bluuebunny/crossref_metadata_2025_split huggingface dataset:
NMR solution structure of Apis mellifera chymotrypsin/cathepsin G inhibitor‐1 (AMCI‐1): Structural similarity with Ascaris protease inhibitors
Authors: Cierpicki Tomasz , Bania Jacek , Otlewski Jacek 
Published: February 2009

AbstractThe three‐dimensional structure of the 56 residue polypeptide Apis mellifera chymotrypsin/cathepsin G inhibitor 1 (AMCI‐1) isolated from honey bee hemolymph was calculated based on 730 experimental NMR restraints. It consists of two approximately perpendicular β‐sheets, several turns, and a long exposed loop that includes the protease binding site. The lack of extensive secondary structure features or hydrophobic core is compensated by the presence of five disulfide bridges that stabilize both the protein scaffold and the binding loop segment. A detailed analysis of the protease binding loop conformation reveals that it is similar to those found in other canonical serine protease inhibitors. The AMCI‐1 structure exhibits a common fold with a novel family of inhibitors from the intestinal parasitic worm Ascaris suum. The pH‐induced conformational changes in the binding loop region observed in the Ascaris inhibitor ATI are absent in AMCI‐1. Similar binding site sequences and structures strongly suggest that the lack of the conformational change can be attributed to a Glu → Gln substitution at the P1′ position in AMCI‐1, compared to ATI. Analysis of amide proton temperature coefficients shows very good correlation with the presence of hydrogen bond donors in the calculated AMCI‐1 structure.
Change in backbone torsion angle distribution on protein folding
Authors: Petrescu Andrei‐José , Calmettes Patrick , Durand Dominique , Receveur Veronique , Smith Jeremy C. 
Published: February 2009

AbstractUnderstanding protein folding requires the determination of the configurational space accessible to the protein at different stages in folding. Here, computer simulation analysis of small angle neutron scattering results is used to probe the change in the distribution of configurations on strong denaturation of a globular protein, phosphoglycerate kinase, in 4 M guanidine hydrochloride solution. To do this atomic‐detail ensembles of the unfolded protein chain are modeled and their scattering profiles compared with the experiment. The local conformational statistics are found to strongly influence the experimental intensity at scattering vectors between 0.05 and 0.3 Å−1. Denaturation leads to a reduction in the protein atom‐pair distance distribution function over the ∼3‐15 Å region that is associated with a quantifiable shift in the backbone torsional angle (φ,ψ) distribution toward the β region of the Ramachandran plot.
Structural requirements of pyrroloquinoline quinone dependent enzymatic reactions
Authors: Oubrie Arthur , Dijkstra Bauke W. 
Published: February 2009

AbstractOn the basis of crystal structures of the pyrroloquinoline quinone (PQQ) dependent enzymes methanol dehydrogenase (MDH) and soluble glucose dehydrogenase (s‐GDH), different catalytic mechanisms have been proposed. However, several lines of biochemical and kinetic evidence are strikingly similar for both enzymes. To resolve this discrepancy, we have compared the structures of these enzymes in complex with their natural substrates in an attempt to bring them in line with a single reaction mechanism. In both proteins, PQQ is located in the center of the molecule near the axis of pseudo‐symmetry. In spite of the absence of significant sequence homology, the overall binding of PQQ in the respective active sites is similar. Hydrogen bonding interactions are made with polar protein side chains in the plane of the cofactor, whereas hydrophobic stacking interactions are important below and above PQQ. One Arg side chain and one calcium ion are ligated to the ortho‐quinone group of PQQ in an identical fashion in either active site, in agreement with their proposed catalytic function of polarizing the PQQ C5‐O5 bond. The substrates are bound in a similar position above PQQ and within hydrogen bond distance of the putative general bases Asp297 (MDH) and His144 (s‐GDH). On the basis of these similarities, we propose that MDH and s‐GDH react with their substrates through an identical mechanism, comprising general base‐catalyzed hydride transfer from the substrate to PQQ and subsequent tautomerization of the PQQ intermediate to reduced PQQ.
Optimization of the catalytic properties of Aspergillus fumigatus phytase based on the three‐dimensional structure
Authors: Tomschy Andrea , Tessier Michel , Wyss Markus , Brugger Roland , Broger Clemens , Schnoebelen Line , Van Loon Adolphus P.G.M. , Pasamontes Luis 
Published: February 2009

AbstractPreviously, we determined the DNA and amino acid sequences as well as biochemical and biophysical properties of a series of fungal phytases. The amino acid sequences displayed 49‐68% identity between species, and the catalytic properties differed widely in terms of specific activity, substrate specificity, and pH optima. With the ultimate goal to combine the most favorable properties of all phytases in a single protein, we attempted, in the present investigation, to increase the specific activity of Aspergillus fumigatus phytase. The crystal structure of Aspergillus niger NRRL 3135 phytase known at 2.5 A resolution served to specify all active site residues. A multiple amino acid sequence alignment was then used to identify nonconserved active site residues that might correlate with a given favorable property of interest. Using this approach, Gln27 of A. fumigatus phytase (amino acid numbering according to A. niger phytase) was identified as likely to be involved in substrate binding and/or release and, possibly, to be responsible for the considerably lower specific activity (26.5 vs. 196 U[mg protein]−1 at pH 5.0) of A. fumigatus phytase when compared to Aspergillus terreus phytase, which has a Leu at the equivalent position. Site‐directed mutagenesis of Gln27 of A. fumigatus phytase to Leu in fact increased the specific activity to 92.1 U(mg protein)−1, and this and other mutations at position 27 yielded an interesting array of pH activity profiles and substrate specificities. Analysis of computer models of enzyme‐substrate complexes suggested that Gln27 of wild‐type A. fumigatus phytase forms a hydrogen bond with the 6‐phosphate group of myo‐inositol hexakisphosphate, which is weakened or lost with the amino acid substitutions tested. If this hydrogen bond were indeed responsible for the differences in specific activity, this would suggest product release as the rate‐limiting step of the A. fumigatus wild‐type phytase reaction.
The dihydrolipoyl acyltransferase (BCE2) subunit of the plant branched‐chain α‐ketoacid dehydrogenase complex forms a 24‐mer core with octagonal symmetry
Authors: Mooney Brian P. , Henzl Michael T. , Randall Douglas D. , Miernyk Jan A. 
Published: February 2009

AbstractLittle is known of the plant branched‐chain α‐ketoacid dehydrogenase complex. We have undertaken a detailed study of the structure of the dihydrolipoyl acyltransferase (BCE2) subunit that forms the core of the complex, to which two other enzymes attach. Mature Arabidopsis thaliana BCE2 was expressed in Escherichia coli. The soluble recombinant protein was purified using a Superose 6 size‐exclusion column to >90% homogeneity and was catalytically active. The recombinant protein formed a stable complex with a native molecular mass of 0.95 MDa and an S coefficient of 19.4, consistent with formation of a 24‐mer. Negative‐staining transmission electron microscopy of the recombinant protein confirmed that BCE2 forms a core with octagonal symmetry. Despite divergence of mammalian and plant BCE2s, there is clearly conservation of structure that is independent of primary sequence.
Van't Hoff enthalpies without baselines
Authors: John Deborah M. , Weeks Kevin M. 
Published: February 2009

AbstractAnalysis of thermal melting curves represents one important approach for evaluating protein stability and the consequences of amino acid substitution on protein structure. By use of the van't Hoff relationship, the differential melting curve can be robustly fit to only three parameters, two of which are the underlying physical constants of melting temperature (Tm) and van't Hoff enthalpy (ΔHvH). Calculated Tm and ΔHvH values are insensitive to the choice of pre‐ and post‐transition baselines. Consequently, the method accurately computes Tm and ΔHvH for extremely truncated data sets, in the complete absence of baseline information, and for proteins with low melting temperatures, where the traditional direct approach routinely fails. Moreover, agreement between ΔHvH values obtained using points derived from pre‐vs. post‐transition data provide an independent method for detecting some classes of non‐two‐state transitions. Finally, fitting of the differential denaturation curve should prove useful for analysis of abbreviated data sets obtained from high throughput array analysis of protein stability.
Improving the quality of twilight‐zone alignments
Authors: Jaroszewski Lukasz , Godzik Adam , Rychlewski Leszek 
Published: February 2009

AbstractSeveral recent publications illustrated advantages of using sequence profiles in recognizing distant homologies between proteins. At the same time, the practical usefulness of distant homology recognition depends not only on the sensitivity of the algorithm, but also on the quality of the alignment between a prediction target and the template from the database of known proteins. Here, we study this question for several supersensitive protein algorithms that were previously compared in their recognition sensitivity (Rychlewski et al., 2000). A database of protein pairs with similar structures, but low sequence similarity is used to rate the alignments obtained with several different methods, which included sequence–sequence, sequence–profile, and profile–profile alignment methods. We show that incorporation of evolutionary information encoded in sequence profiles into alignment calculation methods significantly increases the alignment accuracy, bringing them closer to the alignments obtained from structure comparison.In general, alignment quality is correlated with recognition and alignment score significance. For every alignment method, alignments with statistically significant scores correlate with both correct structural templates and good quality alignments. At the same time, average alignment lengths differ in various methods, making the comparison between them difficult. For instance, the alignments obtained by FFAS, the profile–profile alignment algorithm developed in our group are always longer that the alignments obtained with the PSI‐BLAST algorithms. To address this problem, we develop methods to truncate or extend alignments to cover a specified percentage of protein lengths. In most cases, the elongation of the alignment by profile–profile methods is reasonable, adding fragments of similar structure. The examples of erroneous alignment are examined and it is shown that they can be identified based on the model quality.
NMR investigation of the interaction of the inhibitor protein Im9 with its partner DNase
Authors: Boetzel Ruth , Czisch Michael , Kaptein Robert , Hemmings Andrew M. , James Richard , Kleanthous Colin , Moore Geoffrey R. 
Published: February 2009

AbstractThe bacterial toxin colicin E9 is secreted by producing Escherichia coli cells with its 9.5 kDa inhibitor protein Im9 bound tightly to its 14.5 kDa C‐terminal DNase domain. Double‐ and triple‐resonance NMR spectra of the 24 kDa complex of uniformly 13C and 15N labeled Im9 bound to the unlabeled DNase domain have provided sufficient constraints for the solution structure of the bound Im9 to be determined. For the final ensemble of 20 structures, pairwise RMSDs for residues 3–84 were 0.76 ± 0.14 Å for the backbone atoms and 1.36 ± 0.15 Å for the heavy atoms. Representative solution structures of the free and bound Im9 are highly similar, with backbone and heavy atom RMSDs of 1.63 and 2.44 Å, respectively, for residues 4–83, suggesting that binding does not cause a major conformational change in Im9. The NMR studies have also allowed the DNase contact surface on Im9 to be investigated through changes in backbone chemical shifts and NOEs between the two proteins determined from comparisons of 1H‐1H‐13C NOESY‐HSQC spectra with and without 13C decoupling. The NMR‐defined interface agrees well with that determined in a recent X‐ray structure analysis with the major difference being that a surface loop of Im9, which is at the interface, has a different conformation in the solution and crystal structures. Tyr54, a key residue on the interface, is shown to exhibit NMR characteristics indicative of slow rotational flipping. A mechanistic description of the influence binding of Im9 has on the dynamic behavior of E9 DNase, which is known to exist in two slowly interchanging conformers in solution, is proposed.
Thermodynamic dissection of the binding energetics of KNI‐272, a potent HIV‐1 protease inhibitor
Authors: Velazquez‐Campoy Adrian , Luque Irene , Todd Matthew J. , Milutinovich Mark , Kiso Yoshiaki , Freire Ernesto 
Published: February 2009

AbstractKNI‐272 is a powerful HIV‐1 protease inhibitor with a reported inhibition constant in the picomolar range. In this paper, a complete experimental dissection of the thermodynamic forces that define the binding affinity of this inhibitor to the wild‐type and drug‐resistant mutant V82F/I84V is presented. Unlike other protease inhibitors, KNI‐272 binds to the protease with a favorable binding enthalpy. The origin of the favorable binding enthalpy has been traced to the coupling of the binding reaction to the burial of six water molecules. These bound water molecules, previously identified by NMR studies, optimize the atomic packing at the inhibitor/protein interface enhancing van der Waals and other favorable interactions. These interactions offset the unfavorable enthalpy usually associated with the binding of hydrophobic molecules. The association constant to the drug resistant mutant is 100–500 times weaker. The decrease in binding affinity corresponds to an increase in the Gibbs energy of binding of 3–3.5 kcal/mol, which originates from less favorable enthalpy (1.7 kcal/mol more positive) and entropy changes. Calorimetric binding experiments performed as a function of pH and utilizing buffers with different ionization enthalpies have permitted the dissection of proton linkage effects. According to these experiments, the binding of the inhibitor is linked to the protonation/deprotonation of two groups. In the uncomplexed form these groups have pKs of 6.0 and 4.8, and become 6.6 and 2.9 in the complex. These groups have been identified as one of the aspartates in the catalytic aspartyl dyad in the protease and the isoquinoline nitrogen in the inhibitor molecule. The binding affinity is maximal between pH 5 and pH 6. At those pH values the affinity is close to 6 × 1010 M−1 (Kd = 16 pM). Global analysis of the data yield a buffer‐ and pH‐independent binding enthalpy of −6.3 kcal/mol. Under conditions in which the exchange of protons is zero, the Gibbs energy of binding is −14.7 kcal/mol from which a binding entropy of 28 cal/K mol is obtained. Thus, the binding of KNI‐272 is both enthalpically and entropically favorable. The structure‐based thermodynamic analysis indicates that the allophenylnorstatine nucleus of KNI‐272 provides an important scaffold for the design of inhibitors that are less susceptible to resistant mutations.
Structure‐based prediction of binding peptides to MHC class I molecules: Application to a broad range of MHC alleles
Authors: Schueler‐Furman Ora , Altuvia Yael , Sette Alessandro , Margalit Hanah 
Published: February 2009

AbstractSpecific binding of antigenic peptides to major histocompatibility complex (MHC) class I molecules is a prerequisite for their recognition by cytotoxic T‐cells. Prediction of MHC‐binding peptides must therefore be incorporated in any predictive algorithm attempting to identify immunodominant T‐cell epitopes, based on the amino acid sequence of the protein antigen. Development of predictive algorithms based on experimental binding data requires experimental testing of a very large number of peptides. A complementary approach relies on the structural conservation observed in crystallographically solved peptide‐MHC complexes. By this approach, the peptide structure in the MHC groove is used as a template upon which peptide candidates are threaded, and their compatibility to bind is evaluated by statistical pairwise potentials. Our original algorithm based on this approach used the pairwise potential table of Miyazawa and Jernigan (Miyazawa S, Jernigan RL, 1996, J Mol Biol 256:623–644) and succeeded to correctly identify good binders only for MHC molecules with hydrophobic binding pockets, probably because of the high emphasis of hydrophobic interactions in this table. A recently developed pairwise potential table by Betancourt and Thirumalai (Betancourt MR, Thirumalai D, 1999, Protein Sci 8:361–369) that is based on the Miyazawa and Jernigan table describes the hydrophilic interactions more appropriately. In this paper, we demonstrate how the use of this table, together with a new definition of MHC contact residues by which only residues that contribute exclusively to sequence specific binding are included, allows the development of an improved algorithm that can be applied to a wide range of MHC class I alleles.
Structure of streptococcal pyrogenic exotoxin A reveals a novel metal cluster
Authors: Earhart Cathleen A. , Vath Gregory M. , Roggiani Manuela , Schlievert Patrick M. , Ohlendorf Douglas H. 
Published: February 2009

AbstractThe streptococcal pyrogenic toxins A, B, and C (SPEA, SPEB, and SPEC) are responsible for the fever, rash, and other toxicities associated with scarlet fever and streptococcal toxic shock syndrome. This role, together with the ubiquity of diseases caused by Streptococcus pyogenes, have prompted structural analyses of SPEA by several groups. Papageorgiou et al. (1999) have recently reported the structure of SPEA crystallized in the absence of zinc. Zinc has been shown to be important in the ability of some staphylococcal and streptococcal toxins to stimulate proliferation of CD4+ T‐cells. Since cadmium is more electron dense than zinc and typically binds interchangeably, we grew crystals in the presence of 10 mM CdCl2. Crystals have been obtained in three space groups, and the structure in the P212121 crystal form has been refined to 1.9 Å resolution. The structural analysis revealed an identical tetramer as well as a novel tetrahedral cluster of cadmium in all three crystal forms on a disulfide loop encompassing residues 87–98. No cadmium was bound at the site homologous to the zinc site in staphylococcal enterotoxins C (SECs) despite the high structural homology between SPEA and SECs. Subsequent soaking of crystals grown in the presence of cadmium in 10 mM ZnCl2 showed that zinc binds in this site (indicating it can discriminate between zinc and cadmium ions) using the three ligands (Asp77, His106, and His 110) homologous to the SECs plus a fourth ligand (Glu33).
Review article: small intestinal bacterial overgrowth, bile acid malabsorption and gluten intolerance as possible causes of chronic watery diarrhoea
Authors: FAN X. , SELLIN J. H. 
Published: February 2009

SummaryBackground  Chronic watery diarrhoea is one of the most common symptoms prompting GI evaluation. Recently, new diagnostic considerations have emerged as possible factors in chronic diarrhoea.Aim  To review available data regarding diagnosis and treatment of chronic diarrhoea with an emphasis on bacterial overgrowth and bile acid malabsorption.Methods  A systematic search of the English language literature of chronic diarrhoea was performed focused on three possible aetiologies of diarrhoea: small intestinal bacterial overgrowth (SIBO), idiopathic bile salt malabsorption (IBAM), gluten responsive enteropathy.Results  Recent studies suggest that SIBO and bile acid malabsorption may have been underestimated as possible causes of chronic watery diarrhoea. Gluten intolerance with negative coeliac serology is a contentious possible cause of watery diarrhoea, but requires further research before acceptance as an entity.Conclusion  In patients with otherwise unexplained chronic watery diarrhoea, small intestinal bacterial overgrowth and bile salt malabsorption should be considered and investigated.
Genetic diversity of dog breeds: within‐breed diversity comparing genealogical and molecular data
Authors: Leroy G. , Verrier E. , Meriaux J. C. , Rognon X. 
Published: February 2009

SummaryThe genetic diversity of 61 dog breeds raised in France was investigated. Genealogical analyses were performed on the pedigree file of the French kennel club. A total of 1514 dogs were also genotyped using 21 microsatellite markers. For animals born from 2001 to 2005, the average coefficient of inbreeding ranged from 0.2% to 8.8% and the effective number of ancestors ranged from 9 to 209, according to the breed. The mean value of heterozygosity was 0.62 over all breeds (range 0.37–0.77). At the breed level, few correlations were found between genealogical and molecular parameters. Kinship coefficients and individual similarity estimators were, however, significantly correlated, with the best mean correlation being found for the Lynch & Ritland estimator (r = 0.43). According to both approaches, it was concluded that special efforts should be made to maintain diversity for three breeds, namely the Berger des Pyrénées, Braque Saint‐Germain and Bull Terrier.
Genetic diversity of dog breeds: between‐breed diversity, breed assignation and conservation approaches
Authors: Leroy G. , Verrier E. , Meriaux J. C. , Rognon X. 
Published: February 2009

SummaryGenetic relationships between 61 dog breeds were investigated, using a sampling of 1514 animals and a panel of 21 microsatellite markers. Based on the results from distance‐based and Bayesian methods, breed constituted the main genetic structure, while groups including genetically close breeds showed a very weak structure. Depending on the method used, between 85.7% and 98.3% of dogs could be assigned to their breed, with large variations according to the breed. However, breed heterozygosity influenced assignment results differently according to the method used. Within‐breed and between‐breed diversity variations when breeds were removed were highly negatively correlated (r = −0.963, P < 0.0001), because of the genetic structure of the breed set.