−1 s−1, at pH 7 and μ = 0. 02 M). Brownian dynamics simulations give rise to identical enzyme‐substrate association rates for both wild‐type and mutant enzymes, ruling out the possibility that enhancement of the activity is due to pure electrostatic factors. Comparative analysis of the experimental catalytic rate of the quadruple and single mutants reveals the nonadditivity of the mutation effects, indicating that the hyperefficiency of the mutant is due to a decrease of the energy barrier and/or to an alternative pathway for the diffusion of superoxide within the active site channel. At physiological ionic strength the catalytic rate of the mutant at neutral pH is similar to that of the wild‐type enzyme as it is to the catalytic rate pH dependence. Moreover, mutation effects are additive. These results show that, at physiological salt conditions, electrostatic loop charged residues do not influence the diffusion pathway of the substrate and, if concomitantly neutralized, are not essential for high catalytic efficiency of the enzyme, pointing out the role of the metal cluster and of the invariant Arg141 in determining the local electrostatic forces facilitating the diffusion of the substrate towards the active site.
Monitoring metal ion flux in reactions of metallothionein and drug‐modified metallothionein by electrospray mass spectrometry
Authors: Zaia Joseph , Fabris Daniele , Wei Dong , Karpel Richard L. , Fenselau Catherine 
Published: February 2009

AbstractThe capabilities of electrospray ionization mass spectrometry are demonstrated for monitoring the flux of metal ions out of and into the metalloprotein rabbit liver metallothionein and, in one example, chlorambucil‐alkylated metallothionein. Metal ion transfers may be followed as the reactions proceed in situ to provide kinetic information. More uniquely to this technique, metal ion stoichiometries may be determined for reaction intermediates and products. Partners used in these studies include EDTA, carbonic anhydrase, a zinc‐bound hexamer of insulin, and the core domain of bacteriophage T4 gene 32 protein, a binding protein for single‐stranded DNA.
The orientation and dynamics of substance P in lipid environments
Authors: Keire David A. , Kobayashi Mitsuo 
Published: February 2009

AbstractThe membrane‐associated conformation of substance P (RPKPQQFFGLM‐NH2) has been previously proposed to be the NK1‐receptor‐active conformation. In this work, NMR methods are applied to explore the orientation and dynamics of substance P at lipid surfaces for which the peptide's three‐dimensional structure had been previously determined. Here the presence of dodecylphosphocholine (DPC) or sodium dodecylsulfate (SDS) micelles has been found to cause sequence specific changes in the acid‐ and base‐catalyzed amide proton exchange rates relative to the solution state values. On binding of substance P to SDS micelles, the FFG portion showed the largest decreases in the base‐catalyzed amide exchange rates. Similar sequence‐specific changes in substance P are observed in the presence of DPC micelles, albeit at much weaker levels due to fast exchange between free and bound forms of the peptide. These differences are attributed to the location of the amide protons either in the surface double layer (via electrostatic effect) or inserted into the polar head group region of the micelles (via low dielectric). The sequence‐specific effects of micelle association were also observed in the homonuclear nonselective spin‐lattice relaxation time; these, in combination with spin‐spin relaxation times, were used to calculate correlation times for the backbone amide protons. These data combined with paramagnetic broadening observations on peptide protons in the presence of spin‐labeled lipids yield a detailed model of the interaction of substance P with lipid surfaces.
Cation‐π (Na+‐Trp) interactions in the crystal structure of tetragonal lysozyme
Authors: Wouters Johan 
Published: February 2009

AbstractExperimental evidence of a cation‐π interaction between a sodium cation (Na+) and the indole ring of residue Trpl23 in a structure (2. 0 Å) of hen egg‐white lysozyme is presented. The geometry of the metal ion‐π interaction observed in the protein structure (distance between the aromatic plane and the cation ∼4 Å) is consistent with geometries observed among small molecules crystal structures and quantum chemistry ab initio calculations. The present crystal structure of lysozyme provides unique structural information about the geometry of binding of cations to π systems in proteins. It shows that the metal ion‐π interaction within proteins is not significantly different from similar bindings found in small molecules and that it can be modeled by theoretical methods.
Solution structure of barley lipid transfer protein complexed with palmitate. Two different binding modes of palmitate in the homologous maize and barley nonspecific lipid transfer proteins
Authors: Lerche Mathilde H. , Poulsen Flemming M. 
Published: February 2009

AbstractThe structure of a nonspecific lipid transfer protein from barley (ns‐LTPbarley) in complex with palmitate has been determined by NMR spectroscopy. The structure has been compared to the structure of ns‐LTPbarley in the absence of palmitate, to the structure of ns‐LTPbarley in complex with palmitoyl coenzyme A, to the structure ofns‐LTPmaize in its free form, and to the maize protein complexed with palmitate. Binding of palmitate only affects the structure of ns‐LTPbarley moderately in contrast to the binding of palmitoyl coenzyme A, which leads to a considerable expansion of the protein. The modes of binding palmitate to the maize and barley protein are different. Although in neither case there are major conformational changes in the protein, the orientation of the palmitate in the two proteins is exactly opposite.
Crystal structure of the effector‐binding domain of the trehalose‐repressor of escherichia coli, a member of the LacI family, in its complexes with inducer trehalose‐6‐phosphate and noninducer trehalose
Authors: Hars Ulrike , Horlacher Reinhold , Boos Winfried , Welte Wolfram , Diederichs Kay 
Published: February 2009

AbstractThe crystal structure of the Escherichia coli trehalose repressor (TreR) in a complex with its inducer trehalose‐6‐phosphate was determined by the method of multiple isomorphous replacement (MIR) at 2.5 Å resolution, followed by the structure determination of TreR in a complex with its noninducer trehalose at 3.1 Å resolution. The model consists of residues 61 to 315 comprising the effector binding domain, which forms a dimer as in other members of the LacI family. This domain is composed of two similar subdomains each consisting of a central β‐sheet sandwiched between α‐helices. The effector binding pocket is at the interface of these subdomains. In spite of different physiological functions, the crystal structures of the two complexes of TreR turned out to be virtually identical to each other with the conformation being similar to those of the effector binding domains of the LacI and PurR in complex with their effector molecules. According to the crystal structure, the noninducer trehalose binds to a similar site as the trehalose portion of trehalose‐6‐phosphate. The binding affinity for the former is lower than for the latter. The noninducer trehalose thus binds competitively to the repressor. Unlike the phosphorylated inducer molecule, it is incapable of blocking the binding of the repressor headpiece to its operator DNA. The ratio of the concentrations of trehalose‐6‐phosphate and trehalose thus is used to switch between the two alternative metabolic uses of trehalose as an osmoprotectant and as a carbon source.