bluuebunny/crossref_metadata_2025 huggingface dataset:
Conservative mutation met8 → leu affects the folding process and structural stability of squash trypsin inhibitor CMTI‐I
Authors: Zhukov Igor , Bierzynaski Andrzej , Jaroszewski Lukasz
Published: October 2010

Protein molecules can accommodate a large number of mutations without noticeable effects on their stability and folding kinetics. On the other hand, some mutations can have quite strong effects on protein conformational properties. Such mutations either destabilize secondary structures, e.g., α‐helices, are incompatible with close packing of protein hydrophobic cores, or lead to disruption of some specific interactions such as disulfide cross links, salt bridges, hydrogen bonds, or aromatic‐aromatic contacts. The Met8 → Leu mutation in CMTI‐I results in significant destabilization of the protein structure. This effect could hardly be expected since the mutation is highly conservative, and the side chain of residue 8 is situated on the protein surface. We show that the protein destabilization is caused by rearrangement of a hydrophobic cluster formed by side chains of residues 8, Ile6, and Leu17 that leads to partial breaking of a hydrogen bond formed by the amide group of Leu17 with water and to a reduction of a hydrophobic surface buried within the cluster. The mutation perturbs also the protein folding. In aerobic conditions the reduced wild‐type protein folds effectively into its native structure, whereas more then 75% of the mutant molecules are trapped in various misfolded species. The main conclusion of this work is that conservative mutations of hydrophobic residues can destabilize a protein structure even if these residues are situated on the protein surface and partially accessible to water. Structural rearrangement of small hydrophobic clusters formed by such residues can lead to local changes in protein hydration, and consequently, can affect considerably protein stability and folding process.
An interdomain distance in cardiac troponin c determined by fluorescence spectroscopy
Authors: Dong Wen‐Ji , Robinson John M. , Xing Jun , Cheung Herbert C. , Umeda Patrick K.
Published: October 2010

The distance between Ca2+‐binding site III in the C‐terminal domain and Cys35 in the N‐terminal domain in cardiac muscle troponin C (cTnC) was determined with a single‐tryptophan mutant using bound Tb3+ as the energy donor and iodoacetamidotetramethylrhodamine linked to the cysteine residue as energy acceptor. The luminescence of bound Tb3+ was generated through sensitization by the tryptophan located in the 12‐residue binding loop of site III upon irradiation at 295 nm, and this sensitized luminescence was the donor signal transferred to the acceptor. In the absence of bound cation at site II, the mean interdomain distance was found to be 48–49 Å regardless of whether the cTnC was unbound or bound to cardiac troponin I, or reconstituted into cardiac troponin. These results suggest that cTnC retains its overall length in the presence of bound target proteins. The distribution of the distances was wide (half‐width >9 Å) and suggests considerable interdomain flexibility in isolated cTnC, but the distributions became narrower for cTnC in the complexes with the other subunits. In the presence of bound cation at the regulatory site II, the interdomain distance was shortened by 6 Å for cTnC, but without an effect on the half‐width. The decrease in the mean distance was much smaller or negligible when cTnC was complexed with cTnI or cTnI and cTnT under the same conditions. Although free cTnC has considerable interdomain flexibility, this dynamics is slightly reduced in troponin. These results indicate that the transition from the relaxed state to an activated state in cardiac muscle is not accompanied by a gross alteration of the cTnC conformation in cardiac troponin.
Microscopic stability of cold shock protein a examined by NMR native state hydrogen exchange as a function of urea and trimethylamine N‐oxide
Authors: Jaravine Victor A. , Rathgeb‐Szabo Klara , Alexandrescu Andrei T.
Published: October 2010

Native state hydrogen exchange of cold shock protein A (CspA) has been characterized as a function of the denaturant urea and of the stabilizing agent trimethylamine N‐oxide (TMAO). The structure of CspA has five strands of β‐sheet. Strands β1‐β4 have strongly protected amide protons that, based on experiments as a function of urea, exchange through a simple all‐or‐none global unfolding mechanism. By contrast, the protection of amide protons from strand β5 is too weak to measure in water. Strand β5 is hydrogen bonded to strands β3 and β4, both of which afford strong protection from solvent exchange. Gaussian network model (GNM) simulations, which assume that the degree of protection depends on tertiary contact density in the native structure, accurately predict the strong protection observed in strands β1‐β4 but fail to account for the weak protection in strand β5. The most conspicuous feature of strand β5 is its low sequence hydrophobicity. In the presence of TMAO, there is an increase in the protection of strands b̊1‐β4, and protection extends to amide protons in more hydrophilic segments of the protein, including strand β5 and the loops connecting the β‐strands. TMAO stabilizes proteins by raising the free energy of the denatured state, due to highly unfavorable interactions between TMAO and the exposed peptide backbone. As such, the stabilizing effects of TMAO are expected to be relatively independent of sequence hydrophobicity. The present results suggest that the magnitude of solvent exchange protection depends more on solvent accessibility in the ensemble of exchange susceptible conformations than on the strength of hydrogen‐bonding interactions in the native structure.
Resolution of ligand positions by site‐directed tryptophan fluorescence in tear lipocalin
Authors: Gasymov Oktay K. , Abduragimov Adil R. , Yusifov Taleh N. , Glasgow Ben J.
Published: October 2010

The lipocalin superfamily of proteins functions in the binding and transport of a variety of important hydrophobic molecules. Tear lipocalin is a promiscuous lipid binding member of the family and serves as a paradigm to study the molecular determinants of ligand binding. Conserved regions in the lipocalins, such as the G strand and the F‐G loop, may play an important role in ligand binding and delivery. We studied structural changes in the G strand of holo‐ and apo‐tear lipocalin using spectroscopic methods including circular dichroism analysis and site‐directed tryptophan fluorescence. Apo‐tear lipocalin shows the same general structural characteristics as holo‐tear lipocalin including alternating periodicity of a β‐strand, orientation of amino acid residues 105, 103, 101, and 99 facing the cavity, and progressive depth in the cavity from residues 105 to 99. For amino acid residues facing the internal aspect of cavity, the presence of a ligand is associated with blue shifted spectra. The collisional rate constants indicate that these residues are not less exposed to solvent in holo‐tear lipocalin than in apo‐tear lipocalin. Rather the spectral blue shifts may be accounted for by a ligand induced rigidity in holo‐TL. Amino acid residues 94 and 95 are consistent with positions in the F‐G loop and show greater exposure to solvent in the holo‐ than the apo‐proteins. These findings are consistent with the general hypothesis that the F‐G loop in the holo‐proteins of the lipocalin family is available for receptor interactions and delivery of ligands to specific targets. Site‐directed tryptophan fluorescence was used in combination with a nitroxide spin labeled fatty acid analog to elucidate dynamic ligand interactions with specific amino acid residues. Collisional quenching constants of the nitroxide spin label provide evidence that at least three amino acids of the G strand residues interact with the ligand. Stern‐Volmer plots are inconsistent with a ligand that is held in a static position in the calyx, but rather suggest that the ligand is in motion. The combination of site‐directed tryptophan fluorescence with quenching by nitroxide labeled species has broad applicability in probing specific interactions in the solution structure of proteins and provides dynamic information that is not attainable by X‐ray crystallography.
Role of the switch II region in the conformational transition of activation of Ha‐ras‐p21
Authors: Díaz José Fernando , Escalona María Milagrosa , Kuppens Steven , Engelborghs Yves
Published: October 2010

The role of the switch II region in the conformational transition of activation of Ha‐ras‐p21 has been investigated by mutating residues predicted to act as hinges for the conformational transition of this loop (Ala59, Gly60, and Gly75) (Diaz JF, Wroblowski B, Schlitter J, Engelborghs Y, 1997, Proteins 28:434–451), as well as mutating the catalytic residue Gln61. The proposed mutations of the hinge residues decrease the rate of the conformational transition of activation as measured by the binding of BeF3‐ to the GDP‐p21 complex. Also, the thermodynamic parameters of the binding reaction are altered by a factor between three and five, depending on the temperature. (Due to changes in activation and reaction enthalpies, partially compensated by entropy changes.) The control mutation Q61H in which only the catalytic residue is changed has only a limited effect on the kinetic rate constants of the conformational transition and on the thermodynamic parameters of the reaction. The fact that mutations of the hinge residues of the switch II region affect both the binding of the phosphate analog and the conformational transition of activation indicates that the switch II is implicated both in the early and the late states of the transition.
Cleaved antitrypsin polymers at atomic resolution
Authors: Dunstone Michelle A. , Rossjohn Jamie , Feil Susanne C. , Parker Michael W. , Dai Weiwen , Whisstock James C. , Pike Robert N. , Bottomley Stephen P. , Le Bonniec Bernard F.
Published: October 2010

α1‐Antitrypsin deficiency, which can lead to both emphysema and liver disease, is a result of the accumulation of α1 antitrypsin polymers within the hepatocyte. A wealth of biochemical and biophysical data suggests that α1‐antitrypsin polymers form via insertion of residues from the reactive center loop of one molecule into the β‐sheet of another. However, this long‐standing hypothesis has not been confirmed by direct structural evidence. Here, we describe the first crystallographic evidence of a β‐strand linked polymer form of α1‐antitrypsin: the crystal structure of a cleaved α1‐antitrypsin polymer.
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

In 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

A 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

NMR 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

The 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

The 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

Search 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

Effects 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

Staphylococcal 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

The 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

The 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

The 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

At 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

Using 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.
Novel disulfide engineering in human carbonic anhydrase II using the PAIRWISE side‐chain geometry database
Authors: Burton Randall E. , Oas Terrence G. , Fterke Carol A. , Hunt Jennifer A.
Published: October 2010

An analysis of the pairwise side‐chain packing geometries of cysteine residues observed in high‐resolution protein crystal structures indicates that cysteine pairs have pronounced orientational preferences due to the geometric constraints of disulfide bond formation. A potential function was generated from these observations and used to evaluate models for novel disulfide bonds in human carbonic anhydrase II (HCAII). Three double‐cysteine variants of HCAII were purified and the effective concentrations of their thiol groups were determined by titrations with glutathione and dithiothreitol. The effects of the cysteine mutations on the native state structure and stability were characterized by circular dichroism, enzymatic activity, sulfonamide binding, and guanidine hydrochloride titration. These analyses indicate that the PAIRWISE potential is a good predictor of the strength of the disulfide bond itself, but the overall structural and thermodynamic effects on the protein are complicated by additional factors. In particular, the effects of cysteine substitutions on the native state and the stabilization of compact nonnative states by the disulfide can override any stabilizing effect of the cross‐link.
Chemical denaturation and elevated folding temperatures are required for wild‐type activity and stability of recombinant Methanococcus jannaschii 20S proteasome
Authors: Frankenberg Rob J. , Hsu Tina S. , Yakota Hisao , Kim Rosalind , Clark Douglas S.
Published: October 2010

The 20S proteasome from the extreme thermophile Methanococcus jannaschii (Mj) was purified and sequenced to facilitate production of the recombinant proteasome in E. coli. The recombinant proteasome remained in solution at a purity level of 80–85% (according to SDS PAGE) following incubation of cell lysates at 70°C. Temperature–activity profiles indicated that the temperature optima of the wild‐type and recombinant enzymes differed substantially, with optimal activities occurring at 119°C and 95°C, respectively. To ameliorate this discrepancy, two recombinant enzyme preparations were produced, each of which included denaturation of the proteasome by 4 M urea followed by high‐temperature (85°C) dialysis. The wild‐type temperature optimum was restored, but only if proteasome subunits were denatured and refolded prior to assembly (a preparation designated as α & β). In contrast, when proteasome assembly preceded denaturation (designated α + β) the optimum temperature was raised to a lesser degree. Moreover, the α & β and α + β preparations had apparent thermal half‐lives at 114°C of 54.2 and 26.2 min, respectively, and the thermostability of the less stable enzyme was more sensitive to a reduction in pH. Attainment of wild‐type activity and stability thus required the proper folding of both the α‐ and β‐subunits prior to proteasome assembly. Consistent with this behavior, dual‐scanning calorimetry (DSC) measurements revealed differences in the reassembly efficiency of the two proteasome preparations. The ability to produce structural conformers with dramatically different thermal optima and thermostabilities may facilitate the determination of molecular forces and structural motifs responsible for enzyme thermostablity and high‐temperature activity.
Aberrant mobility phenomena of the DNA repair protein XPA
Authors: Iakoucheva Lilia M. , Kimzey Amy L. , Masselon Christophe D. , Smith Richard D. , Dunker A. Keith , Ackerman Eric J.
Published: October 2010

The DNA repair protein XPA recognizes a wide variety of bulky lesions and interacts with several other proteins during nucleotide excision repair. We recently identified regions of intrinsic order and disorder in full lengthXenopusXPA (xXPA) protein using an experimental approach that combined time‐resolved trypsin proteolysis and electrospray ionization interface coupled to a Fourier transform ion cyclotron resonance (ESI‐FTICR) mass spectrometry (MS). MS data were consistent with the interpretation that xXPA contains no post‐translational modifications. Here we characterize the discrepancy between the calculated molecular weight (31 kDa) for xXPA and its apparent molecular weight on SDS‐PAGE (multiple bands from ∼40–45 kDa) and gel filtration chromatography (∼92 kDa), as well as the consequences of DNA binding on its anomalous mobility. Iodoacetamide treatment of xXPA prior to SDS‐PAGE yielded a single 42‐kDa band, showing that covalent modification of Cys did not correct aberrant mobility. Determination of sulfhydryl content in xXPA with Ellman's reagent revealed that all nine Cys in active protein are reduced. Unexpectedly, structural constraints induced by intramolecular glutaraldehyde crosslinks in xXPA produced a ∼32‐kDa monomer in closer agreement with its calculated molecular weight. To investigate whether binding to DNA alters xXPA's anomalous migration, we used gel filtration chromatography. For the first time, we purified stable complexes of xXPA and DNA ± cisplatin ± mismatches. xXPA showed at least 10‐fold higher affinity for cisplatin DNA ± mismatches compared to undamaged DNA ± mismatches. In all cases, DNA binding did not correct xXPA's anomalous migration. To test predictions that a Glu‐rich region (EEEEAEE) and/or disordered N‐ and C‐terminal domains were responsible for xXPA's aberrant mobility, the molecular weights of partial proteolytic fragments from ∼5 to 25 kDa separated by reverse‐phase HPLC and precisely determined by ESI‐FTICR MS were correlated with their migration on SDS‐PAGE. Every partial tryptic fragment analyzed within this size range exhibited 10%–50% larger molecular weights than expected. Thus, both the disordered domains and the Glu‐rich region in xXPA are primarily responsible for the aberrant mobility phenomena.
The effect of elevated temperature on spawning of introduced pumpkinseed Lepomis gibbosus in Europe
Authors: Ziȩba G. , Fox M. G. , Copp G. H.
Published: October 2010

In an experimental comparison of pumpkinseed Lepomis gibbosus reproduction under ambient and climate change water temperature regimes, spawning occurred earlier in the season, which is likely to lead to greater young‐of‐the‐year survival with concomitant implications in the U.K. under warmer climatic conditions.
Dopamine release in ventral striatum during Iowa Gambling Task performance is associated with increased excitement levels in pathological gambling
Authors: Linnet Jakob , Møller Arne , Peterson Ericka , Gjedde Albert , Doudet Doris
Published: September 2010

Aims  Gambling excitement is believed to be associated with biological measures of pathological gambling. Here, we tested the hypothesis that dopamine release would be associated with increased excitement levels in Pathological Gamblers compared with Healthy Controls.

Design  Pathological Gamblers and Healthy Controls were experimentally compared in a non‐gambling (baseline) and gambling condition.

Measurements  We used Positron Emission Tomography (PET) with the tracer raclopride to measure dopamine D 2/3 receptor availability in the ventral striatum during a non‐gambling and gambling condition of the Iowa Gambling Task (IGT). After each condition participants rated their excitement level.

Setting  Laboratory experiment.

Participants  18 Pathological Gamblers and 16 Healthy Controls.

Findings  Pathological Gamblers with dopamine release in the ventral striatum had significantly higher excitement levels than Healthy Controls despite lower IGT performance. No differences in excitement levels and IGT performance were found between Pathological Gamblers and Healthy Controls without dopamine release. Pathological Gamblers showed a significant correlation between dopamine release and excitement level, while no such interaction was found in Healthy Controls.

Conclusions  In pathological gamblers dopamine release in the ventral striatum appears to be associated with increased excitement levels despite lower IGT performance. The results might suggest a ‘double deficit’ function of dopamine in pathological gambling, where dopamine release reinforces maladaptive gambling through increasing excitement levels, reducing inhibition of risky decisions, or a combination of both. These findings may have implications for the understanding of dopamine in pathological gambling and other forms of addiction.
Identification of potential host plant mimics of CLAVATA3/ESR (CLE)‐like peptides from the plant‐parasitic nematode Heterodera schachtii
Authors: WANG JIANYING , REPLOGLE AMY , HUSSEY RICHARD , BAUM THOMAS , WANG XIAOHONG , DAVIS ERIC L. , MITCHUM MELISSA G.
Published: October 2010

In this article, we present the cloning of two CLAVATA3/ESR (CLE)‐like genes, HsCLE1 and HsCLE2, from the beet cyst nematode Heterodera schachtii, a plant‐parasitic cyst nematode with a relatively broad host range that includes the model plant Arabidopsis. CLEs are small secreted peptide ligands that play important roles in plant growth and development. By secreting peptide mimics of plant CLEs, the nematode can developmentally reprogramme root cells for the formation of unique feeding sites within host roots for its own benefit. Both HsCLE1 and HsCLE2 encode small secreted polypeptides with a conserved C‐terminal CLE domain sharing highest similarity to Arabidopsis CLEs 1–7. Moreover, HsCLE2 contains a 12‐amino‐acid CLE motif that is identical to AtCLE5 and AtCLE6. Like all other plant and nematode CLEs identified to date, HsCLEs caused wuschel‐like phenotypes when overexpressed in Arabidopsis, and this activity was abolished when the proteins were expressed without the CLE motif. HsCLEs could also function in planta without a signal peptide, highlighting the unique, yet conserved function of nematode CLE variable domains in trafficking CLE peptides for secretion. In a direct comparison of HsCLE2 overexpression phenotypes with those of AtCLE5 and AtCLE6, similar shoot and root phenotypes were observed. Exogenous application of 12‐amino‐acid synthetic peptides corresponding to the CLE motifs of HsCLEs and AtCLE5/6 suggests that the function of this class of CLEs may be subject to complex endogenous regulation. When seedlings were grown on high concentrations of peptide (10 µm), root growth was suppressed; however, when seedlings were grown on low concentrations of peptide (0.1 µm), root growth was stimulated. Together, these findings indicate that AtCLEs1–7 may be the target peptides mimicked by HsCLEs to promote parasitism.
Level of psychological distress and social support among patients with limb fractures in Hong Kong
Authors: Lam Kin , Chan Sally WC , Lam Simon C
Published: October 2010

Aims.  To explore the relationship between psychological distress and social support and examine its association with various socio‐demographic and clinical characteristics.

Background.  Psychological distress is a common experience in patients after limb fractures. In this regard, a lower level of psychological distress is associated with better social support among patients with various types of diseases. However, there is a paucity of studies examining the relationship between social support and psychological distress among patients with limb fractures. More so, no such study has been conducted in the Chinese population.

Design.  A cross‐sectional and correlational design.

Method.  A convenience sample of participants with upper limb fractures (n = 43) or lower limb fractures (n = 43) was recruited from two orthopaedic wards of a regional acute public hospital in Hong Kong. Data were obtained through face‐to‐face interview. The Impact of Event Scale was used to measure the level of psychological distress, while the Social Support Questionnaire Short‐Form (SSQ6) was used to measure social support.

Results.  Approximately two‐thirds of the participants (n = 54) suffered from medium to high level of psychological distress. Majority of them were fairly satisfied with their social support, with their children being the most common support persons. Furthermore, there was no significant difference in the level of distress between the participants with lower or upper limb fractures. No significant correlation was found between psychological distress and social support. Those who were women, had lower education level and lower monthly income were significantly associated with a higher level of psychological distress.

Relevance to clinical practice.  Patients with fractured limb suffered from a high level of psychological distress. Therefore, a comprehensive assessment of psychological distress should be incorporated as routine assessment. Also, assessing the socio‐demographic characteristics of patients should enable nurses to identify patients at risk of developing a high level of psychological distress.