bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of the lateral channel in catalase HPII of Escherichia coli
Authors: Sevinc M. Serdal , Maté Maria J. , Switala Jack , Fita Ignacio , Loewen Peter C.
Published: October 2010

The heme‐containing catalase HPII of Escherichia coli consists of a homotetramer in which each subunit contains a core region with the highly conserved catalase tertiary structure, to which are appended N‐ and C‐terminal extensions making it the largest known catalase. HPII does not bind NADPH, a cofactor often found in catalases. In HPII, residues 585–590 of the C‐terminal extension protrude into the pocket corresponding to the NADPH binding site in the bovine liver catalase. Despite this difference, residues that define the NADPH pocket in the bovine enzyme appear to be well preserved in HPII. Only two residues that interact ionically with NADPH in the bovine enzyme (Asp212 and His304) differ in HPII (Glu270 and Glu362), but their mutation to the bovine sequence did not promote nucleotide binding. The active‐site heme groups are deeply buried inside the molecular structure requiring the movement of substrate and products through long channels. One potential channel is about 30 Å in length, approaches the heme active site laterally, and is structurally related to the branched channel associated with the NADPH binding pocket in catalases that bind the dinucleotide. In HPII, the upper branch of this channel is interrupted by the presence of Arg260 ionically bound to Glu270. When Arg260 is replaced by alanine, there is a threefold increase in the catalytic activity of the enzyme. Inhibitors of HPII, including azide, cyanide, various sulfhydryl reagents, and alkylhydroxylamine derivatives, are effective at lower concentration on the Ala260 mutant enzyme compared to the wild‐type enzyme. The crystal structure of the Ala260 mutant variant of HPII, determined at 2.3 Å resolution, revealed a number of local structural changes resulting in the opening of a second branch in the lateral channel, which appears to be used by inhibitors for access to the active site, either as an inlet channel for substrate or an exhaust channel for reaction products.
Comparison of protein–protein interactions in serine protease‐inhibitor and antibody‐antigen complexes: Implications for the protein docking problem
Authors: Jackson Richard M.
Published: October 2010

The protein‐protein interaction energy of 12 nonhomologous serine protease‐inhibitor and 15 antibody‐antigen complexes is calculated using a molecular mechanics formalism and dissected in terms of the main‐chain vs. side‐chain contribution, nonrotameric side‐chain contributions, and amino acid residue type involvement in the interface interaction.

There are major differences in the interactions of the two types of protein‐protein complex. Protease‐inhibitor complexes interact predominantly through a main‐chain‐main‐chain mechanism while antibody‐antigen complexes interact predominantly through a side‐chain‐side‐chain or a side‐chain‐main‐chain mechanism. However, there is no simple correlation between the main‐chain‐main‐chain interaction energy and the percentage of main‐chain surface area buried on binding. The interaction energy is equally effected by the presence of nonrotameric side‐chain conformations, which constitute ∼20% of the interaction energy. The ability to reproduce the interface interaction energy of the crystal structure if original side‐chain conformations are removed from the calculation is much greater in the protease‐inhibitor complexes than the antibody‐antigen complexes. The success of a rotameric model for protein‐protein docking appears dependent on the extent of the main‐chain‐main‐chain contribution to binding.

Analysis of (1) residue type and (2) residue pair interactions at the interface show that antibody‐antigen interactions are very restricted with over 70% of the antibody energy attributable to just six residue types (Tyr > Asp > Asn > Ser > Glu > Trp) in agreement with previous studies on residue propensity. However, it is found here that 50% of the antigen energy is attributable to just four residue types (Arg = Lys > Asn > Asp). On average just 12 residue pair interactions (6%) contribute over 40% of the favorable interaction energy in the antibody‐antigen complexes, with charge‐charge and charge/polar‐tyrosine interactions being prominent. In contrast protease inhibitors use a diverse set of residue types and residue pair interactions.
Actin surface structure revealed by antibody imprints: Evaluation of phage‐display analysis of anti‐actin antibodies
Authors: Jesaitis Algirdas J. , Gizachew Dawit , Dratz Edward A. , Siemsen Daniel W. , Stone Kim Clawson , Burritt James B.
Published: October 2010

Phage‐display peptide library analysis of an anti‐F actin polyclonal antibody identified 12 amino acid residues of actin that appear, in its X‐ray crystal structure, to be grouped together in a surface accessible conformational epitope. Phage epitope mapping was carried out by isolating immune complexes containing members of the J404 nonapeptide phage‐display library formed in diluted antiserum and isolated on a protein A affinity matrix. Immunoreactive clones were grown as plaques, replica plated onto nitrocellulose, and labeled with anti‐actin immune serum. One hundred and forty‐four positively staining clones identified in this way were sequenced. Of these, 54 displayed peptides with sequence similarities. When the most abundantly selected sequence, KQTWQQLWD, was produced as a synthetic peptide and derivatized to ovalbumin, the complex was strongly recognized by the antiserum on Western blots and inhibited the binding of the antibody to immobilized F‐actin by 60%. A scrambled version of this sequence WQDK WLQTQ, when coupled to ovalbumin, was not recognized by the antiserum and minimally inhibited binding of antiserum to immobilized F‐actin by 10%. KQTWQQLWD contained four residues that corresponded, in frame, to a highly conserved six residue region of the chicken β‐actin sequence 351TFQQMW356 (identical residues are shown in bold). Examination of the rabbit skeletal muscle X‐ray crystal structure suggested that within a 15 å radius of W356, nine additional residues were arranged on the actin surface in such a way that they could be mimicked by several of the selected phage sequences with root‐mean‐square deviation fits of 2.1–2.5 å. We conclude that phage‐display analysis can provide information about the relative location of amino acids on the surfaces of proteins using antibody imprints of the protein surface structure.
Genome analysis: Assigning protein coding regions to three‐dimensional structures
Authors: Salamov Asaf A. , Suwa Makiko , Orengo Christine A. , Swindells Mark B.
Published: October 2010

We describe the results of a procedure for maximizing the number of sequences that can be reliably linked to a protein of known three‐dimensional structure. Unlike other methods, which try to increase sensitivity through the use of fold recognition software, we only use conventional sequence alignment tools, but apply them in a manner that significantly increases the number of relationships detected. We analyzed 11 genomes and found that, depending on the genome, between 23 and 32% of the ORFs had significant matches to proteins of known structure. In all cases, the aligned region consisted of either >100 residues or >50% of the smaller sequence. Slightly higher percentages could be attained if smaller motifs were also included. This is significantly higher than most previously reported methods, even those that have a fold‐recognition component. We survey the biochemical and structural characteristics of the most frequently occurring proteins, and discuss the extent to which alignment methods can realistically assign function to gene products.
Nucleotide binding to the G12V‐mutant of Cdc42 investigated by X‐ray diffraction and fluorescence spectroscopy: Two different nucleotide states in one crystal
Authors: Rudolph Markus Georg , Wittinghofer Alfred , Vetter Ingrid Renate
Published: October 2010

The 2.5 Å crystal structure of the full length human placental isoform of the Gly12 to Val mutant Cdc42 protein (Cdc42(G12V)) bound to both GDP/Mg2+ and GDPNH2 (guanosine‐5′‐diphospho‐β‐amidate) is reported. The crystal contains two molecules in the asymmetric unit, of which one has bound GDP/Mg2+, while the other has bound GDPNH2 without a Mg2+ ion. Crystallization of the protein was induced via hydrolysis of the Cdc42 · GppNHp complex by the presence of contaminating alkaline phosphatase activity in combination with the crystallization conditions. This prompted us to compare the binding characteristics of GDPNH2 vs. GDP. The amino group of GDPNH2 drastically reduces the affinity to Cdc42 in comparison with that of GDP, causes the loss of the Mg2+ ion, and apparently also increases the conformational flexibility of the protein as seen in the crystal. Both the switch I and switch II regions are visible in the electron density of the GDP‐bound molecule, but not in the molecule bound to GDPNH2. The C‐terminus containing the CaaX‐motif is partly ordered in both molecules due to an intramolecular disulfide bond formed between Cys105/Cys188 and Cys305/Cys388, respectively.
Nucleolar protein B23 has molecular chaperone activities
Authors: Szebeni Attila , Olson Mark O.J.
Published: October 2010

Protein B23 is an abundant, multifunctional nucleolar phosphoprotein whose activities are proposed to play a role in ribosome assembly. Szebeni et al. (1997) showed stimulation of nuclear import in vitro by protein B23 and suggested that this effect was due to a molecular chaperone‐like activity. Protein B23 was tested for chaperone activities using several protein substrates. The temperature‐dependent and ‐independent aggregation of the HIV‐1 Rev protein was measured using a zero angle light scattering (turbidity) assay. Protein B23 inhibited the aggregation of the Rev protein, with the amount of inhibition proportional to the concentration of B23 added. This activity was saturable with nearly complete inhibition when the molar ratio of B23:Rev was slightly above one. Protein B23 also protected liver alcohol dehydrogenase (LADH), carboxypeptidase A, citrate synthase, and rhodanese from aggregation during thermal denaturation and preserved the enzyme activity of LADH under these conditions. In addition, protein B23 was able to promote the restoration of activity of LADH previously denatured with guanidine‐HCl. Protein B23 preferentially bound denatured substrates and exposed hydrophobic regions when complexed with denatured proteins. Thus, by several criteria, protein B23 behaves like a molecular chaperone; these activities may be related to its role in ribosome biogenesis.
Computational studies on mutant protein stability: The correlation between surface thermal expansion and protein stability
Authors: Palma Rocio , Curmi Paul M.G.
Published: October 2010

Thermal stability of mutant proteins has been investigated using temperature dependent molecular dynamics (MD) simulations in vacuo. The numerical modeling was aimed at mimicking protein expansion upon heating. After the conditions for an expanding protein accessible surface area were established for T4 lysozyme and barnase wild‐type proteins, MD simulations were carried out under the same conditions using the crystal structures of several mutant proteins. The computed thermal expansion of the accessible surface area of mutant proteins was found to be strongly correlated with their experimentally measured stabilities. A similar, albeit weaker, correlation was observed for model mutant proteins. This opens the possibility of obtaining stability information directly from protein structure.
Disulfide engineering at the dimer interface of lactobacillus casei thymidylate synthase: Crystal structure of the T155C/E188C/C244T mutant
Authors: Velanker S.S. , Gokhale R.S. , Ray Soumya S. , Gopal B. , Parthasarathy S. , Santi D.V. , Balaram P. , Murthy M.R.N.
Published: October 2010

The crystal structure of a covalently cross‐linked Lactobacillus casei thymidylate synthase has been determined at 2.8 å resolution. The sites for mutation to achieve the bis‐disulfide linked dimer were identified using the disulfide modeling program MODIP. The mutant so obtained was found to be remarkably thermostable. This increase in stability has been reasoned to be entirely a consequence of the covalent gluing between the two subunits.
Substrate‐assisted catalysis: Molecular basis and biological significance
Authors: Dall'Acqua William , Carter Paul
Published: October 2010

Substrate‐assisted catalysis (SAC) is the process by which a functional group in a substrate contributes to catalysis by an enzyme. SAC has been demonstrated for representatives of three major enzyme classes: serine proteases, GTPases, and type II restriction endonucleases, as well as lysozyme and hexose‐1‐phosphate uridylyltransferase. Moreover, structure‐based predictions of SAC have been made for many additional enzymes. Examples of SAC include both naturally occurring enzymes such as type II restriction endonucleases as well as engineered enzymes including serine proteases. In the latter case, a functional group from a substrate can substitute for a catalytic residue replaced by site‐directed mutagenesis. From a protein engineering perspective, SAC provides a strategy for drastically changing enzyme substrate specificity or even the reaction catalyzed. From a biological viewpoint, SAC contributes significantly to the activity of some enzymes and may represent a functional intermediate in the evolution of catalysis. This review focuses on advances in engineering enzyme specificity and activity by SAC, together with the biological significance of this phenomenon.
Affinity cleavage at the divalent metal site of porcine NAD‐specific isocitrate dehydrogenase
Authors: Huang Yu‐Chu , Soundar Sambanthamurthy , Colman Roberta F.
Published: October 2010

A divalent metal ion, such as Mn2+, is required for the catalytic reaction and allosteric regulation of pig heart NAD‐dependent isocitrate dehydrogenase. The enzyme is irreversibly inactivated and cleaved by Fe2+ in the presence of O2 and ascorbate at pH 7.0. Mn2+ prevents both inactivation and cleavage. Nucleotide ligands, such as NAD, NADPH, and ADP, neither prevent nor promote inactivation or cleavage of the enzyme by Fe2+. The NAD‐specific isocitrate dehydrogenase is composed of three distinct subunits in the ratio 2α:1 β:1 γ. The results indicate that the oxidative inactivation and cleavage are specific and involve the 40 kDa α subunit of the enzyme. A pair of major peptides is generated during Fe2+ inactivation: 29.5 + 10.5 kDa, as determined by SDS‐PAGE. Amino‐terminal sequencing reveals that these peptides arise by cleavage of the Val262‐His263 bond of the α subunit. No fragments are produced when enzyme is incubated with Fe2+ and ascorbate under denaturing conditions in the presence of 6 M urea, indicating that the native structure is required for the specific cleavage. These results suggest that His263 of the α subunit may be a ligand of the divalent metal ion needed for the reaction catalyzed by isocitrate dehydrogenase. Isocitrate enhances the inactivation of enzyme caused by Fe2+ in the presence of oxygen, but prevents the cleavage, suggesting that inactivation occurs by a different mechanism when metal ion is bound to the enzyme in the presence of isocitrate: oxidation of cysteine may be responsible for the rapid inactivation in this case. Affinity cleavage caused by Fe2+ implicates α as the catalytic subunit of the multisubunit porcine NAD‐dependent isocitrate dehydrogenase.
Apoflavodoxin (un) folding followed at the residue level by NMR
Authors: Van Mierlo Carlo P.M. , Van Den Oever Jos M.P. , Steensma Elles
Published: October 2010

The denaturant‐induced (un)folding of apoflavodoxin from Azotobacter vinelandii has been followed at the residue level by NMR spectroscopy. NH groups of 21 residues of the protein could be followed in a series of 1H‐15N heteronuclear single‐quantum coherence spectra recorded at increasing concentrations of guanidinium hydrochloride despite the formation of protein aggregate. These NH groups are distributed throughout the whole apoflavodoxin structure. The midpoints of unfolding determined by NMR coincide with the one obtained by fluorescence emission spectroscopy. Both techniques give rise to unfolding curves with transition zones at significantly lower denaturant concentrations than the one obtained by circular dichroism spectroscopy. The NMR (un)folding data support a mechanism for apoflavodoxin folding in which a relatively stable intermediate is involved. Native apoflavodoxin is shown to cooperatively unfold to a molten globule‐like state with extremely broadened NMR resonances. This initial unfolding step is slow on the NMR chemical shift timescale. The subsequent unfolding of the molten globule is faster on the NMR chemical shift timescale and the limited appearance of 1H‐15N HSQC cross peaks of unfolded apoflavodoxin in the denaturant range studied indicates that it is noncooperative.
Amide proton hydrogen exchange rates for sperm whale myoglobin obtained from 15N‐1H NMR spectra
Authors: Cavagnero Silvia , Thériault Yves , Narula Surinder S. , Dyson H. Jane , Wright Peter E.
Published: October 2010

The hydrogen exchange behavior of exchangeable protons in proteins can provide important information for understanding the principles of protein structure and function. The positions and exchange rates of the slowly‐exchanging amide protons in sperm whale myoglobin have been mapped using 15N‐1H NMR spectroscopy. The slowest‐exchanging amide protons are those that are hydrogen bonded in the longest helices, including members of the B, E, and H helices. Significant protection factors were observed also in the A, C, and G helices, and for a few residues in the D and F helices. Knowledge of the identity of slowly‐exchanging amide protons forms the basis for the extensive quench‐flow kinetic folding experiments that have been performed for myoglobin, and gives insights into the tertiary interactions and dynamics in the protein.
X‐ray crystal structure and molecular dynamics simulations of silver hake parvalbumin (Isoform B)
Authors: Richardson Robert C. , King Nancy M. , Sun Hongmao , Nelson Donald J. , Harrington Daniel J. , Royer William E.
Published: October 2010

Parvalbumins constitute a class of calcium‐binding proteins characterized by the presence of several helix‐loop‐helix (EF‐hand) motifs. In a previous study (Revett SP, King G, Shabanowitz J, Hunt DF, Hartman KL, Laue TM, Nelson DJ, 1997, Protein Sci 7:2397–2408), we presented the sequence of the major parvalbumin isoform from the silver hake (Merluccius bilinearis) and presented spectroscopic and structural information on the excised “EF‐hand” portion of the protein. In this study, the X‐ray crystal structure of the silver hake major parvalbumin has been determined to high resolution, in the frozen state, using the molecular replacement method with the carp parvalbumin structure as a starting model. The crystals are orthorhombic, space group C2221, with a = 75.7 Å, b = 80.7 Å, and c = 42.1 Å. Data were collected from a single crystal grown in 15% glycerol, which served as a cryoprotectant for flash freezing at ‐188 °C. The structure refined to a conventional R‐value of 21% (free R 25%) for observed reflections in the range 8 to 1.65 Å [I > 2σ(I)]. The refined model includes an acetylated amino terminus, 108 residues (characteristic of a β parvalbumin lineage), 2 calcium ions, and 114 water molecules per protein molecule. The resulting structure was used in molecular dynamics (MD) simulations focused primarily on the dynamics of the ligands coordinating the Ca2+ ions in the CD and EF sites. MD simulations were performed on both the fully Ca2+ loaded protein and on a Ca2+ deficient variant, with Ca2+ only in the CD site. There was substantial agreement between the MD and X‐ray results in addressing the issue of mobility of key residues in the calcium‐binding sites, especially with regard to the side chain of Ser55 in the CD site and Asp92 in the EF site.
2.9 Å crystal structure of ligand‐free tryptophanyl‐tRNA synthetase: Domain movements fragment the adenine nucleotide binding site
Authors: Ilyin Valentin A. , Temple Brenda , Hu Mei , Carter Charles W. , Li Genpei , Yin Yuhui , Vachette Patrice
Published: October 2010

The crystal structure of ligand‐free tryptophanyl‐tRNA synthetase (TrpRS) was solved at 2.9 Å using a combination of molecular replacement and maximum‐entropy map/phase improvement. The dimeric structure (R = 23.7, Rfree = 26.2) is asymmetric, unlike that of the TrpRS tryptophanyl‐5′AMP complex (TAM; Doublie S, Bricogne G, Gilmore CJ, Carter CW Jr, 1995, Structure 3:17–31). In agreement with small‐angle solution X‐ray scattering experiments, unliganded TrpRS has a conformation in which both monomers open, leaving only the tryptophan‐binding regions of their active sites intact. The amino terminal αA‐helix, TIGN, and KMSKS signature sequences, and the distal helical domain rotate as a single rigid body away from the dinucleotide‐binding fold domain, opening the AMP binding site, seen in the TAM complex, into two halves. Comparison of side‐chain packing in ligand‐free TrpRS and the TAM complex, using identification of nonpolar nuclei (Ilyin VA, 1994, Protein Eng 7:1189–1195), shows that significant repacking occurs between three relatively stable core regions, one of which acts as a bearing between the other two. These domain rearrangements provide a new structural paradigm that is consistent in detail with the“induced‐fit” mechanism proposed for TyrRS by Fersht et al. (Fersht AR, Knill‐Jones JW, Beduelle H, Winter G, 1988, Biochemistry 27:1581–1587). Coupling of ATP binding determinants associated with the two catalytic signature sequences to the helical domain containing the presumptive anticodon‐binding site provides a mechanism to coordinate active‐site chemistry with relocation of the major tRNA binding determinants.
Characterization of the functional role of asp 141, asp 194, and asp464 residues in the Mn2+‐l‐malate binding of pigeon liver malic enzyme
Authors: Chou Wei‐Yuan , Chang Hwei‐Ping , Huang Chien‐Hsiun , Kuo Cheng‐Chin , Chang Gu‐Gang , Tong Liang
Published: October 2010

Pigeon liver malic enzyme was inactivated and cleaved at Asp141, Asp194, and Asp464 by the Cu2+‐ascorbate system in acidic environment. Site‐specific mutagenesis was performed at these putative metal‐binding sites. Three point mutants, D141N, D194N, and D464N; three double mutants, D(141,194)N, D(194,464)N, and D(141,464)N; and a triple mutant, D(141,194,464)N; as well as the wild‐type malic enzyme (WT) were successfully cloned and expressed inEscherichia colicells. All recombinant enzymes, except the triple mutant, were purified to apparent homogeneity by successive Q‐Sepharose and adenosine‐2′,5′‐bisphosphate‐agarose columns. The mutants showed similar apparentKm,nadpvalues to that of the WT. TheKm, Mal value was increased in the D141N and D194N mutants. TheKm, Mnvalue, on the other hand, was increased only in the D141N mutant by 14‐fold, corresponding to ∼ 1.6 kcal/mol for the Asp141‐Mn2+binding energy. Substrate inhibition by L‐malate was only observed in WT, D464N, and D(141,464)N. Initial velocity experiments were performed to derive the various kinetic parameters. The possible interactions between Asp141, Asp 194, and Asp464 were analyzed by the double‐mutation cycles and triple‐mutation box. There are synergistic weakening interactions between Asp141 and Asp194 in the metal binding that impel the D(141,194)N double mutant to an overall specificity constant [kcat/(Kd, MnKm, MalKm, NADP)] at least four orders of magnitude smaller than the WT value. This difference corresponds to an increase of 6.38 kcal/mol energy barrier for the catalytic efficiency. Mutation at Asp464, on the other hand, has partial additivity on the mutations at Asp141 and Asp194. The overall specificity constants for the double mutants D(194,464)N and D(141,464)N or the triple mutant D(141,194,464)N were decreased by only 10‐ to 100‐fold compared to the WT. These results strongly suggest the involvement of Asp141 in the Mn2+‐L‐malate binding for the pigeon liver malic enzyme. The Asp194 and Asp464, which may be oxidized by nonspecific binding of Cu2+, are involved in the Mn2+‐L‐malate binding or catalysis indirectly by modulating the binding affinity of Asp141 with the Mn2+.
Structure‐based thermodynamic analysis of the dissociation of protein phosphatase‐1 catalytic subunit and microcystin‐LR docked complexes
Authors: Lavigne Pierre , Willard Leigh , Sykes Brian D. , Bagu John R. , Boyko Robert , Holmes Charles E. B.
Published: October 2010

The relationship between the structure of a free ligand in solution and the structure of its bound form in a complex is of great importance to the understanding of the energetics and mechanism of molecular recognition and complex formation. In this study, we use a structure‐based thermodynamic approach to study the dissociation of the complex between the toxin microcystin‐LR (MLR) and the catalytic domain of protein phosphatase‐1 (PP‐lc) for which the crystal structure of the complex is known. We have calculated the thermodynamic parameters (enthalpy, entropy, heat capacity, and free energy) for the dissociation of the complex from its X‐ray structure and found the calculated dissociation constant (4.0 × 10−11) to be in excellent agreement with the reported inhibitory constant (3.9 × 10−11). We have also calculated the thermodynamic parameters for the dissociation of 47 PP‐1c:MLR complexes generated by docking an ensemble of NMR solution structures of MLR onto the crystal structure of PP‐1c. In general, we observe that the lower the root‐mean‐square deviation (RMSD) of the docked complex (compared to the X‐ray complex) the closer its free energy of dissociation (δG°d) is to that calculated from the X‐ray complex. On the other hand, we note a significant scatter between the δG°d and the RMSD of the docked complexes. We have identified a group of seven docked complexes with δG°d values very close to the one calculated from the X‐ray complex but with significantly dissimilar structures. The analysis of the corresponding enthalpy and entropy of dissociation shows a compensation effect suggesting that MLR molecules with significant structural variability can bind PP‐1c and that substantial conformational flexibility in the PP‐1c:MLR complex may exist in solution.
Substrate‐ and pH‐dependent contribution of oxyanion binding site to the catalysis of prolyl oligopeptidase, a paradigm of the serine oligopeptidase family
Authors: Szeltner Zoltán , Renner Veronika , Polgár László
Published: October 2010

Prolyl oligopeptidase, an enzyme implicated in memory disorders, is a member of a new serine peptidase family. Crystallographic studies (Fülöp et al., 1998) revealed a novel oxyanion binding site containing a tyrosine residue, Tyr473. To study the importance of Tyr473 OH, we have produced prolyl oligopeptidase and its Tyr473Phe variant in Escherichia coli. The specificity rate constant, kcat/Km, for the modified enzyme decreased by a factor of 8–40 with highly specific substrates, Z‐Gly‐Pro‐Nap, and a fluorogenic octapeptide. With these compounds, the decline in kcat was partly compensated for by reduction in Km, a difference from the extensively studied subtilisin. With the less specific suc‐Gly‐Pro‐Nap, the Km value, which approximates Ks, was not significantly changed, resulting in greater diminution (∼500‐fold) in kcat/Km. The second‐order rate constant for the reaction with Z‐Pro‐prolinal, a slow tight‐binding transition‐state analogue inhibitor, and the Kj values for a slow substrate and two product‐like inhibitors were not significantly affected by the Tyr473 OH group. The mechanism of transition‐state stabilization was markedly dependent upon the nature of substrate and varied with pH as the enzyme interconverted between its two catalytically competent forms.
Contribution of proton linkage to the thermodynamic stability of the major cold‐shock protein of Escherichia coli CspA
Authors: Petrosian Susan A. , Makhatadze George I.
Published: October 2010

The stability of protein is defined not only by the hydrogen bonding, hydrophobic effect, van der Waals interactions, and salt bridges. Additional, much more subtle contributions to protein stability can arise from surface residues that change their properties upon unfolding. The recombinant major cold shock protein of Escherichia coli CspA an all‐β protein unfolds reversible in a two‐state manner, and behaves in all other respects as typical globular protein. However, the enthalpy of CspA unfolding strongly depends on the pH and buffer composition. Detailed analysis of the unfolding enthalpies as a function of pH and buffers with different heats of ionization shows that CspA unfolding in the pH range 5.5–9.0 is linked to protonation of an amino group. This amino group appears to be the N‐terminal α‐amino group of the CspA molecule. It undergoes a 1.6 U shift in pKa values between native and unfolded states. Although this shift in pKa is expected to contribute ∼5 kJ/mol to CspA stabilization energy the experimentally observed stabilization is only ∼1 kJ/mol. This discrepancy is related to a strong enthalpy—entropy compensation due, most likely, to the differences in hydration of the protonated and deprotonated forms of the α‐amino group.
Entropic barriers, transition states, funnels, and exponential protein folding kinetics: A simple model
Authors: Bicout D. J. , Szabo Attila
Published: October 2010

This paper presents an analytically tractable model that captures the most elementary aspect of the protein folding problem, namely that both the energy and the entropy decrease as a protein folds. In this model, the system diffuses within a sphere in the presence of an attractive spherically symmetric potential. The native state is represented by a small sphere in the center, and the remaining space is identified with unfolded states. The folding temperature, the time‐dependence of the populations, and the relaxation rate are calculated, and the folding dynamics is analyzed for both golf‐course and funnel‐like energy landscapes. This simple model allows us to illustrate a surprising number of concepts including entropic barriers, transition states, funnels, and the origin of single exponential relaxation kinetics.
Cytochrome c folds through a smooth funnel
Authors: Panda Markandeswar , Benavides‐Garcia Maria G. , Pierce Michael M. , Nall Barry T.
Published: October 2010

A dominant feature of folding of cytochrome c is the presence of nonnative His‐heme kinetic traps, which either pre‐exist in the unfolded protein or are formed soon after initiation of folding. The kinetically trapped species can constitute the majority of folding species, and their breakdown limits the rate of folding to the native state. A temperature jump (T‐jump) relaxation technique has been used to compare the unfolding/folding kinetics of yeast iso‐2 cytochrome c and a genetically engineered double mutant that lacks His‐heme kinetic traps, H33N, H39K iso‐2. The results show that the thermodynamic properties of the transition states are very similar. A single relaxation time τobs is observed for both proteins by absorbance changes at 287 nm, a measure of solvent exclusion from aromatic residues. At temperatures near Tm, the midpoint of the thermal unfolding transitions, τobs is four to eight times faster for H33N, H39K iso‐2 (τobs ∼ 4–10 ms) than for iso‐2 (τobs ∼ 20–30 ms). T‐jumps show that there are no kinetically unresolved (τ < 1–3 mus t‐jump dead time) “burst” phases for either protein. using a two‐state model, the folding (kf) and unfolding (ku) rate constants and the thermodynamic activation parameters ΔG‡f G‡u H‡f H‡u S‡f S‡u are evaluated by fitting the data to a function describing the temperature dependence of the apparent rate constant kobs (= τ−1obs−1) = kf + ku. The results show that there is a small activation enthalpy for folding, suggesting that the barrier to folding is largely entropic. In the “new view,” a purely entropic kinetic barrier to folding is consistent with a smooth funnel folding landscape.
Direct evidence by H/D exchange and ESI‐MS for transient unproductive domain interaction in the refolding of an antibody scFv fragment
Authors: Jäger Marcus , Plückthun Andreas
Published: October 2010

The refolding kinetics of a single‐chain Fv (scFv) fragment, derived from a stabilized mutant of the phosphorylcholine binding antibody McPC603, was investigated by H/D exchange and ESI‐MS and compared with the folding kinetics of its constituting domains VH and VL. Both VH and VL adopt essentially native‐like exchange protection within the dead time of the manual‐mixing H/D exchange experiment (10 s) and in the case of VL, which contains two cis‐prolines in the native conformation, this fast protection is independent of proline cis/trans isomerization. At the earliest time point resolvable by manual mixing, fewer deuterons are protected in the scFv fragment than in the two isolated domains together, despite the fact that the scFv fragment is significantly more stable than VL and VH. Full H/D exchange protection in the scFv fragment is gained on a time scale of minutes. This means that the domains in the scFv fragment do not refold independently. Rather, they associate prematurely and in nonnative form, a kinetic trap. Unproductive domain association is observed both after equilibrium‐ and short‐term denaturation. For the equilibrium‐denatured scFv fragment, whose native structure formation is dependent on a cis conformation of an interface proline in VL, this cis/trans isomerization reaction proceeds about one order in magnitude more slowly than the escape from the trap to a conformation where full H/D exchange protection is already achieved. We interpret these data in terms of a general kinetic scheme involving intermediates with and without domain association.
Oxygen binding by α(Fe2+)2β(Ni2+)2 hemoglobin crystals
Authors: Bruno Stefano , Bettati Stefano , Manfredini Michele , Mozzarelli Andrea , Bolognesi Martino , Deriu Daniela , Rosano Camillo , Tsuneshige Antonio , Yonetani Takashi , Henry Eric R.
Published: October 2010

Oxygen binding by hemoglobin fixed in the T state either by crystallization or by encapsulation in silica gels is apparently noncooperative. However, cooperativity might be masked by different oxygen affinities of α and β subunits. Metal hybrid hemoglobins, where the noniron metal does not bind oxygen, provide the opportunity to determine the oxygen affinities of α and β hemes separately. Previous studies have characterized the oxygen binding by α(Ni2+)2β(Fe2+)2 crystals. Here, we have determined the three‐dimensional (3D) structure and oxygen binding of α(Fe2+)2β(Ni2+)2 crystals grown from polyethylene glycol solutions. Polarized absorption spectra were recorded at different oxygen pressures with light polarized parallel either to the b or c crystal axis by single crystal microspectrophotometry. The oxygen pressures at 50% saturation (p50s) are 95 ± 3 and 87 ± 4 Torr along the b and c crystal axes, respectively, and the corresponding Hill coefficients are 0.96 ± 0.06 and 0.90 ± 0.03. Analysis of the binding curves, taking into account the different projections of the α hemes along the optical directions, indicates that the oxygen affinity of α1 hemes is 1.3‐fold lower than α2 hemes. Inspection of the 3D structure suggests that this inequivalence may arise from packing interactions of the Hb tetramer within the monoclinic crystal lattice. A similar inequivalence was found for the β subunits of α(Ni2+)2β(Fe2+)2 crystals. The average oxygen affinity of the α subunits (p50 = 91 Torr) is about 1.2‐fold higher than the β subunits (p50 =110 Torr). In the absence of cooperativity, this heterogeneity yields an oxygen binding curve of Hb A with a Hill coefficient of 0.999. Since the binding curves of Hb A crystals exhibit a Hill coefficient very close to unity, these findings indicate that oxygen binding by T‐state hemoglobin is noncooperative, in keeping with the Monod, Wyman, and Changeux model.
NMR solution structure of the θ subunit of DNA polymerase III from Escherichia coli
Authors: Keniry Max A. , Berthon Hilary A. , Yang Ji Yeon , Miles Caroline S. , Dixon Nicholas E.
Published: October 2010

The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits (α, €, and θ). The θ subunit is the smallest, but the least understood of the three. As a first step in a program aimed at understanding its function, the structure of the θ subunit has been determined by triple‐resonance multidimensional NMR spectroscopy. Although only a small protein, θ was difficult to assign fully because approximately one‐third of the protein is unstructured, and some sections of the remaining structured parts undergo intermediate intramolecular exchange. The secondary structure was deduced from the characteristic nuclear Overhauser effect patterns, the 3JHNα coupling constants and the consensus chemical shift index. The C‐terminal third of the protein, which has many charged and hydrophilic amino acid residues, has no well‐defined secondary structure and exists in a highly dynamic state. The N‐terminal two‐thirds has three helical segments (Gln10‐Asp19, Glu38‐Glu43, and His47‐Glu54), one short extended segment (Pro34‐Ala37), and a long loop (Ala20‐Glu29), of which part may undergo intermediate conformational exchange. Solution of the three‐dimensional structure by NMR techniques revealed that the helices fold in such a way that the surface of θ is bipolar, with one face of the protein containing most of the acidic residues and the other face containing most of the long chain basic residues. Preliminary chemical shift mapping experiments with a domain of the € subunit have identified a loop region (Ala20‐Glu29) in θ as the site of association with €.
The sarcosine effect on protein stability: A case of nonadditivity?
Authors: Ibarra‐Molero Beatriz , Del Pino Isabel M. Plaza , Souhail Badredine , Hammou Hassan O. , Sanchez‐Ruiz Jose M.
Published: October 2010

We have used differential scanning calorimetry to determine the effect of low concentrations (C = 0–2 M) of the osmolyte sarcosine on the Gibbs energy changes (δG) for the unfolding of hen‐egg‐white lysozyme, ribonuclease A, and ubiq‐uitin, under the same buffer and pH conditions. We have also computed this effect on the basis of the additivity assumption and using published values of the transfer Gibbs energies for the amino acid side chains and the peptide backbone unit. The values thus predicted for the slope ∂ΔLG/∂C agree with the experimental ones, but only if the unfolded state is assumed to be compact (that is, if the accessibility to solvent of the unfolded state is modeled using segments excised from native structures). The additivity‐based calculations predict similar ∂ΔLG/∂C values for the three proteins studied. We point out that, to the extent that this approximate constancy of ∂ΔLG/∂C holds, osmolyte‐induced increases in denaturation temperature will be larger for proteins with low unfolding enthalpy (small proteins that bury a large proportion of apolar surface). The experimental results reported here are consistent with this hypothesis.
Infection of Arabidopsis thaliana by Phytophthora parasitica and identification of variation in host specificity
Authors: WANG YAN , MENG YULING , ZHANG MENG , TONG XINMENG , WANG QINHU , SUN YINYIN , QUAN JUNLI , GOVERS FRANCINE , SHAN WEIXING
Published: October 2010

Oomycete pathogens cause severe damage to a wide range of agriculturally important crops and natural ecosystems. They represent a unique group of plant pathogens that are evolutionarily distant from true fungi. In this study, we established a new plant–oomycete pathosystem in which the broad host range pathogen Phytophthora parasitica was demonstrated to be capable of interacting compatibly with the model plant Arabidopsis thaliana. Water‐soaked lesions developed on leaves within 3 days and numerous sporangia formed within 5 days post‐inoculation of P. parasitica zoospores. Cytological characterization showed that P. parasitica developed appressoria‐like swellings and penetrated epidermal cells directly and preferably at the junction between anticlinal host cell walls. Multiple haustoria‐like structures formed in both epidermal cells and mesophyll cells 1 day post‐inoculation of zoospores. Pathogenicity assays of 25 A. thaliana ecotypes with six P. parasitica strains indicated the presence of a natural variation in host specificity between A. thaliana and P. parasitica. Most ecotypes were highly susceptible to P. parasitica strains Pp014, Pp016 and Pp025, but resistant to strains Pp008 and Pp009, with the frequent appearance of cell wall deposition and active defence response‐based cell necrosis. Gene expression and comparative transcriptomic analysis further confirmed the compatible interaction by the identification of up‐regulated genes in A. thaliana which were characteristic of biotic stress. The established A. thaliana–P. parasitica pathosystem expands the model systems investigating oomycete–plant interactions, and will facilitate a full understanding of Phytophthora biology and pathology.
The health of hospitals and lessons from history: public health and sanitary reform in the Dublin hospitals, 1858–1898
Authors: Fealy Gerard M , McNamara Martin S , Geraghty Ruth
Published: October 2010

Aims and objectives.  The aim was to examine, critically, 19th century hospital sanitary reform with reference to theories about infection and contagion.

Background.  In the nineteenth century, measures to control epidemic diseases focused on providing clean water, removing waste and isolating infected cases. These measures were informed by the ideas of sanitary reformers like Chadwick and Nightingale, and hospitals were an important element of sanitary reform.

Design.  Informed by the paradigmatic tradition of social history, the study design was a historical analysis of public health policy.

Method.  Using the methods of historical research, documentary primary sources, including official reports and selected hospital archives and related secondary sources, were consulted.

Results.  Emerging theories about infection were informing official bodies like the Board of Superintendence of Dublin Hospitals in their efforts to improve hospital sanitation. The Board secured important reforms in hospital sanitation, including the provision of technically efficient sanitary infrastructure.

Conclusions.  Public health measures to control epidemic infections are only as effective as the state of knowledge of infection and contagion and the infrastructure to support sanitary measures.

Relevance to clinical practice.  Today, public mistrust about the safety of hospitals is reminiscent of that of 150 years ago, although the reasons are different and relate to a fear of contracting antimicrobial‐resistant infections. A powerful historical lesson from this study is that resistance to new ideas can delay progress and improved sanitary standards can allay public mistrust. In reforming hospital sanitation, policies and regulations were established – including an inspection body to monitor and enforce standards – the benefits of which provide lessons that resonate today. Such practices, especially effective independent inspection, could be adapted for present‐day contexts and re‐instigated where they do not exist. History has much to offer contemporary policy development and practice reform and is a relevant method for health professionals.
Injuries, falls and accidents among adults with intellectual disabilities. Prospective cohort study
Authors: Finlayson J. , Morrison J. , Jackson A. , Mantry D. , Cooper S.‐A.
Published: October 2010

Background  Injuries are among the leading causes of death and disability in the world and a major public health concern. Falls are a common cause. Young persons with intellectual disabilities (ID) have a higher rate and different pattern of injuries than the general population, but little is known regarding adults.

Methods  The aim of this study was to determine the incidence and types of injuries experienced by a community‐based cohort of adults with ID (n = 511) in a 12‐month period. Face‐to‐face interviews were conducted with participants 2 years after they had first been recruited into a longitudinal study.

Results  Incidence of at least one injury in a 12‐month period was 20.5% (105), of which 12.1% (62) was because of falls. Incident injury was predicted by having epilepsy and not having autism. Incident fall injury was predicted by urinary incontinence, while Down syndrome reduced risk.

Conclusions  Adults with ID do experience a higher rate of injuries and falls when compared with the general population. The results of this study highlight this, and hence the need to work towards the development of interventions for injury and falls prevention in this population.
The effect of configuration on VSWM performance of Down syndrome individuals
Authors: Carretti B. , Lanfranchi S.
Published: October 2010

Background  Recent studies have demonstrated that individuals with Down syndrome (DS) are poorer than controls in spatial‐simultaneous tasks, but not in spatial‐sequential tasks. To explain this finding, it has been suggested that the simultaneous visuo‐spatial working memory deficit of individuals with DS could be due to the request for processing more than one item at a time. The present study examines the possibility of reducing the difficulties encountered by DS individuals on spatial‐simultaneous tasks by proposing a task that uses structured material.

Method  Two tasks were administered to 20 children and adolescents with DS, and 20 controls matched for verbal mental age. The tasks involved memorising and recalling filled‐cell positions in a series of matrices in which an increasing number of cells (1–7) were filled. In the structured condition the filled cells were displayed to form a pattern, whereas in the random condition they were randomly arranged.

Results and Conclusions  Results showed that individuals with DS are able to take advantage of structured material for raising their performance, but to a less extent than typically developing children.
Association of caspase‐9 and RUNX3 with inflammatory bowel disease
Authors: Guo C. , Ahmad T. , Beckly J. , Cummings J. R. F. , Hancock L. , Geremia A. , Cooney R. , Pathan S. , Jewell D. P.
Published: October 2010

Previous linkage studies have identified a region at 1p36 as the susceptibility locus (IBD7) of inflammatory bowel disease (IBD). The objective of this study was to investigate whether polymorphisms of caspase‐9 (CASP9) gene and RUNX3 are associated with IBD susceptibility and clinical phenotypes. We studied 555 Crohn's disease (CD) and 651 ulcerative colitis (UC) patients recruited from a single UK center. A total of 964 healthy Caucasian subjects were recruited as controls from general practitioner well person clinics in Oxfordshire. Fourteen single nucleotide polymorphisms (SNPs) of CASP9 and 11 SNPs of RUNX3 were genotyped using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) (homogenous MassEXTEND, hME, Sequenom™, Sequenom Inc., San Diego, CA). Linkage disequilibrium (LD) and haplotype association analysis were performed using 2ld and phase v2.0 software. No association of individual SNPs of CASP9 or RUNX3 with UC or CD was identified. The rs1052571 of CASP9 was associated with severe UC [P = 0.0034, odds ratio (OR) = 1.957, 95% confidence interval (CI) = 1.240–3.088]. Significant haplotype associations between CASP9 and IBD were identified, while no association of RUNX3 haplotypes with either UC or CD was found. Our findings suggested that CASP9 gene might be another IBD susceptibility gene.