bluuebunny/crossref_metadata_2025 huggingface dataset:
The de novo design of a rubredoxin‐like fe site
Authors: Farinas Edgardo , Regan Lynne
Published: February 2009

A redox center similar to that of rubredoxin was designed into the 56 amino acid immunoglobulin binding B1 domain of Streptococcals protein G. The redox center in rubredoxin contains an iron ion tetrahedrally coordinated by four cysteine residues, [Fe(S‐Cys)4]−1,‐2. The design criteria for the target site included taking backbone movements into account, tetrahedral metal‐binding, and maintaining the structure and stability of the wild‐type protein. The optical absorption spectrum of the Co(II) complex of the metal‐binding variant is characteristic of tetrahedral chelation by four cysteine residues. Circular dichroism and nuclear magnetic resonance measurements reveal that the metal‐free and Cd(II)‐bound forms of the variant are folded correctly and are stable. The Fe(III) complex of the metal‐binding mutant reproduces the optical and the electron paramagnetic resonance spectra of oxidized rubredoxin. This demonstrates that the engineered protein chelates Fe(III) in a tetrahedral array, and the resulting center is similar to that of oxidized rubredoxin.
The structure of serine hydroxymethyltransferase as modeled by homology and validated by site‐directed mutagenesis
Authors: Pascarella Stefano , Angelaccio Sebastiana , Contestabile Roberto , Fratte Sonia Delle , Salvo Martino Di , Bossa Francesco
Published: February 2009

We describe a model for the three‐dimensional structure of E. coli serine hydroxymethyltransferase based on its sequence homology with other PLP enzymes of theα‐family and whose tertiary structures are known. The model suggests that certain amino acid residues at the putative active site of the enzyme can adopt specific roles in the catalytic mechanism. These proposals were supported by analysis of the properties of a number of site‐directed mutants. New active site features are also proposed for further experimental testing.
Compactness of the kinetic molten globule of bovineα‐lactalbumin: A dynamic light scattering study
Authors: Gast Klaus , Zirwer Dietrich , MÜller‐Frohne Marlies , Damaschun Gregor
Published: February 2009

During folding of globular proteins, the molten globule state was observed as an equilibrium intermediate under mildly denaturing conditions as well as a transient intermediate in kinetic refolding experiments. While the high compactness of the equilibrium intermediate ofα‐lactalbumin has been verified, direct measurements of the compactness of the kinetic intermediate have not been reported until now. Our dynamic light scattering measurements provide a complete set of the hydrodynamic dimensions of bovineα‐lactalbumin in different conformational states, particularly in the kinetic molten globule state. The Stokes radii for the native, kinetic molten globule, equilibrium molten globule, and unfolded states are 1.91, 1.99, 2.08, and 2.46 nm, respectively. Therefore, the kinetic intermediate appears to be even more compact than its equilibrium counterpart. Remarkable differences in the concentration dependence of the Stokes radius exist revealing strong attractive but repulsive intermolecular interactions in the kinetic and equilibrium molten globule states, respectively. This underlines the importance of extrapolation to zero protein concentration in measurements of the molecular compactness.
The role of quaternary interactions on the stability and activity of ascorbate peroxidase
Authors: Mandelman David , Li Huiying , Poulos Thomas L. , Schwarz Frederick P.
Published: February 2009

Point mutations at the dimer interface of the homodimeric enzyme ascorbate peroxidase (APx) were constructed to assess the role of quaternary interactions in the stability and activity of APx. Analysis of the APx crystal structure shows that Glu 112 forms a salt bridge with Lys20 and Arg24 of the opposing subunit near the axis of dyad symmetry between the subunits. Two point mutants, E112A and E112K, were made to determine the effects of a neutral (alanine) and repulsive (lysine) mutation on dimerization, stability, and activity. Gel filtration analysis indicated that the ratio of the monomer to dimer increased as the dimer interface interactions went from attractive to repulsive. Differential scanning calorimetry (DSC) data exhibited a decrease in both the transition temperature (Tm) and enthalpy of unfolding (ΔHc) with Tm = 58.3 ± 0.5 °C, 56.0 ± 0.8 °C, and 53.0 ± 0.9 °C and ΔHc = 245 ± 29 kcal/mol, 199 ± 38 kcal/mol, and 170 ± 25 kcal/mol for wild‐type APx, E112A, and E112K, respectively. Similar changes were observed based on thermal melting curves obtained by absorption spectroscopy. No change in enzyme activity was found for the E112A mutant, and only a 25% drop in activity was observed for the El 12K mutant which demonstrates that the non‐Michaelis Menten kinetics of APx is not due to the APx oligomeric structure. The cryogenic crystal structures of the wild‐type and mutant proteins show that mutation induced changes are limited to the dimer interface including an alteration in solvent structure.
Structural characterizations of nonpeptidic thiadiazole inhibitors of matrix metalloproteinases reveal the basis for stromelysin selectivity
Authors: Finzel B. C. , Baldwin E. T. , Bryant G. L. , Hess G. F. , Wilks J. W. , Trepod C. M. , Mott J. E. , Marshall V. P. , Petzold G. L. , Poorman R. A. , O'Sullivan T. J. , Schostarez H. J. , Mitchell M. A.
Published: February 2009

The binding of two 5‐substituted‐l,3,4‐thiadiazole‐2‐thione inhibitors to the matrix metalloproteinase stromelysin (MMP‐3) have been characterized by protein crystallography. Both inhibitors coordinate to the catalytic zinc cation via an exocyclic sulfur and lay in an unusual position across the unprimed (P1‐P3) side of the proteinase active site. Nitrogen atoms in the thiadiazole moiety make specific hydrogen bond interactions with enzyme structural elements that are conserved across all enzymes in the matrix metalloproteinase class. Strong hydrophobic interactions between the inhibitors and the side chain of tyrosine‐155 appear to be responsible for the very high selectivity of these inhibitors for stromelysin. In these enzyme/inhibitor complexes, the S1′ enzyme subsite is unoccupied. A conformational rearrangement of the catalytic domain occurs that reveals an inherent flexibility of the substrate binding region leading to speculation about a possible mechanism for modulation of stromelysin activity and selectivity.
Construction, expression, and characterization of a novel fully activated recombinant single‐chain hepatitis C virus protease
Authors: Taremi S. Shane , Beyer Brian , Maher Maureen , Yao Nanhua , Prosise Winifred , Weber Patricia C. , Malcolm Bruce A.
Published: February 2009

Efficient proteolytic processing of essential junctions of the hepatitis C virus (HCV) polyprotein requires a heterodimeric complex of the NS3 bifunctional protease/helicase and the NS4A accessory protein. A single‐chain recombinant form of the protease has been constructed in which NS4A residues 21‐32 (GSVVIVGRIILS) were fused in frame to the amino terminus of the NS3 protease domain (residues 3ndash181) through a tetrapeptide linker. The single‐chain recombinant protease has been overexpressed as a soluble protein in E. coli and purified to homogeneity by a combination of metal chelate and size‐exclusion chromatography. The single‐chain recombinant protease domain shows full proteolytic activity cleaving the NS5A‐5B synthetic peptide substrate, DTEDVVCCSMSYTWTGK with a Km and kcat of 20.0 ± 2.0 μM and 9.6 ± 2.0 min−1, respectively; parameters identical to those of the authentic NS31_631/NS4A1_54 protein complex generated in eukaryotic cells (Sali DL et al., 1998, Biochemistry 37:3392‐3401).
Structural characterization of the entire 1.3S subunit of transcarboxylase from Propionibacterium shermanii
Authors: Reddy D. Venkat , Rothemund Sven , Sönnichsen Frank D. , Shenoy Bhami C. , Carey Paul R.
Published: February 2009

Transcarboxylase (TC) from Propionibacterium shermanii, a biotin‐dependent enzyme, catalyzes the transfer of a carboxyl group from methylmalonyl‐CoA to pyruvate in two partial reactions. Within the multisubunit enzyme complex, the 1.3S subunit functions as the carboxyl group carrier. The 1.3S is a 123‐amino acid polypeptide (12.6 kDa), to which biotin is covalently attached at Lys 89. We have expressed 1.3S in Escherichia coli with uniform 15N labeling. The backbone structure and dynamics of the protein have been characterized in aqueous solution by three‐dimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy. The secondary structure elements in the protein were identified based on NOE information, secondary chemical shifts, homonuclear 3Jhnhα coupling constants, and amide proton exchange data. The protein contains a predominantly disordered N‐terminal half, while the C‐terminal half is folded into a compact domain comprising eight β‐strands connected by short loops and turns. The topology of the C‐terminal domain is consistent with the fold found in both carboxyl carrier and lipoyl domains, to which this domain has approximately 26‐30% sequence similarity.
Topological mimicry and epitope duplication in the guanylyl cyclase C receptor
Authors: Nandi Animesh , Visweswariah Sandhya S. , Suguna K. , Surolia Avadhesha
Published: February 2009

Guanylyl cyclase C (GCC) is the receptor for the gastrointestinal hormones, guanylin, and uroguanylin, in addition to the bacterial heat‐stable enterotoxins, which are one of the major causes of watery diarrhea the world over. GCC is expressed in intestinal cells, colorectal tumor tissue and tumors originating from metastasis of the colorectal carcinoma. We have earlier generated a monoclonal antibody to human GCC, GCC:B10, which was useful for the immunohisto‐chemical localization of the receptor in the rat intestine (Nandi A et al., 1997, J Cell Biochem 66:500‐511), and identified its epitope to a 63‐amino acid stretch in the intracellular domain of GCC. In view of the potential that this antibody has for the identification of colorectal tumors, we have characterized the epitope for GCC:B10 in this study. Overlapping peptide synthesis indicated that the epitope was contained in the sequence HIPPENIFPLE. This sequence was unique to GCC, and despite a short stretch of homology with serum amyloid protein and pertussis toxin, no cross reactivity was detected. The core epitope was delineated using a random hexameric phage display library, and two categories of sequences were identified, containing either a single, or two adjacent proline residues. No sequence identified by phage display was identical to the epitope present in GCC, indicating that phage sequences represented mimotopes of the native epitope. Alignment of these sequences with HIPPENIFPLE suggested duplication of the recognition motif, which was confirmed by peptide synthesis. These studies allowed us not only to define the requirements of epitope recognition by GCC:B10 monoclonal antibody, but also to describe a novel means of epitope recognition involving topological mimicry and probable duplication of the cognate epitope in the native guanylyl cyclase C receptor sequence.
Effects of pressure on the structure of metmyoglobin: Molecular dynamics predictions for pressure unfolding through a molten globule intermediate
Authors: Floriano Wely B. , Nascimento Marco A. C. , Domont Gilberto B. , Goddard William A.
Published: February 2009

We investigated the pathway for pressure unfolding of metmyoglobin using molecular dynamics (MD) for a range of pressures (0. 1 MPa to 1. 2 GPa) and a temperature of 300 K. We find that the unfolding of metmyoglobin proceeds via a two‐step mechanism native → molten globule intermediate → unfolded, where the molten globule forms at 700 MPa. The simulation describes qualitatively the experimental behavior of metmyoglobin under pressure. We find that unfolding of the alpha‐helices follows the sequence of migrating hydrogen bonds (i, i + 4) → (i, i + 2).
Toward a universal inhibitor of retroviral proteases: Comparative analysis of the interactions of LP‐130 complexed with proteases from HIV‐1, FIV, and EIAV
Authors: Kervinen Jukka , Lubkowski Jacek , Zdanov Alexander , Bhatt Deepa , Dunn Ben M. , Hui Kwan Y. , Powell David J. , Kay John , Wlodawer Alexander , Gustchina Alla
Published: February 2009

One of the major problems encountered in antiviral therapy against AIDS is the emergence of viral variants that exhibit drug resistance. The sequences of proteases (PRs) from related retroviruses sometimes include, at structurally equivalent positions, amino acids identical to those found in drug‐resistant forms of HIV‐1 PR. The statine‐based inhibitor LP‐130 was found to be a universal, nanomolar‐range inhibitor against all tested retroviral PRs. We solved the crystal structures of LP‐130 in complex with retroviral PRs from HIV‐1, feline immunodeficiency virus, and equine infectious anemia virus and compared the structures to determine the differences in the interactions between the inhibitor and the active‐site residues of the enzymes. This comparison shows an extraordinary similarity in the binding modes of the inhibitor molecules. The only exceptions are the different conformations of naphthylalanine side chains at the P3/P3′ positions, which might be responsible for the variation in the Ki values. These findings indicate that successful inhibition of different retroviral PRs by LP‐130 is achieved because this compound can be accommodated without serious conformational differences, despite the variations in the type of residues forming the active‐site region. Although strong, specific interactions between the ligand and the enzyme might improve the potency of the inhibitor, the absence of such interactions seems to favor the universality of the compound. Hence, the ability of potential anti‐AIDS drugs to inhibit multiple retroviral PRs might indicate their likelihood of not eliciting drug resistance. These studies may also contribute to the development of a small‐animal model for preclinical testing of antiviral compounds.
The equilibrium unfolding of Azotobacter vinelandii apoflavodoxin II occurs via a relatively stable folding intermediate
Authors: Van Mierlo Carlo P. M. , Van Dongen Walter M. A. M. , Vergeldt Frank , Van Berkel Willem J. H. , Steensma Elles
Published: February 2009

A flavodoxin from Azotobacter vinelandii is chosen as a model system to study the folding of α/ß doubly wound proteins. The guanidinium hydrochloride induced unfolding of apoflavodoxin is demonstrated to be reversible. Apoflavodoxin thus can fold in the absence of the FMN cofactor. The unfolding curves obtained for wild‐type, C69A and C69S apoflavodoxin as monitored by circular dichroism and fluorescence spectroscopy do not coincide. Apoflavodoxin unfolding occurs therefore not via a simple two‐state mechanism. The experimental data can be described by a three‐state mechanism of apoflavodoxin equilibrium unfolding in which a relatively stable intermediate is involved. The intermediate species lacks the characteristic tertiary structure of native apoflavodoxin as deduced from fluorescence spectroscopy, but has significant secondary structure as inferred from circular dichroism spectroscopy. Both spectroscopic techniques show that thermally‐induced unfolding of apoflavodoxin also proceeds through formation of a similar molten globule‐like species. Thermal unfolding of apoflavodoxin is accompanied by anomalous circular dichroism characteristics: the negative ellipticity at 222 nM increases in the transition zone of unfolding. This effect is most likely attributable to changes in tertiary interactions of aromatic side chains upon protein unfolding. From the presented results and hydrogen/deuterium exchange data, a model for the equilibrium unfolding of apoflavodoxin is presented.
pH‐Induced conformational changes of membrane‐bound influenza hemagglutinin and its effect on target lipid bilayers
Authors: Gray Cameron , Tamm Lukas K.
Published: February 2009

Influenza virus hemagglutinin (HA) has served as a paradigm for both pH‐dependent and ‐independent viral membrane fusion. Although large conformational changes were observed by X‐ray crystallography when soluble fragments of HA were subjected to fusion‐pH conditions, it is not clear whether the same changes occur in membrane‐bound HA, what the spatial relationship is between the conformationally changed HA and the target and viral membranes, and in what way HA perturbs the target membrane at low pH. We have taken a spectroscopic approach using an array of recently developed FTIR techniques to address these questions. Difference attenuated total reflection FTIR spectroscopy was employed to reveal reversible and irreversible components of the pH‐induced conformational change of the membrane‐bound bromelain fragment of HA, BHA. Additional proteolytic fragments of BHA were produced which permitted a tentative assignment of the observed changes to the HA1 and HA2 subunits, respectively. The membrane‐bound HA1 subunit undergoes a reversible conformational change, which most likely involves the loss of a small proportion of ß‐sheet at low pH. BHA was found to undergo a partially reversible tilting motion relative to the target membrane upon exposure to pH 5, indicating a previously undescribed hinge near the anchoring point to the target membrane. Time‐resolved amide H/D exchange experiments revealed a more dynamic (tertiary) structure of membrane‐bound BHA and its HA2, but not its HA1, subunit. Finally BHA and, to a lesser degree, HA1 perturbed the lipid bilayer of the target membrane at the interface, as assessed by spectral changes of the lipid ester carbonyl groups. These results are discussed in the context of a complementary study of HA that was bound to viral membranes through its transmembrane peptide (Gray C, Tamm LK, 1997, Protein Sci 6: 1993–2006). A distinctive role for the HA1 subunit in the conformational change of HA becomes apparent from these combined studies.
Addition of side‐chain interactions to 310‐helix/coil and α‐helix/310‐helix/coil theory
Authors: Sun Jia Ke , Doig Andrew J.
Published: February 2009

An increasing number of experimental and theoretical studies have demonstrated the importance of the 310‐helix/α‐helix/coil equilibrium for the structure and folding of peptides and proteins. One way to perturb this equilibrium is to introduce side‐chain interactions that stabilize or destabilize one helix. For example, an attractive i, i + 4 interaction, present only in the α‐helix, will favor the α‐helix over 310, while an i, i + 4 repulsion will favor the 310‐helix over α. To quantify the 310/α/coil equilibrium, it is essential to use a helix/coil theory that considers the stability of every possible conformation of a peptide. We have previously developed models for the 310‐helix/coil and 310‐helix/α‐helix/coil equilibria. Here we extend this work by adding i, i + 3 and i, i + 4 side‐chain interaction energies to the models. The theory is based on classifying residues into α‐helical, 310‐helical, or nonhelical (coil) conformations. Statistical weights are assigned to residues in a helical conformation with an associated helical hydrogen bond, a helical conformation with no hydrogen bond, an N‐cap position, a C‐cap position, or the reference coil conformation plus i, i + 3 and i, i + 4 side‐chain interactions. This work may provide a framework for quantitatively rationalizing experimental work on isolated 310‐helices and mixed 310‐/α‐helices and for predicting the locations and stabilities of these structures in peptides and proteins. We conclude that strong i, i + 4 side‐chain interactions favor α‐helix formation, while the 310‐helix population is maximized when weaker i, i + 4 side‐chain interactions are present.
Identification of tryptophan oxidation products in bovine α‐crystallin
Authors: Finley Eric L. , Dillon James , Crouch Rosalie K. , Schey Kevin L.
Published: February 2009

Oxidation is known to affect the structure, activity, and rate of degradation of proteins, and is believed to contribute to a variety of pathological conditions. Metal‐catalyzed oxidation (MCO) is a primary oxidizing system in many cell types. In this study, the oxidative effects of a MCO system (the Fenton reaction) on the structure of the tryptophan residues of α‐crystallin were determined. Tandem mass spectrometry (MS/MS) was utilized to identify specific tryptophan and methionine oxidation products in the bovine α‐crystallin sequence. After oxidative exposure, α‐crystallin was digested with trypsin, and the resulting peptides were fractionated by reverse‐phase HPLC. Structural analysis by mass spectrometry revealed that tryptophan 9 of αA‐ and tryptophan 60 of αB‐crystallin were each converted into hydroxytryptophans (HTRP), N‐formylkynurenine (NFK), and kynurenine (KYN). However, only HTRP and KYN formation were detected at residue 9 of αB‐crystallin. Oxidation of methionine 1 of αA‐ and methionine 1 and 68 of αB‐crystallin was also detected. The products NFK and KYN are of particular importance in the lens, as they themselves are photosensitizers that can generate reactive oxygen species (ROS) upon UV light absorption. The unambiguous identification of HTRP, NFK, and KYN in intact α‐crystallin represents the first structural proof of the formation of these products in an intact protein, and provides a basis for detailed structural analysis of oxidized proteins generated in numerous pathological conditions.
Global analysis of the thermal and chemical denaturation of the N‐terminal domain of the ribosomal protein L9 in H2O and D2O. Determination of the thermodynamic parameters, ΔH°, ΔS°, and ΔC°p, and evaluation of solvent isotope effects
Authors: Kuhlman Brian , Raleigh Daniel P.
Published: February 2009

The stability of the N‐terminal domain of the ribosomal protein L9, NTL9, from Baccilus stearothermophilus has been monitored by circular dichroism at various temperatures and chemical denaturant concentrations in H2O and D2O. The basic thermodynamic parameters for the unfolding reaction, ΔH°, ΔS°, and ΔC°p, were determined by global analysis of temperature and denaturant effects on stability. The data were well fit by a model that assumes stability varies linearly with denaturant concentration and that uses the Gibbs‐Helmholtz equation to model changes in stability with temperature. The results obtained from the global analysis are consistent with information obtained from individual thermal and chemical denaturations. NTL9 has a maximum stability of 3. 78±0. 25 kcal mol−1 at 14°C. ΔH°(25°C) for protein unfolding equals 9. 9±0. 7 kcal mol−1 and TΔS°(25 °C) equals 6. 2±0. 6 kcal mol−1. Cp equals 0. 53±0. 06 kcal mol−1 deg−1. There is a small increase in stability when D2O is substituted for H2O. Based on the results from global analysis, NTL9 is 1. 06±0. 60 kcal mol−1 more stable in D2O at 25 °C and Tm is increased by 5. 8±3. 6°C in D2O. Based on the results from individual denaturation experiments, NTL9 is 0. 68±0. 68 kcal mol−1 more stable in D2O at 25°C and Tm is increased by 3. 5±2. 1°C in D2O. Within experimental error there are no changes in ΔH°(25°C) when D2O is substituted for H2O.
Prediction by a neural network of outer membrane β‐strand protein topology
Authors: Diederichs Kay , Freigang Jörg , Umhau Stephan , Zeth Kornelius , Breed Jason
Published: February 2009

An artificial neural network (NN) was trained to predict the topology of bacterial outer membrane (OM) ß‐strand proteins. Specifically, the NN predicts the z‐coordinate of Cα atoms in a coordinate frame with the outer membrane in the xy‐plane, such that low z‐values indicate periplasmic turns, medium z‐values indicate transmembrane ß‐strands, and high z‐values indicate extracellular loops. To obtain a training set, seven OM proteins (porins) with structures known to high resolution were aligned with their pores along the z‐axis. The relationship between Cα z‐values and topology was thereby established. To predict the topology of other OM proteins, all seven porins were used for the training set. Z‐values (topologies) were predicted for two porins with hitherto unknown structure and for OM proteins not belonging to the porin family, all with insignificant sequence homology to the training set. The results of topology prediction compare favorably with experimental topology data.
Sequential assignment of 1H, 15N, 13C resonances and secondary structure of human calmodulin‐like protein determined by NMR spectroscopy
Authors: Qian Hong , Rogers Michael S. , Schleucher Jürgen , Edlund Ulf , Strehler Emanuel E. , Sethson Ingmar
Published: February 2009

Human calmodulin‐like protein (CLP) is closely related to vertebrate calmodulin, yet its unique cell specific expression pattern, overlapping but divergent biochemical properties, and specific target proteins suggest that it is not an isoform of calmodulin. To gain insight into the structural differences that may underlie the difference target specificities and biochemical properties of CLP when compared to calmodulin, we determined the sequential backbone assignment and associated secondary structure of 144 out of the 148 residues of Ca2+‐CLP by using multinuclear multidimensional NMR spectroscopy. Despite a very high overall degree of structural similarity between CLP and calmodulin, a number of significant differences were found mainly in the length of α‐helices and in the central nonhelical flexible region. Interestingly, the regions of greatest primary sequence divergence between CLP and calmodulin in helices III and VIII displayed only minor secondary structure differences. The data suggest that the distinct differences in target specificity and biochemical properties of CLP and calmodulin result from the sum of several minor structural and side‐chain changes spread over multiple domains in these proteins.
Surface salt bridges stabilize the GCN4 leucine zipper
Authors: Spek Erik J. , Bui Au H. , Lu Min , Kallenbach Neville R.
Published: February 2009

We present a study of the role of salt bridges in stabilizing a simplified tertiary structural motif, the coiled‐coil. Changes in GCN4 sequence have been engineered that introduce trial patterns of single and multiple salt bridges at solvent exposed sites. At the same sites, a set of alanine mutants was generated to provide a reference for thermodynamic analysis of the salt bridges. Introduction of three alanines stabilizes the dimer by 1. 1 kcal/mol relative to the wild‐type. An arrangement corresponding to a complex type of salt bridge involving three groups stabilizes the dimer by 1. 7 kcal/mol, an apparent elevation of the melting temperature relative to wild type of about 22°C. While identifying local from nonlocal contributions to protein stability is difficult, stabilizing interactions can be identified by use of cycles. Introduction of alanines for side chains of lower helix propensity and complex salt bridges both stabilize the coiled‐coil, so that combining the two should yield melting temperatures substantially higher than the starting species, approaching those of thermophilic sequences.
Proteolysis as a measure of the free energy difference between cytochrome c and its derivatives
Authors: Wang L. , Kallenbach N. R.
Published: February 2009

Limited cleavage of oxidized and reduced horse heart cytochrome c (Cyt c) and the azide complex of Cyt c by proteinase K at room temperature yields a single cut within the central loop (36–60 in the sequence). Using an assay that allows spectroscopic evaluation of the fraction of intact protein as a function of time, together with a simple kinetic model for proteolysis, fluctuation opening of the loop can be related to the free energy of the corresponding protein. This allows us to estimate quantitatively the free energy difference between the oxidized form of Cyt c and other states using proteolysis as a probe. The results we obtain indicate that oxidized Cyt c is 2. 0 kcal mol−1 less stable than the reduced form, and 0. 07 kcal mol−1 is more stable than the Cyt c: azide complex at 25°C. These values agree in magnitude with results from hydrogen exchange and unfolding studies, suggesting that the stability of a protein can be directly related to its structural dynamics.
Evidence that peroxiredoxins are novel members of the thioredoxin fold superfamily
Authors: Schröder Ewald , Ponting Chris P.
Published: February 2009

Peroxiredoxins catalyze reduction of hydrogen peroxide or alkyl peroxide, to water or the corresponding alcohol. Detailed analysis of their sequences indicates that these enzymes possess a thioredoxin (Trx)‐like fold and consequently are homologues of both thioredoxin and glutathione peroxidase (GPx). Sequence‐ and structure‐based multiple sequence alignments indicate that the peroxiredoxin active site cysteine and GPx active site selenocysteine are structurally equivalent. Homologous peroxiredoxin and GPx enzymes are predicted to catalyze equivalent reactions via similar reaction intermediates.
HOMSTRAD: A database of protein structure alignments for homologous families
Authors: Mizuguchi Kenji , Deane Charlotte M. , Blundell Tom L. , Overington John P.
Published: February 2009

We describe a database of protein structure alignments for homologous families. The database HOMSTRAD presently contains 130 protein families and 590 aligned structures, which have been selected on the basis of quality of the X‐ray analysis and accuracy of the structure. For each family, the database provides a structure‐based alignment derived using COMPARER and annotated with JOY in a special format that represents the local structural environment of each amino acid residue. HOMSTRAD also provides a set of superposed atomic coordinates obtained using MNYFIT, which can be viewed with a graphical user interface or used for comparative modeling studies. The database is freely available on the World Wide Web at: http://www‐cryst. bioc. cam. ac. uk/homstrad/, with search facilities and links to other databases.
A homology identification method that combines protein sequence and structure information
Authors: Yu Lihua , White James V. , Smith Temple F.
Published: February 2009

A new method is presented for identifying distantly related homologous proteins that are unrecognizable by conventional sequence comparison methods. The method combines information about functionally conserved sequence patterns with information about structure context. This information is encoded in stochastic discrete state‐space models (DSMs) that comprise a new family of hidden Markov models. The new models are called sequence‐pattern‐embedded DSMs (pDSMs). This method can identify distantly related protein family members with a high sensitivity and specificity. The method is illustrated with trypsin‐like serine proteases and globins. The strategy for building pDSMs is presented. The method has been validated using carefully constructed positive and negative control sets. In addition to the ability to recognize remote homologs, pDSM sequence analysis predicts secondary structures with higher sensitivity, specificity, and Q3 accuracy than DSM analysis, which omits information about conserved sequence patterns. The identification of trypsin‐like serine proteases in new genomes is discussed.
Pulmonary surfactant‐associated polypeptide C in a mixed organic solvent transforms from a monomeric α‐helical state into insoluble β‐sheet aggregates
Authors: Szyperski Thomas , Wüthrich Kurt , Vandenbussche Guy , Ruysschaert Jean‐Marie , Curstedt Tore , Johansson Jan
Published: February 2009

In the 35‐residue pulmonary surfactant‐associated lipopolypeptide C (SP‐C), the stability of the valyl‐rich α‐helix comprising residues 9–34 has been monitored by circular dichroism, nuclear magnetic resonance, and Fourier transform infrared spectroscopy in both a mixed organic solvent and in phospholipid micelles. The α‐helical form of SP‐C observed in freshly prepared solutions in a mixed solvent of CHCl3/CH3OH/0.1 M HCl 32:64:5 (v/v/v) at 10°C undergoes within a few days an irreversible transformation to an insoluble aggregate that contains β‐sheet secondary structure. Hydrogen exchange experiments revealed that this conformational transition proceeds through a transition state with an Eyring free activation enthalpy of about 100 kJ mol–1, in which the polypeptide segment 9–27 largely retains a helical conformation. In dodecylphosphocholine micelles, the helical form of SP‐C was maintained after seven weeks at 50 °C. The α‐helical form of SP‐C thus seems to be the thermodynamically most stable state in this micellar environment, whereas its presence in freshly prepared samples in the aforementioned mixed solvent is due to a high kinetic barrier for unfolding. These observations support a previously proposed pathway for in vivo synthesis of SP‐C through proteolytic processing from a 21‐kDa precursor protein.
Recognition of partially‐folded mitochondrial malate dehydrogenase by GroEL. Steady and time‐dependent emission anisotropy measurements
Authors: Churchich Jorge E.
Published: February 2009

The binding of partially‐folded mitochondrial malate dehydrogenase (mMDH) to GroEL was assessed by steady and nanosecond emission spectroscopy. Partially‐folded intermediates of mMDH show significant residual secondary structure when examined by CD spectroscopy in the far UV. They bind the extrinsic fluorescent probe ANS and the protein‐ANS complexes display a rotational correlation time of 19 ns. Similar rotational correlation time (ø = 18.6 ns) was determined for partially‐folded species tagged with anthraniloyl. GroEL recognizes partially‐folded species with a KD ⋍ 60 nM. The rotational correlation time of the complex, i.e., GroEL‐mMDH‐ANT, approaches a value of 280 ns in the absence of ATP. Reactivation of mMDH‐ANT by addition of GroEL and ATP brings about a significant decrease in the observed rotational correlation time. The results indicate that partially‐folded malate dehydrogenase is rigidly trapped by GroEL in the absence of ATP, whereas addition of ATP facilitates reactivation and release of folded conformations endowed with catalytic activity.
Cupryphans, metal‐binding, redox‐active, redesigned conopeptides
Authors: Barba Marco , Sobolev Anatoli P. , Romeo Cristina , Schininà M. Eugenia , Pietraforte Donatella , Mannina Luisa , Musci Giovanni , Polticelli Fabio
Published: February 2009

Contryphans are bioactive peptides, isolated from the venom of marine snails of the genus Conus, which are characterized by the short length of the polypeptide chain and the high degree of unusual post‐translational modifications. The cyclization of the polypeptide chain through a single disulphide bond, the presence of two conserved Pro residues, and the epimerization of a Trp/Leu residue confer to Contryphans a stable and well‐defined structure in solution, conserved in all members of the family, and tolerant to multiple substitutions. The potential of Contryphans as scaffolds for the design of redox‐active (macro)molecules was tested by engineering a copper‐binding site on two different variants of the natural peptide Contryphan‐Vn. The binding site was designed by computational modeling, and the redesigned peptides were synthesized and characterized by optical, fluorescence, electron spin resonance, and nuclear magnetic resonance spectroscopy. The novel peptides, named Cupryphan and Arg–Cupryphan, bind Cu2+ ions with a 1:1 stoichiometry and a Kd in the 100 nM range. Other divalent metals (e.g., Zn2+ and Mg2+) are bound with much lower affinity. In addition, Cupryphans catalyze the dismutation of superoxide anions with an activity comparable to other nonpeptidic superoxide dismutase mimics. We conclude that the Contryphan motif represents a natural robust scaffold which can be engineered to perform different functions, providing additional means for the design of catalytically active mini metalloproteins.
Triad of polar residues implicated in pH specificity of acidic mammalian chitinase
Authors: Olland Andrea M. , Strand James , Presman Eleonora , Czerwinski Robert , Joseph‐McCarthy Diane , Krykbaev Rustem , Schlingmann Gerhard , Chopra Rajiv , Lin Laura , Fleming Margaret , Kriz Ron , Stahl Mark , Somers William , Fitz Lori , Mosyak Lidia
Published: February 2009

Acidic mammalian chitinase (AMCase) is a mammalian chitinase that has been implicated in allergic asthma. One of only two active mammalian chinases, AMCase, is distinguished from other chitinases by several unique features. Here, we present the novel structure of the AMCase catalytic domain, both in the apo form and in complex with the inhibitor methylallosamidin, determined to high resolution by X‐ray crystallography. These results provide a structural basis for understanding some of the unique characteristics of this enzyme, including the low pH optimum and the preference for the β‐anomer of the substrate. A triad of polar residues in the second‐shell is found to modulate the highly conserved chitinase active site. As a novel target for asthma therapy, structural details of AMCase activity will help guide the future design of specific and potent AMCase inhibitors.
Lone pair ··· π interactions between water oxygens and aromatic residues: Quantum chemical studies based on high‐resolution protein structures and model compounds
Authors: Jain Alok , Ramanathan Venkatnarayan , Sankararamakrishnan Ramasubbu
Published: February 2009

The π electron cloud of aromatic centers is known to be involved in several noncovalent interactions such as CH···π, OH···π, and π···π interactions in biomolecules. Lone‐pair (lp) ··· π interactions have gained attention recently and their role in biomolecular structures is being recognized. In this article, we have carried out systematic analysis of high‐resolution protein structures and identified more than 400 examples in which water oxygen atoms are in close contact (distance < 3.5 Å) with the aromatic centers of aromatic residues. Three different methods were used to build hydrogen atoms and we used a consensus approach to find out potential candidates for lp···π interactions between water oxygen and aromatic residues. Quantum mechanical calculations at MP2/6‐311++G(d,p) level on model systems based on protein structures indicate that majority of the identified examples have energetically favorable interactions. The influence of water hydrogen atoms was investigated by sampling water orientations as a function of two parameters: distance from the aromatic center and the angle between the aromatic plane and the plane formed by the three water atoms. Intermolecular potential surfaces were constructed using six model compounds representing the four aromatic amino acids and 510 different water orientations for each model compound. Ab initio molecular orbital calculations at MP2/6‐311++G(d,p) level show that the interaction energy is favorable even when hydrogen atoms are farthest from the aromatic plane while water oxygen is pointing toward the aromatic center. The strength of such interaction depends upon the distance of water hydrogen atoms from the aromatic substituents. Our calculations clearly show that the lp···π interactions due to the close approach of water oxygen and aromatic center are influenced by the positions of water hydrogen atoms and the aromatic substituents.
C‐terminal domain of SARS‐CoV main protease can form a 3D domain‐swapped dimer
Authors: Zhong Nan , Zhang Shengnan , Xue Fei , Kang Xue , Zou Peng , Chen Jiaxuan , Liang Chao , Rao Zihe , Jin Changwen , Lou Zhiyong , Xia Bin
Published: February 2009

SARS coronavirus main protease (Mpro) plays an essential role in the extensive proteolytic processing of the viral polyproteins (pp1a and pp1ab), and it is an important target for anti‐SARS drug development. We have reported that both the Mpro C‐terminal domain alone (Mpro‐C) and the N‐finger deletion mutant of Mpro (Mpro‐Δ7) exist as a stable dimer and a stable monomer (Zhong et al., J Virol 2008; 82:4227‐4234). Here, we report structures of both Mpro‐C monomer and dimer. The structure of the Mpro‐C monomer is almost identical to that of the C‐terminal domain in the crystal structure of Mpro. Interestingly, the Mpro‐C dimer structure is characterized by 3D domain‐swapping, in which the first helices of the two protomers are interchanged and each is enwrapped by four other helices from the other protomer. Each folding subunit of the Mpro‐C domain‐swapped dimer still has the same general fold as that of the Mpro‐C monomer. This special dimerization elucidates the structural basis for the observation that there is no exchange between monomeric and dimeric forms of Mpro‐C and Mpro‐Δ7.
CPDadh: A new peptidase family homologous to the cysteine protease domain in bacterial MARTX toxins
Authors: Pei Jimin , Lupardus Patrick J. , Garcia K. Christopher , Grishin Nick V.
Published: February 2009

A cysteine protease domain (CPD) has been recently discovered in a group of multifunctional, autoprocessing RTX toxins (MARTX) and Clostridium difficile toxins A and B. These CPDs (referred to as CPDmartx) autocleave the toxins to release domains with toxic effects inside host cells. We report identification and computational analysis of CPDadh, a new cysteine peptidase family homologous to CPDmartx. CPDadh and CPDmartx share a Rossmann‐like structural core and conserved catalytic residues. In bacteria, domains of the CPDadh family are present at the N‐termini of a diverse group of putative cell‐cell interaction proteins and at the C‐termini of some RHS (recombination hot spot) proteins. In eukaryotes, catalytically inactive members of the CPDadh family are found in cell surface protein NELF (nasal embryonic LHRH factor) and some putative signaling proteins.
Structure of dystrophia myotonica protein kinase
Authors: Elkins Jonathan M. , Amos Ann , Niesen Frank H. , Pike Ashley C.W. , Fedorov Oleg , Knapp Stefan
Published: February 2009

Dystrophia myotonica protein kinase (DMPK) is a serine/threonine kinase composed of a kinase domain and a coiled‐coil domain involved in the multimerization. The crystal structure of the kinase domain of DMPK bound to the inhibitor bisindolylmaleimide VIII (BIM‐8) revealed a dimeric enzyme associated by a conserved dimerization domain. The affinity of dimerisation suggested that the kinase domain alone is insufficient for dimerisation in vivo and that the coiled‐coil domains are required for stable dimer formation. The kinase domain is in an active conformation, with a fully‐ordered and correctly positioned αC helix, and catalytic residues in a conformation competent for catalysis. The conserved hydrophobic motif at the C‐terminal extension of the kinase domain is bound to the N‐terminal lobe of the kinase domain, despite being unphosphorylated. Differences in the arrangement of the C‐terminal extension compared to the closely related Rho‐associated kinases include an altered PXXP motif, a different conformation and binding arrangement for the turn motif, and a different location for the conserved NFD motif. The BIM‐8 inhibitor occupies the ATP site and has similar binding mode as observed in PDK1.
Structural characterization of unphosphorylated STAT5a oligomerization equilibrium in solution by small‐angle X‐ray scattering
Authors: Bernadó Pau , Pérez Yolanda , Blobel Jascha , Fernández‐Recio Juan , Svergun Dmitri I. , Pons Miquel
Published: February 2009

Signal transducer and activator of transcription (STAT) proteins play a crucial role in the activation of gene transcription in response to extracellular stimuli. The regulation and activity of these proteins require a complex rearrangement of the domains. According to the established models, based on crystallographic data, STATs convert from a basal antiparallel inactive dimer into a parallel active one following phosphorylation. The simultaneous analysis of small‐angle X‐ray scattering data measured at different concentrations of unphosphorylated human STAT5a core domain unambiguously identifies the simultaneous presence of a monomer and a dimer. The dimer is the minor species but could be structurally characterized by SAXS in the presence of the monomer using appropriate computational tools and shown to correspond to the antiparallel assembly. The equilibrium is governed by a moderate dissociation constant of Kd ∼ 90 μM. Integration of these results with previous knowledge of the N‐terminal domain structure and dissociation constants allows the modeling of the full‐length protein. A complex network of intermolecular interactions of low or medium affinity is suggested. These contacts can be eventually formed or broken to trigger the dramatic modifications in the dimeric arrangement needed for STAT regulation and activity.
Jasmonate‐ and salicylate‐mediated plant defense responses to insect herbivores, pathogens and parasitic plants
Authors: Smith Jordan L , De Moraes Consuelo M , Mescher Mark C
Published: February 2009

While extensive research has examined plant defense responses to attack by herbivores and pathogens, plant responses to parasitism by other plants are not well characterized. The expression of induced plant defenses is mediated by complex signaling networks in which the plant hormones jasmonic acid (JA) and salicylic acid (SA) play key roles. In general, JA‐mediated signaling pathways are implicated in the regulation of antiherbivore defenses, while the SA pathway is associated with defense responses against pathogens. However, there are many exceptions to this basic framework, and recent work suggests that interactions between the JA and SA pathways may play important roles in fine‐tuning defense responses. The few studies that have addressed plant responses to parasitism by other plants suggest that both salicylates and jasmonates can mediate effective defenses. Here, a review is given of recent work on these defense signaling molecules with relevance for understanding their role in plant responses to parasitism. Copyright © 2009 Society of Chemical Industry
The Year of the Rat ends—time to fight hunger!
Authors: Meerburg Bastiaan G , Singleton Grant R , Leirs Herwig
Published: February 2009

This paper investigates the importance of ecologically based rodent management in the light of the current food crisis, and the potential effects of this approach on the position of the undernourished. Hunger and food prices are on the rise owing to shortages that can be traced to reasons such as climatic extremes, use of crops for biofuels, reduced growth in yields which lag behind population growth, reduced world stocks and lack of sufficient investment in maintaining the irrigation infrastructure. For the undernourished this is problematic as they are most vulnerable to the rise in food prices. Very often, agricultural experts focus on an increase in agricultural production to reduce food prices. It is postulated in this article that almost 280 million undernourished could additionally benefit if more attention were paid to reducing pre‐ and post‐harvest losses by rodents. Moreover, rodent‐borne diseases would decrease, diseases that can be catastrophic to the livelihoods of the poorest of the poor. Copyright © 2009 Society of Chemical Industry
Inheritance mode and realized heritability of resistance to imidacloprid in the brown planthopper, Nilaparvata lugens (Stål) (Homoptera: Delphacidae)
Authors: Wang Yan Hua , Liu Xu Gan , Zhu Yu Cheng , Wu Sheng Gan , Li Shu Yong , Chen Wen Ming , Shen Jin Liang
Published: February 2009

BACKGROUND: The brown planthopper, Nilaparvata lugens (Stål), is a serious pest that causes enormous losses to the rice crop in Asia. The genetic basis of imidacloprid resistance was investigated in N. lugens.

RESULTS: The resistant strain, selected for imidacloprid resistance from a field population of N. lugens collected from Nanjing, Jiangsu Province, China, showed a 964‐fold resistance compared with the laboratory strain. Progenies of reciprocal crosses (F1 and F1′) showed similar dose–mortality responses (LC50) to imidacloprid, and also exhibited a similar degree of dominance (D), 0.58 for F1 and 0.63 for F1′. Chi‐square analyses of self‐bred and backcross progenies (F2, F2′ and BC respectively) rejected the hypothesis for a single gene control of the resistance. The estimated realized heritability (h2) of imidacloprid resistance was 0.1141 in the resistant strain of N. lugens.

CONCLUSION: The results showed that imidacloprid resistance in N. lugens was autosomal and was expressed as an incompletely dominant trait, probably controlled by multiple genes. Copyright © 2009 Society of Chemical Industry