bluuebunny/crossref_metadata_2025_split huggingface dataset:
An additional aromatic interaction improves the thermostability and thermophilicity of a mesophilic family 11 xylanase: Structural basis and molecular study
Authors: Georis Jacques , Esteves Frederic De Lemos , Lamotte‐Brasseur Josette , Bougnet Viviane , Giannotta Fabrizio , Frère Jean‐Marie , Devreese Bart , Granier Benoict 
Published: October 2010

AbstractIn a general approach to the understanding of protein adaptation to high temperature, molecular models of the closely related mesophilic Streptomyces sp. S38 Xyl1 and thermophilic Thermomonospora fusca TfxA family 11 xylanases were built and compared with the three‐dimensional (3D) structures of homologous enzymes. Some of the structural features identified as potential contributors to the higher thermostability of TfxA were introduced in Xyl1 by site‐directed mutagenesis in an attempt to improve its thermostability and thermophilicity. A new Y11‐Y16 aromatic interaction, similar to that present in TfxA and created in Xyl1 by the T11Y mutation, improved both the thermophilicity and thermostability. Indeed, the optimum activity temperature (70 vs. 60°C) and the apparent Tm were increased by about 9°C, and the mutant was sixfold more stable at 57°C. The combined mutations A82R/F168H/N169D/δ170 potentially creating a R82‐D169 salt bridge homologous to that present in TfxA improved the thermostability but not the thermophilicity. Mutations R82/D170 and S33P seemed to be slightly destabilizing and devoid of influence on the optimal activity temperature of Xyl1. Structural analysis revealed that residues Y11 and Y16 were located on β‐strands B1 and B2, respectively. This interaction should increase the stability of the N‐terminal part of Xyl1. Moreover, Y11 and Y16 seem to form an aromatic continuum with five other residues forming putative subsites involved in the binding of xylan (+3, +2, +1, −1, −2). Y11 and Y16 might represent two additional binding subsites (−3, −4) and the T11Y mutation could thus improve substrate binding to the enzyme at higher temperature and thus the thermophilicity of Xyl1.
Characterization of a discontinuous epitope of the human immunodeficiency virus (HIV) core protein p24 by epitope excision and differential chemical modification followed by mass spectrometric peptide mapping analysis
Authors: Hochleitner Elisabeth O. , Borchers Christoph , Parker Carol , Bienstock Rachelle J. , Tomer Kenneth B. 
Published: October 2010

AbstractA combination of epitope excision, epitope extraction, and differential chemical modification followed by mass spectrometric peptide mapping was used for the characterization of a discontinuous epitope that is recognized by the mouse anti‐HIV‐p24 monoclonal antibody 5E2.A3. In epitope excision, the protein is first bound to an immobilized antibody and then digested with proteolytic enzymes. In epitope extraction, the protein is first digested and subsequently allowed to react with the antibody. After epitope excision of the p24–5E2.A3 complex with endoproteinase Lys‐C, a large fragment remained affinity bound corresponding to amino acids 1–158 of HIV‐p24 (fragment 1–158). Further digestion, however, resulted in loss of affinity. Moreover, no affinity‐bound fragments were observed after an epitope extraction experiment. These data from the epitope excision and extraction experiments suggest that the epitope is discontinuous. For the further characterization of the epitope, amino groups in the epitope‐containing fragment were acetylated in both the affinity bound and free states followed by mass spectrometric analysis. Two successive acetylation reactions were performed: (1) the first used a low molar excess of acetic anhydride, and (2) the second, after separation from the antibody, a high molar excess of its hexadeuteroderivative. This isotopic labeling procedure, in combination with high resolution mass spectrometry, allowed the precise determination of relative reactivities of amino groups. In this study, no differences were observed in the ranking of the relative reactivities of five lysine residues. However, the N‐terminal amino group was found to be part of the discontinuous epitope.
Analysis of the dynamic properties of Bacillus circulans xylanase upon formation of a covalent glycosyl‐enzyme intermediate
Authors: Connelly Gregory P. , Withers Stephen G. , Mcintosh Lawrence P. 
Published: October 2010

AbstractNMR spectroscopy was used to search for mechanistically significant differences in the local mobility of the main‐chain amides of Bacillus circulans xylanase (BCX) in its native and catalytically competent covalent glycosyl‐enzyme intermediate states. 15N T1, T2, and 15N{1H} NOE values were measured for ∼120 out of 178 peptide groups in both the apo form of the protein and in BCX covalently modified at position Glu78 with a mechanism‐based 2‐deoxy‐2‐fluoro‐β‐xylobioside inactivator. Employing the model‐free formalism of Lipari and Szabo, the measured relaxation parameters were used to calculate a global correlation time (m) for the protein in each form (9.2 ± 0.2 ns for apo‐BCX; 9.8 ± 0.3 ns for the modified protein), as well as individual order parameters for the main‐chain NH bond vectors. Average values of the order parameters for the protein in the apo and complexed forms were S2 = 0.86 ± 0.04 and S2 = 0.91 ± 0.04, respectively. No correlation is observed between these order parameters and the secondary structure, solvent accessibility, or hydrogen bonding patterns of amides in either form of the protein. These results demonstrate that the backbone of BCX is well ordered in both states and that formation of the glycosyl‐enzyme intermediate leads to little change, in any, in the dynamic properties of BCX on the time scales sampled by 15N‐NMR relaxation measurements.
Early intermediates in the PDI‐assisted folding of ribonuclease A
Authors: Vinci Floriana , Ruoppolo Margherita , Pucci Piero , Freedman Robert B. , Marino Gennaro 
Published: October 2010

AbstractThe oxidative refolding of ribonuclease A has been investigated in several experimental conditions using a variety of redox systems. All these studies agree that the formation of disulfide bonds during the process occurs through a nonrandom mechanism with a preferential coupling of certain cysteine residues. We have previously demonstrated that in the presence of glutathione the refolding process occurs through the reiteration of two sequential reactions: a mixed disulfide with glutathione is produced first which evolves to form an intramolecular S‐S bond. In the same experimental conditions, protein disulfide isomerase (PDI) was shown to catalyze formation and reduction of mixed disulfides with glutathione as well as formation of intramolecular S‐S bonds.This paper reports the structural characterization of the one‐disulfide intermediate population during the oxidative refolding of Ribonuclease A under the presence of PDI and glutathione with the aim of defining the role of the enzyme at the early stages of the reaction. The one‐disulfide intermediate population occurring at the early stages of both the uncatalyzed and the PDI‐catalyzed refolding was purified and structurally characterized by proteolytic digestion followed by MALDI‐MS and LC/ESIMS analyses. In the uncatalyzed refolding, a total of 12 disulfide bonds out of the 28 theoretical possible cysteine couplings was observed, confirming a nonrandom distribution of native and nonnative disulfide bonds. Under the presence of PDI, only two additional nonnative disulfides were detected. Semiquantitative LC/ESIMS analysis of the distribution of the S‐S bridged peptides showed that the most abundant species were equally populated in both the uncatalyzed and the catalyzed process.This paper shows the first structural characterization of the one‐disulfide intermediate population formed transiently during the refolding of ribonuclease A in quasi‐physiological conditions that mimic those present in the ER lumen. At the early stages of the process, three of the four native disulfides are detected, whereas the Cys26‐Cys84 pairing is absent. Most of the nonnative disulfide bonds identified are formed by nearest‐neighboring cysteines. The presence of PDI does not significantly alter the distribution of S‐S bonds, suggesting that the ensemble of single‐disulfide species is formed under thermodynamic control.
Alternative modes of binding of proteins with tandem SH2 domains
Authors: O'Brien Ronan , Renzoni Debora , Waterfield Michael D. , Driscoll Paul C. , Ladbury John E. , Handa Raj , Hilyard Kate , Rugman Paul , Layto Meredith 
Published: October 2010

AbstractThe issue of specificity in tyrosine kinase intracellular signaling mediated by src homology 2 (SH2) domains has great importance in the understanding how individual signals maintain their mutual exclusivity and affect downstream responses. Several proteins contain tandem SH2 domains that, on interacting with their ligand, provide a higher level of specificity than can be afforded by the interaction of a single SH2 domain. In this study, we focus on the comparison of two proteins ZAP70 and the p85 subunit of PI 3‐kinase, which although distinctly different in function and general structure, possess tandem SH2 domains separated by a linker region and which bind to phosphorylated receptor molecules localized to the cell membrane. Binding studies using isothermal titration calorimetry show that these two proteins interact with peptides mimicking their physiological ligands in very different ways. In the case of the SH2 domains from ZAP70, they interact with a stoichiometry of unity, while p85 is able to make two distinct interactions, one with a stoichiometry of 1:1 and the other with two p85 molecules interacting with one receptor. The observation of two different modes of binding of p85 might be important in providing different cellular responses based on fluctuating intracellular concentration regimes of this protein. Thermodynamic data on both proteins suggest that a conformational change occurs on binding. On investigation of this structural change using a truncated form of p85 (including just the two SH2 domains and the inter‐SH2 region), both NMR and circular dichroism spectroscopic studies failed to show significant changes in secondary structure. This suggests that any conformational change associated with binding is small and potentially limited to loop regions of the protein.
Optimal region of average side‐chain entropy for fast protein folding
Authors: Galzitskaya Oxana V. , Surin Alexei K. , Nakamura Haruki 
Published: October 2010

AbstractSearch and study the general principles that govern kinetics and thermodynamics of protein folding generates new insight into the factors that control this process. Here, we demonstrate based on the known experimental data and using theoretical modeling of protein folding that side‐chain entropy is one of the general determinants of protein folding. We show for proteins belonging to the same structural family that there exists an optimal relationship between the average side‐chain entropy and the average number of contacts per residue for fast folding kinetics. Analysis of side‐chain entropy for proteins that fold without additional agents demonstrates that there exists an optimal region of average side‐chain entropy for fast folding. Deviation of the average side‐chain entropy from the optimal region results in an anomalous protein folding process (prions, α‐lytic protease, subtilisin, some DNA‐binding proteins). Proteins with high or low side‐chain entropy would have extended unfolded regions and would require some additional agents for complete folding. Such proteins are common in nature, and their structure properties have biological importance.
Steady state and time resolved effects of guanidine hydrochloride on the structure of humicola lanuginosa lipase revealed by fluorescence spectroscopy
Authors: Zhu Keng , Jutila Arimatti , Kinnunen PAAVO K J. 
Published: October 2010

AbstractEffects of guanidine hydrochloride (GdnHCl) on the structure and dynamics of wild‐type Humicola lanuginosa lipase (HLL) and its two mutants were studied. The latter were S146A (with the active site Ser replaced by Ala) and the single Trp mutant W89m, with substitutions W117F, W221H, and W260H. Steady‐state, stopped‐flow, and time‐resolved laser‐induced fluorescence spectroscopy were carried out as a function of [GdnHCl]. The maximum emission wavelength and fluorescence lifetimes revealed the microenvironment of the tryptophan(s) in these lipases to become more polar upon increasing [GdnHCl]. However, significant extent of tertiary structure in GdnHCl is suggested by the observation that both wild‐type HLL and W89m remain catalytically active at rather high GdnHCl concentrations of >6 and 4.0 M, respectively. Changes in steady‐state emission anisotropy, as well as variation in rotational correlation times and residual anisotropy values, demonstrate that upon increasing [GdnHCl] the structure of the lipases became more loose, with increasing amplitude of structural fluctuations. Finally, intermediate states in the course of exposure of the proteins to GdnHCl were revealed by stopped‐flow fluorescence measurements.
Structural similarities and differences in staphylococcus aureus exfoliative toxins A and B as revealed by their crystal structures
Authors: Papageorgiou Anastassios C. , Acharya K. Ravi , Plano LISA R. W. , Collins Carleen M. 
Published: October 2010

AbstractStaphylococcal aureus epidermolytic toxins (ETs) A and B are responsible for the induction of staphylococcal scalded skin syndrome, a disease of neonates and young children. The clinical features of this syndrome vary from localized blisters to severe exfoliation affecting most of the body surface. Comparison of the crystal structures of two subtypes of ETs‐rETA (at 2.0 Å resolution), rETB (at 2.8 Å resolution), and an active site variant of rETA, Ser195Ala at 2.0 Å resolution has demonstrated that their overall topology resembles that of a “trypsin‐like” serine protease, but with significant differences at the N‐ and C‐termini and loop regions. The details of the catalytic site in both ET structures are very similar to those in glutamate‐specific serine proteases, suggesting a common catalytic mechanism. However, the “oxyanion hole,” which is part of the catalytic sites of glutamate specific serine proteases, is in the closed or inactive conformation for rETA, yet in the open or active conformation for rETB. The ETs contain a unique amphipathic helix at the N‐terminus, and it appears to be involved in optimizing the conformation of the catalytic site residues. Determination of the structure of the rETA catalytic site variant, Ser195Ala, showed no significant perturbation at the active site, establishing that the loss of biological and esterolytic activity can be attributed solely to disruption of the catalytic serine residue. Finally, the crystal structure of ETs, together with biochemical data and mutagenesis studies, strongly confirms the classification of these molecules as “serine proteases” rather than “superantigens.”
Role of residue Y99 in tissue plasminogen activator
Authors: Vindigni Alessandro , Winfield Mollie , Ayala Youhna M. , Cera Enrico Di 
Published: October 2010

AbstractThe crystal structure of the fibrinolytic enzyme tissue plasminogen activator (tPA) shows that the bulky side chain of Y99 hinders access to the active site by partially occluding the S2 site and may be responsible for the low catalytic activity of tPA toward plasminogen. We have tested the role of Y99 by replacing it with Leu, the residue found in more proficient proteases like trypsin and thrombin. The Y99L replacement results in an increase in the kcat/Km for chromogenic substrates due to enhanced diffusion into the active site. The increase is modest (threefold) for substrates specific for tPA that carry Pro or Gly at P2, but reaches 80‐fold for less specific substrates carrying Arg at P2. On the other hand, the Y99L mutation has no effect on the activity of tPA toward the natural substrate plasminogen, that carries Gly at P2, and reduces more than 10‐fold the inhibition of tPA by plasminogen activator inhibitor‐1 (PAI‐1), that carries Ala at P2. We conclude that the steric hindrance provided by Y99 in the crystal structure affects mostly nonphysiological substrates with bulky residues at P2. In addition, residue Y99 plays an active role in the recognition of PAI‐1, but not plasminogen. Mutations of Y99 could therefore afford a resistance to inhibition by PAI‐1 without compromising the fibrinolytic potency of tPA, a result of potential therapeutic relevance.
A helix‐turn motif in the C‐terminal domain of histone H1
Authors: Vila Roger , Ponte Imma , Suau Pedro , jiménez M. Angeles , Rico Manuel 
Published: October 2010

AbstractThe structural study of peptides belonging to the terminal domains of histone H1 can be considered as a step toward the understanding of the function of H1 in chromatin. The conformational properties of the peptide Ac‐EPKRSVAFKKT KKEVKKVATPKK (CH‐1), which belongs to the C‐terminal domain of histone Hl° (residues 99–121) and is adjacent to the central globular domain of the protein, were examined by means of 1H‐NMR and circular dichroism. In aqueous solution, CH‐1 behaved as a mainly unstructured peptide, although turn‐like conformations in rapid equilibrium with the unfolded state could be present. Addition of trifluoroethanol resulted in a substantial increase of the helical content. The helical limits, as indicated by (i, i + 3) nuclear Overhauser effect (NOE) cross correlations and significant up‐field conformational shifts of the Cα protons, span from Pro100 to Val 116, with Glu99 and Ala117 as N‐ and C‐caps. A structure calculation performed on the basis of distance constraints derived from NOE cross peaks in 90% trifluoroethanol confirmed the helical structure of this region. The helical region has a marked amphipathic character, due to the location of all positively charged residues on one face of the helix and all the hydrophobic residues on the opposite face. The peptide has a TPKK motif at the C‐terminus, following the α‐helical region. The observed NOE connectivities suggest that the TPKK sequence adopts a type (I) β‐turn conformation, a σ‐turn conformation or a combination of both, in fast equilibrium with unfolded states. Sequences of the kind (S/T)P(K/R)(K/R) have been proposed as DNA binding motifs. The CH‐1 peptide, thus, combines a positively charged amphipathic helix and a turn as potential DNA‐binding motifs.
Slow conformational dynamics of an endonuclease persist in its complex with its natural protein inhibitor
Authors: Whittaker Sara B.‐M. , Moore Geoffrey R. , Hemmings Andrew M. , Czisch Michael , Wechselberger Rainer , Kaptein Robert , James Richard , Kleanthous Colin 
Published: October 2010

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 isolated DNase domain uniformly labeled with 13C/15N bound to unlabeled Im9 contain more signals than expected for a single DNase conformer, consistent with the bound DNase being present in more than one form. The presence of chemical exchange cross peaks in 750 MHz 15N‐1H‐15N HSQC‐NOESY‐HSQC spectra for backbone NH groups of Asp20, Lys21, Trp22, Leu23, Lys69, and Asn70 showed that the bound DNase was in dynamic exchange. The rate of exchange from the major to the minor form was determined to be 1.1 ± 0.2 s−1 at 298 K. Previous NMR studies have shown that the free DNase interchanges between two conformers with a forward rate constant of 1.61 ± 0.11 s−1 at 288 K, and that the bound Im9 is fixed in one conformation. The NMR studies of the bound DNase show that Im9 binds similarly to both conformers of the DNase and that the buried Trp22 is involved in the dynamic process. For the free DNase, all NH groups within a 9 Å radius of any point of the Trp22 ring exhibit heterogeneity suggesting that a rearrangement of the position of this side chain is connected with the conformational interchange. The possible functional significance of this feature of the DNase is discussed.
Insect peptides with improved protease‐resistance protect mice against bacterial infection
Authors: Otvos Laszlo , Bokonyi Krisztina , Varga Istvan , Ertl Hildegund C. J. , Hoffmann Ralf , Bulet Philippe , Otvos Balint I. , Wade John D. , Mcmanus Ailsa M. , Craik David J. 
Published: October 2010

AbstractAt a time of the emergence of drug‐resistant bacterial strains, the development of antimicrobial compounds with novel mechanisms of action is of considerable interest. Perhaps the most promising among these is a family of antibacterial peptides originally isolated from insects. These were shown to act in a stereospecific manner on an as‐yet unidentified target bacterial protein. One of these peptides, drosocin, is inactive in vivo due to the rapid decomposition in mammalian sera. However, another family member, pyrrhocoricin, is significantly more stable, has increased in vitro efficacy against Gram‐negative bacterial strains, and if administered alone, as we show here, is devoid of in vitro or in vivo toxicity. At low doses, pyrrhocoricin protected mice against Escherichia coli infection, but at a higher dose augmented the infection of compromised animals. Analogs of pyrrhocoricin were, therefore, synthesized to further improve protease resistance and reduce toxicity. A linear derivative containing unnatural amino acids at both termini showed high potency and lack of toxicity in vivo and an expanded cyclic analog displayed broad activity spectrum in vitro. The bioactive conformation of native pyrrhocoricin was determined by nuclear magnetic resonance spectroscopy, and similar to drosocin, reverse turns were identified as pharmacologically important elements at the termini, bridged by an extended peptide domain. Knowledge of the primary and secondary structural requirements for in vivo activity of these peptides allows the design of novel antibacterial drug leads.
Specificity in substrate binding by protein folding catalysts: Tyrosine and tryptophan residues are the recognition motifs for the binding of peptides to the pancreas‐specific protein disulfide isomerase PDIp
Authors: Ruddock Lloyd W. , Freedman Robert B. , Klappa Peter 
Published: October 2010

AbstractUsing a cross‐linking approach, we recently demonstrated that radiolabeled peptides or misfolded proteins specifically interact in vitro with two luminal proteins in crude extracts from pancreas microsomes. The proteins were the folding catalysts protein disulfide isomerase (PDI) and PDIp, a glycosylated, PDI‐related protein, expressed exclusively in the pancreas. In this study, we explore the specificity of these proteins in binding peptides and related ligands and show that tyrosine and tryptophan residues in peptides are the recognition motifs for their binding by PDIp. This peptide‐binding specificity may reflect the selectivity of PDIp in binding regions of unfolded polypeptide during catalysis of protein folding.