bluuebunny/crossref_metadata_2025 huggingface dataset:
Statistical potentials for fold assessment
Authors: Melo Francisco , Sánchez Roberto , Sali Andrej
Published: June 2010

A protein structure model generally needs to be evaluated to assess whether or not it has the correct fold. To improve fold assessment, four types of a residue‐level statistical potential were optimized, including distance‐dependent, contact, ϕ/Ψ dihedral angle, and accessible surface statistical potentials. Approximately 10,000 test models with the correct and incorrect folds were built by automated comparative modeling of protein sequences of known structure. The criterion used to discriminate between the correct and incorrect models was the Z‐score of the model energy. The performance of a Z‐score was determined as a function of many variables in the derivation and use of the corresponding statistical potential. The performance was measured by the fractions of the correctly and incorrectly assessed test models. The most discriminating combination of any one of the four tested potentials is the sum of the normalized distance‐dependent and accessible surface potentials. The distance‐dependent potential that is optimal for assessing models of all sizes uses both Cα and Cβ atoms as interaction centers, distinguishes between all 20 standard residue types, has the distance range of 30 Å, and is derived and used by taking into account the sequence separation of the interacting atom pairs. The terms for the sequentially local interactions are significantly less informative than those for the sequentially nonlocal interactions. The accessible surface potential that is optimal for assessing models of all sizes uses Cβ atoms as interaction centers and distinguishes between all 20 standard residue types. The performance of the tested statistical potentials is not likely to improve significantly with an increase in the number of known protein structures used in their derivation. The parameters of fold assessment whose optimal values vary significantly with model size include the size of the known protein structures used to derive the potential and the distance range of the accessible surface potential. Fold assessment by statistical potentials is most difficult for the very small models. This difficulty presents a challenge to fold assessment in large‐scale comparative modeling, which produces many small and incomplete models. The results described in this study provide a basis for an optimal use of statistical potentials in fold assessment.
Ecotin: Lessons on survival in a protease‐filled world
Authors: Mcgrath Mary E. , Gillmor Sarah A. , Fletterick Robert J.
Published: June 2010

Ecotin, an Escherichia coli periplasmic protein of 142 amino acids, has been shown to be a potent inhibitor of a group of homologous serine proteases with widely differing substrate recognition. It is highly effective against a number of enzymes, including both pancreatic and neutrophil‐derived elastases, chymotrypsin, trypsin, factor Xa, and kallikrein. Recent structural and functional studies on ecotin and its interactions with different serine proteases have clarified these initial observations and revealed the remarkable features of this protein in inhibiting a strikingly large subset of the chymotrypsin family of serine proteases. The structures of the ecotin:serine protease complexes provide the first examples of protein‐protein recognition where the concept of specificity of interactions needs to be reexamined. The binding sites show a fluidity of protein contacts derived from ecotin's innate flexibility in fitting itself to proteases while strongly interfering with their function.
NMR structure determination of tick anticoagulant peptide (TAP)
Authors: Lim‐Wilby Marguerita S.L. , Vlasuk George P. , Brunck Terence K. , Hallenga Klaas , Maeyer Marc De , Lasters Ignace
Published: June 2010

Tick anticoagulant peptide (TAP) is a potent and selective 60‐amino acid inhibitor of the serine protease Factor Xa (fXa), the penultimate enzyme in the blood coagulation cascade. The structural features of TAP responsible for its remarkable specificity for fXa are unknown, but the binding to its target appears to be unique. The elucidation of the TAP structure may facilitate our understanding of this new mode of serine protease inhibition and could provide a basis for the design of novel fXa inhibitors. Analyses of homo‐ and heteronuclear two‐dimensional NMR spectra (total correlation spectroscopy, nuclear Overhauser effect spectroscopy [NOESY], constant time heteronuclear single quantum correlation spectroscopy [CT‐HSQC], and HSQC‐NOESY; 600 MHz; 1.5 mM TAP; pH 2.5) of unlabeled, 13C‐labeled, and 15N‐labeled TAP provided nearly complete 1H sequence‐specific resonance assignments. Secondary structural elements were identified by characteristic NOE patterns and D2O amide proton‐exchange experiments. A three‐dimensional structure of TAP was generated from 412 NOESY‐derived distance and 47 dihedral angle constraints. The structural elements of TAP are similar in some respects to those of the Kunitz serine protease inhibitor family, with which TAP shares weak sequence homology. This structure, coupled with previous kinetic and biochemical information, confirms previous suggestions that TAP has a unique mode of binding to fXa.
Mechanical deformation enhances catalytic activity of crystalline carboxypeptidase A
Authors: Zenchenko Tatyana A. , Morozov Victor N.
Published: June 2010

A new approach for investigating mechano‐chemical interactions in enzymes is described. The catalytic activity of crystalline crosslinked enzymes subjected to uniaxial deformation has been measured. Extension of monoclinic P21 crystals of carboxypeptidase A along the [010] direction leads to a many‐fold increase in catalytic esterase activity with no changes in the effective Michaelis constant. This increase is interpreted as due to liberation of conformational mobility associated with catalytic activity of the enzyme in the deformed crystal.
Profiles for the analysis of immunoglobulin sequences: Comparison of V gene subgroups
Authors: Harris Linda , Bajorath Jürgen
Published: June 2010

A format for the structure‐oriented analysis of immunoglobulin (Ig) variable region sequences is presented and applied to generate sequence profiles for comparison of heavy‐ and light‐chain subgroups. The profile allows simultaneous evaluation of sequences and structural information and can be used for a number of different applications.
Conformationally constrained analogs of protein kinase inhibitor(6–22)amide: Effect of turn structures in the center of the peptide on inhibition of cAMP‐dependent protein kinase
Authors: Glass David B. , Trewhella Jill , Mitchell Ryan D. , Walsh Donal A.
Published: June 2010

The high‐affinity interaction between protein kinase inhibitor (PKI)(6–22)amide (Thr6‐Tyr‐Ala‐Asp‐Phe‐Ile‐Ala‐Ser‐Gly‐Arg‐Thr‐Gly‐Arg‐Arg‐Asn‐Ala‐Ile22‐NH2) and the catalytic subunit of cAMP‐dependent protein kinase requires both the N‐terminal Thr6 to Ile11 sequence of the inhibitor peptide and its C‐terminal pseudosubstrate site comprised of Arg15 to Ile22. Small angle X‐ray scattering data indicate that PKI(6–22)amide has a compact, rather than extended, structure in solution (Reed J et al., 1989, Biochem J 264:371–380). CD spectroscopic analysis of the PKI peptide led to the suggestion that a β‐turn structure might be located in the ‐Ala12‐Ser‐Gly‐Arg15‐ connecting sequence in the middle of the molecule (Reed J, Kinzel V, Cheng HC, Walsh DA, 1987, Biochemistry 26:7641–7647). To investigate this possibility further, conformationally constrained and flexible analogs of PKI(6–22)amide were synthesized and used to study the structure‐function relationships of this central portion of the inhibitor. (Des12–14)PKI(6–22) amide exhibited over a 200‐fold loss in inhibitory activity. Replacement of the omitted ‐Ala 12‐Ser‐Gly14‐ sequence with aminocaprylic acid yielded an analog that regained more than 90% of the lost binding energy. The D‐alanine14 PKI analog was as potent as the parent peptide, whereas the β‐alanine14 and the sarcosine14 analogs were only 10‐fold less active. Several peptides that promoted a β‐turn structure at residues 12–15 showed about 200‐fold decreases in inhibitory activity. Two constrained analogs that could not assume a β‐turn conformation were only 30‐fold less potent than PKI(6–22)amide. Thus, the structure of the central connecting portion of the PKI peptide, encompassing residues 12–15, greatly influences its ability to effectively bind to and inhibit the catalytic subunit. We conclude, however, that a formal β‐turn at this position is not required and is actually detrimental for a high‐affinity interaction of PKI(6–22)amide with the enzyme. These results are interpreted in light of the Fourier‐transform infrared spectra of the peptide analogs and the crystal structure of the peptide bound at the active site of the protein kinase (Knighton DR et al., 1991b, Science 255:414–420).
Recombinant immunoglobulin variable domains generated from synthetic genes provide a system for in vitro characterization of light‐chain amyloid proteins
Authors: Stevens Priscilla Wilkins , Raffen Rosemarie , Hanson Deborah K. , Deng Ya‐Li , Berrios‐Hammond Maria , Westholm Florence A. , Schiffer Marianne , Stevens Fred J. , Murphy Charles , Solomon Alan , Eulitz Manfred , Wetzel Ronald
Published: June 2010

The primary structural features that render human monoclonal light chains amyloidogenic are presently unknown. To gain further insight into the physical and biochemical factors that result in the pathologic deposition of these proteins as amyloid fibrils, we have selected for detailed study three closely homologous protein products of the light‐chain variable‐region single‐gene family VkIV. Two of these proteins, REC and SMA, formed amyloid fibrils in vivo. The third protein, LEN, was excreted by the patient at levels of 50 g/day with no indication of amyloid deposits. Sequences of amyloidogenic proteins REC and SMA differed from the sequence of the nonpathogenic protein LEN at 14 and 8 amino acid positions, respectively, and these amino acid differences have been analyzed in terms of the three‐dimensional structure of the LEN dimer. To provide a replenishable source of these human proteins, we constructed synthetic genes coding for the REC, SMA, and LEN variable domains and expressed these genes inEscherichia coli. Immunochemical and biophysical comparisons demonstrated that the recombinant VkIV products have tertiary structural features comparable to those of the patient‐derived proteins. This well‐defined set of three clinically characterized humankIV light chains, together with the capability to produce thesekIV proteins recombinantly, provide a system for biophysical and structural comparisons of two different amyloidogenic light‐chain proteins and a nonamyloidogenic protein of the same subgroup. This work lays the foundation for future investigations of the structural basis of light‐chain amyloidogenicity.
Purification and preliminary crystallographic studies of penicillin G acylase from Providencia rettgeri
Authors: Klei Herbert E. , Kelly Judith A. , Daumy Gaston O.
Published: June 2010

Two isoforms of the heterodimeric enzyme penicillin G acylase (EC 3.5.1.11) from Providencia rettgeri ATCC 31052 (strain Brol) were purified to near homogeneity. The isoforms exhibited comparable enzymatic activities but differed slightly in the molecular weight and pI of their respective α‐subunit. The origin of this difference was traced to the partial conversion of the N‐terminal Gln of the α‐subunit to pyrrolidonecarboxylic acid (pyro‐Glu). The boundaries of the mature enzyme within the translated DNA sequence of the wild‐type propeptide (GenBank M86533) were determined. The results conclusively identified the length of the signal peptide and the position of the spacer cleaved from the propeptide to form the active heterodimer. The molecular weights of the α‐ and β‐subunits, based on these termini, were 23.7 and 62.2 kDa, respectively. Both isoforms were crystallized independently as hexagonal bipyramids up to 0.60 mm in diameter in either space group P6122 or P6522 (a = b = 140.5 Å and c = 209.5 Å) from ammonium sulfate solutions buffered by 50 mM potassium phosphate at pH 7.5. The presence of glycerol, although not required, facilitated crystal growth. Native and heavy atom derivative data were collected to 3.0 Å resolution, and the calculation of isomorphous replacement phases is under way.
Structural features of the uniporter/symporter/antiporter superfamily
Authors: Goswitz Visala Chepuri , Brooker Robert J.
Published: June 2010

The uniporter/symporter/antiporter superfamily is an evolutionarily related group of solute transporters. For the entire superfamily, we have used a new predictive program to identify the transmembrane domains. These transmembrane domains were then analyzed with regard to their overall hydrophobicity and amphipathicity. In addition, the lengths of the hydrophilic loops connecting the transmembrane domains were calculated. These data, together with structural information in the literature, were collectively used to produce a general model for the three‐dimensional arrangement of the transmembrane domains.
Deletion mutants of tyrosine hydroxylase identify a region critical for heparin binding
Authors: Daubner S. Colette , Piper M. Michelle
Published: June 2010

Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute a family of tetrahydropterin‐dependent aromatic amino acid hydroxylases. It has been proposed that each hydroxylase is composed of a conserved C‐terminal catalytic domain and an unrelated N‐terminal regulatory domain. Of the three, only tyrosine hydroxylase is activated by heparin and binds to heparin‐Sepharose. A series of N‐terminal deletion mutants of tyrosine hydroxylase has been expressed in Escherichia coli to identify the heparin‐binding site. The mutants lacking the first 32 or 68 amino acids bind to heparin‐Sepharose. The mutant lacking 76 amino acids binds somewhat to heparin‐Sepharose and the proteins lacking 88 or 128 do not bind at all. Therefore, an important segment of the heparin‐binding site must be composed of the region from residues 76 to 90. All of the deletion mutants are active, and the Michaelis constants for pterins and tyrosine are similar among all the mutant and wild‐type enzymes.
S‐ovalbumin, an ovalbumin conformer with properties analogous to those of loop‐inserted serpins
Authors: Huntington James A. , Gettins Peter G. W. , Patston Philip A.
Published: June 2010

Most serpins are inhibitors of serine proteinases and are thought to undergo a conformational change upon complex formation with proteinase that involves partial insertion of the reactive center loop into a β‐sheet of the inhibitor. Ovalbumin, although a serpin, is not an inhibitor of serine proteinases. It has been proposed that this deficiency arises from the presence of a charged residue, arginine, at a critical point (P14) in the reactive center region, which prevents loop insertion into the β‐sheet and thereby precludes inhibitory properties. To test whether loop insertion is prevented in ovalbumin we have examined the properties of two forms of ovalbumin: the native protein and S‐ovalbumin, a form that forms spontaneously from native ovalbumin and has increased stability. Calorimetric measurements showed that S‐ovalbumin was more stable than ovalbumin by about 3 kcal mol−1. CD spectra, which indicated that S‐ovalbumin had less α‐helix than native ovalbumin, and 1H NMR spectra, which indicated very similar overall structures, suggest limited conformational differences between the two forms. From comparison of the susceptibility of the reactive center region of each protein to proteolysis by porcine pancreatic elastase and by subtilisin Carlsberg, we concluded that the limited native‐to‐S conformational change specifically affected the reactive center region. These data are consistent with a structure for S‐ovalbumin in which part of the reactive center loop has inserted into β‐sheet A to give a more stable structure, analogously to other serpins. However, the rate of loop insertion appears to be very much lower than for inhibitory serpins. Thus, although loop insertion does not appear to be prevented by the presence of arginine at P14, it occurs at too slow a rate to compete effectively with the substrate pathway. This alone is sufficient to account for the absence of detectable inhibitory properties in ovalbumin.
Characterizing the microenvironment surrounding protein sites
Authors: Bagley Steven C. , Altman Russ B.
Published: June 2010

Sites are microenvironments within a biomolecular structure, distinguished by their structural or functional role. A site can be defined by a three‐dimensional location and a local neighborhood around this location in which the structure or function exists. We have developed a computer system to facilitate structural analysis (both qualitative and quantitative) of biomolecular sites. Our system automatically examines the spatial distributions of biophysical and biochemical properties, and reports those regions within a site where the distribution of these properties differs significantly from control nonsites. The properties range from simple atom‐based characteristics such as charge to polypeptide‐based characteristics such as type of secondary structure. Our analysis of sites uses non‐sites as controls, providing a baseline for the quantitative assessment of the significance of the features that are uncovered. In this paper, we use radial distributions of properties to study three well‐known sites (the binding sites for calcium, the milieu of disulfide bridges, and the serine protease active site). We demonstrate that the system automatically finds many of the previously described features of these sites and augments these features with some new details. In some cases, we cannot confirm the statistical significance of previously reported features. Our results demonstrate that analysis of protein structure is sensitive to assumptions about background distributions, and that these distributions should be considered explicitly during structural analyses.
Theoretical analysis of the structure of the peptide fasciculin and its docking to acetylcholinesterase
Authors: Van Den Born Harald K. L. , Radić Zoran , Marchot Pascale , Taylor Palmer , Tsigelny Igor
Published: June 2010

The fasciculins are a family of closely related peptides that are isolated from the venom of mambas and exert their toxic action by inhibiting acetylcholinesterase (AChE). Fasciculins belong to the structural family of three‐fingered toxins from Elapidae snake venoms, which include the α‐neurotoxins that block the nicotinic acetylcholine receptor and the cardiotoxins that interact with ceil membranes. The features unique to the known primary and tertiary structures of the fasciculin molecule were analyzed. Loop I contains an arginine at position 11, which is found only in the fasciculins and could form a pivotal anchoring point to AChE. Loop II contains five cationic residues near its tip, which are partly charge‐compensated by anionic side chains in loop III. By contrast, the other three‐fingered toxins show full charge compensation within loop II. The interaction of fasciculin with the recognition site on acetylcholinesterase was investigated by estimating a precollision orientation followed by determination of the buried surface area of the most probable complexes formed, the electrostatic field contours, and the detailed topography of the interaction surface. This approach has led to testable models for the orientation and site of bound fasciculin.
Structure of the Oct‐3 POU‐homeodomain in solution, as determined by triple resonance heteronuclear multidimensional NMR spectroscopy
Authors: Morita Eugene H. , Shirakawa Masahiro , Kyogoku Yoshimasa , Hayashi Fumiaki , Imagawa Masayoshi
Published: June 2010

The POU‐homeodomain (POUH) forms the bipartite DNA‐binding POD domain in association with the POU‐specific domain. The 1H, 15N, and 13C magnetic resonances of the 67‐amino acid long POUH of mouse Oct‐3 have almost completely been assigned, mainly through the combined use of three‐dimensional triple resonance NMR methods. Based on the distance and dihedral angle constraints derived from the NMR data, the solution structure of the POUH domain has been calculated by the ab initio simulated annealing method. The average RMS deviation for all backbone heavy atoms of the 20 best calculated structures for residues 9–53 of the total 67 amino acid residues is 0.44 Å. The POUH domain consists of three α‐helices (helix‐I, 10–20; helix‐II, 28–38; and helix‐III, 42–53), and helices‐II and ‐III form a helix‐turn‐helix motif. In comparison with other classical homeodomains, the folding of the three helices is quite similar. However, the length of helix‐III is fairly short. In the complex of the Oct‐1 POU domain with an octamer site (Klemm JD, et al., 1994, Cell 77:21–32), the corresponding region is involved in helix‐III. The structural difference between these two cases will be discussed.
Thrombin inhibition by cyclic peptides from thrombomodulin
Authors: Lougheed Julie C. , Bowman Carole L. , Meininger David P. , Komives Elizabeth A.
Published: June 2010

Peptides corresponding to the loop regions of the fourth, fifth, and sixth epidermal growth factor (EGF)‐like domains of thrombomodulin (TM) have been synthesized and assayed for thrombin inhibition, as indicated by both inhibition of thrombin‐mediated fibrinogen clotting and inhibition of the association of thrombin with TM that results in protein C activation. Peptides from the fifth EGF‐like domain showed significant inhibition of fibrinogen clotting and protein C activation, whereas peptides from the fourth and sixth EGF‐like domains were weak inhibitors in both assays.

Two structural features were important for inhibitory potency of the peptides from the fifth EGF‐like domain: cyclization by a disulfide bond and attachment of the “tail” amino acids C‐terminal to the disulfide loop. Linear control peptides did not significantly inhibit clotting or protein C activation. The C‐terminal loop alone, the “tail” peptide, or a mixture of the two were at least 10‐fold less potent inhibitors of clotting or protein C activation. A more constrained peptide analog was designed by deletion of an isoleucine within the C5‐C6 disulfide loop, TM52–1+5C. This analog was a better inhibitor in both assay systems, having a Ki, for protein C activation of 26 μM.
Monte Carlo docking with ubiquitin
Authors: Cummings Maxwell D. , Hart Trevor N. , Read Randy J.
Published: June 2010

The development of general strategies for the performance of docking simulations is prerequisite to the exploitation of this powerful computational method. Comprehensive strategies can only be derived from docking experiences with a diverse array of biological systems, and we have chosen the ubiquitin/diubiquitin system as a learning tool for this process. Using our multiple‐start Monte Carlo docking method, we have reconstructed the known structure of diubiquitin from its two halves as well as from two copies of the uncomplexed monomer. For both of these cases, our relatively simple potential function ranked the correct solution among the lowest energy configurations. In the experiments involving the ubiquitin monomer, various structural modifications were made to compensate for the lack of flexibility and for the lack of a covalent bond in the modeled interaction. Potentially flexible regions could be identified using available biochemical and structural information. A systematic conformational search ruled out the possibility that the required covalent bond could be formed in one family of low‐energy configurations, which was distant from the observed dimer configuration. A variety of analyses was performed on the low‐energy dockings obtained in the experiment involving structurally modified ubiquitin. Characterization of the size and chemical nature of the interface surfaces was a powerful adjunct to our potential function, enabling us to distinguish more accurately between correct and incorrect dockings. Calculations with the structure of tetraubiquitin indicated that the dimer configuration in this molecule is much less favorable than that observed in the diubiquitin structure, for a simple monomer‐monomer pair. Based on the analysis of our results, we draw conclusions regarding some of the approximations involved in our simulations, the use of diverse chemical and biochemical information in experimental design and the analysis of docking results, as well as possible modifications to our docking protocol.
Conformational stability of bovine holo and apo adrenodoxin — A scanning calorimetric study
Authors: Burova Tatjana V. , Bernhardt Rita , Pfeil Wolfgang
Published: June 2010

Holo and apo adrenodoxin were studied by differential scanning calorimetry, absorption spectroscopy, limited proteolysis, and size‐exclusion chromatography. To determine the conformational stability of adrenodoxin, a method was found that prevents the irreversible destruction of the iron‐sulfur center. The approach makes use of a buffer solution that contains sodium sulfide and mercaptoethanol. The thermal transition of adrenodoxin takes place at Ttrs = 46–57 °C, depending on the Na2S concentration with a denaturation enthalpy of ΔH = 300–380 kJ/mol. From ΔH versus Ttrs a heat capacity change was determined as ΔCp — 7.5 ± 1.2 kJ/mol/K. The apo protein is less stable than the holo protein as judged by the lower denaturation enthalpy (ΔH = 93 ± 14 kJ/mol at Ttrs = 37.4 ± 3.3 °C) and the higher proteolytic susceptibility. The importance of the iron‐sulfur cluster for the conformational stability of adrenodoxin and some conditions for refolding of the thermally denatured protein are discussed.
Purification and crystallization of benzoylformate decarboxylase
Authors: Hasson Miriam S. , Petsko Gregory A. , Ringe Dagmar , Muscate Angelika , Henehan Gary T.M. , Guidinger Peter F. , Ken Yon George L.
Published: June 2010

A new large‐scale purification method for benzoylformate decarboxylase from Pseudomonas putida has allowed us to undertake an X‐ray crystallographic study of the enzyme. The previously observed instability of the enzyme was overcome by addition of 100 μM thiamine pyrophosphate to buffers used in the purification. The final enzyme preparation was more than 97% pure, as determined by denaturing gel electrophoresis and densitometry. The mobility of the enzyme on a gel filtration column indicates that it is a tetramer of 57‐kDa subunits. Large, single crystals of benzoylformate decarboxylase were grown from solutions of buffered polyethylene glycol 400, pH 8.5. The crystals diffract to beyond 1.6 Å resolution and are stable for days to X‐ray radiation. Analysis of X‐ray data from the crystals, along with the newly determined quaternary structure, identifies the space group as 1222. The unit cell dimensions are a = 82 Å, b = 97 Å, c = 138 Å. An average Vm value for the crystals is consistent with one subunit per asymmetric unit. The subunits of the tetramer must be arranged with tetrahedral 222 symmetry.
Localized solution structure refinement of an F45W variant of ubiquitin using stochastic boundary molecular dynamics and NMR distance restraints
Authors: Laub Paul B. , Khorasanizadeh Sepideh , Roder Heinrich
Published: June 2010

The local structure within an 8‐Å radius around residue 45 of a recombinant F45W variant of human ubiquitin has been determined using 67 interproton distance restraints measured by two‐dimensional proton NMR. Proton chemical shift evidence indicates that structural perturbations due to the F45W mutation are minimal and limited to the immediate vicinity of the site of mutation. Simulated annealing implemented with stochastic boundary molecular dynamics was applied to refine the structure of Trp 45 and 10 neighboring residues. The stochastic boundary method allowed the entire protein to be reassembled from the refined coordinates and the outlying unrefined coordinates with little distortion at the boundary. Refinement began with four low‐energy indole ring orientations of F45W‐substituted wild‐type (WT) ubiquitin crystal coordinates. Distance restraints were derived from mostly long‐range NOE cross peaks with 51 restraints involving the Trp 45 indole ring. Tandem refinements of 64 structures were done using either (1) upper and lower bounds derived from qualitative inspection of NOE cross‐peak intensities or (2) quantitative analysis of cross‐peak heights using the program MARDIGRAS. Though similar to those based on qualitative restraint, structures obtained using quantitative NOE analysis were superior in terms of precision and accuracy as measured by back‐calculated sixth‐root R factors. The six‐membered portion of the indole ring is nearly coincident with the phenyl ring of the WT and the indole NH is exposed to solvent. Accommodation of the larger ring is accompanied by small perturbations in the backbone and a 120° rotation of the x2 dihedral angle of Leu 50.
Amide exchange rates in Escherichia coli acyl carrier protein: Correlation with protein structure and dynamics
Authors: Andrec Michael , Blake Hill R. , Prestegard James H.
Published: June 2010

The acyl carrier protein (ACP) of Escherichia coli is a 77‐amino acid, highly negatively charged three‐helix protein that plays a central role in fatty acid biosynthesis. Previous NMR studies have suggested the presence of multiple conformations and marginally stable secondary structural elements. The stability of these elements is now examined by monitoring amide exchange in apo‐ACP using NMR‐based methods. Because ACP exhibits many rapid exchange rates, application of traditional isotope exchange methods is difficult. In one approach, heteronuclear correlation experiments with pulsed field‐gradient coherence selection have reduced the time needed to collect two‐dimensional 1H‐15N correlation spectra to the point where measurement of exchange of amide protons for deuterium on the timescale of minutes can be made. In another approach, water proton selective inversion‐exchange experiments were performed to estimate the exchange rates of protons exchanging on timescales of less than a second. Backbone amide protons in the region of helix II were found to exchange significantly more rapidly than those in helices I and III, consistent with earlier structural models suggesting a dynamic disruption of the second helix. Highly protected amides occur on faces of the helices that may pack into a hydrophobic core present in a partially disrupted state.
Crystallization of the MS2 translational repressor alone and complexed to bromouridine
Authors: Ni Chao‐Zhou , Hettinga Bradley S. , Wickersham John , Mitchell Richard S. , Williamson Michael M. , Celikel Reha , Ely Kathryn R. , Prangé Thierry , Fourme Roger , Krapcho Karen J. , Thulin Craig , Talbot Phil , Gesteland Raymond F.
Published: June 2010

The coat protein from the MS2 bacteriophage plays a dual role by encapsidating viral RNA and also by binding RNA as a translational repressor. In order to study the isolated dimer in a conformation not influenced by capsid interactions, a mutant molecule was crystallized that is defective in capsid assembly but is an active repressor. The unassembled dimer crystallized in the space group P21212 with a = 76.2, b = 55.7, and c = 28.4 Å. In these crystals, monomers were related by twofold symmetry. When this dimer was co‐crystallized with 5‐bromouridine, crystals formed in space group R3 with a = b = 155.9 Å, c = 29.9 Å, γ = 120°; the dimer was the asymmetric unit.
Quantitative measurements of the cooperativity in an EF‐hand protein with sequential calcium binding
Authors: Linse Sara , Chazin Walter J.
Published: June 2010

Positive cooperativity, defined as an enhancement of the ligand affinity at one site as a consequence of binding the same type of ligand at another site, is a free energy coupling between binding sites. It can be present both in systems with sites having identical ligand affinities and in systems where the binding sites have different affinities. When the sites have widely different affinities such that they are filled with ligand in a sequential manner, it is often difficult to quantify or even detect the positive cooperativity, if it occurs. This study presents verification and quantitative measurements of the free energy coupling between the two calcium binding sites in a mutant form of calbindin D9k. Wild‐type calbindin D9k binds two calcium ions with similar affinities and positive cooperativity — the free energy coupling, ΔΔG, is around ‐8 kJ · mol−1 (Linse S, et al., 1991, Biochemistry 30: 154–162). The mutant, with the substitution Asn 56 → Ala, binds calcium in a sequential manner. In the present work we have taken advantage of the variations among different metal ions in terms of their preferences for the two binding sites in calbindin D9k. Combined studies of the binding of Ca2+, Cd2+, and La3+ have allowed us to conclude that in this mutant ΔΔG < ‐6.4 kJ · mol−1, and that Cd2+ and La3+ also bind to this protein with positive cooperativity. The results justify the use of the (Ca2+), state of the Asn 56 → Ala mutant, as well as the (Cd2+)1 state of the wild type, as models for the half‐saturated states along the two pathways of cooperative Ca2+ binding in calbindin D9k.
Identification of functionally important amino acids in the cellulose‐binding domain of Trichoderma reesei cellobiohydrolase I
Authors: Linder Markus , Mattinen Maija‐Liisa , Kontteli Maarit , Lindeberg Gunnar , Ståhlberg Jerry , Drakenberg Torbjörn , Reinikainen Tapani , Pettersson GÖRan , Annila Arto
Published: June 2010

Cellobiohydrolase I (CBHI) of Trichoderma reesei has two functional domains, a catalytic core domain and a cellulose binding domain (CBD). The structure of the CBD reveals two distinct faces, one of which is flat and the other rough. Several other fungal cellulolytic enzymes have similar two‐domain structures, in which the CBDs show a conserved primary structure. Here we have evaluated the contributions of conserved amino acids in CBHI CBD to its binding to cellulose. Binding isotherms were determined for a set of six synthetic analogues in which conserved amino acids were substituted. Two‐dimensional NMR spectroscopy was used to assess the structural effects of the substitutions by comparing chemical shifts, coupling constants, and NOEs of the backbone protons between the wild‐type CBD and the analogues. In general, the structural effects of the substitutions were minor, although in some cases decreased binding could clearly be ascribed to conformational perturbations. We found that at least two tyrosine residues and a glutamine residue on the flat face were essential for tight binding of the CBD to cellulose. A change on the rough face had only a small effect on the binding and it is unlikely that this face interacts with cellulose directly.
Three‐dimensional structure of the complex of 4‐guanidino‐Neu5Ac2en and influenza virus neuraminidase
Authors: Varghese Joseph N. , Chandana Epa V. , Colman Peter M.
Published: June 2010

The three‐dimensional X‐ray structure of a complex of the potent neuraminidase inhibitor 4‐guanidino‐Neu5Ac2en and influenza virus neuraminidase (Subtype N9) has been obtained utilizing diffraction data to 1.8 Å resolution. The interactions of the inhibitor, solvent water molecules, and the active site residues have been accurately determined. Six water molecules bound in the native structure have been displaced by the inhibitor, and the active site residues show no significant conformational changes on binding. Sialic acid, the natural substrate, binds in a half‐chair conformation that is isosteric to the inhibitor. The conformation of the inhibitor in the active site of the X‐ray structure concurs with that obtained by theoretical calculations and validates the structure‐based design of the inhibitor. Comparison of known high‐resolution structures of neuraminidase subtypes N2, N9, and B shows good structural conservation of the active site protein atoms, but the location of the water molecules in the respective active sites is less conserved. In particular, the environment of the 4‐guanidino group of the inhibitor is strongly conserved and is the basis for the antiviral action of the inhibitor across all presently known influenza strains. Differences in the solvent structure in the active site may be related to variation in the affinities of inhibitors to different subtypes of neuraminidase.
Probing the solution structure of the DNA‐binding protein Max by a combination of proteolysis and mass spectrometry
Authors: Cohen Steven L. , Chait Brian T. , Ferré‐D'Amaré Adrian R. , Burley Stephen K.
Published: June 2010

A simple biochemical method that combines enzymatic proteolysis and matrix‐assisted laser desorption ionization mass spectrometry was developed to probe the solution structure of DNA‐binding proteins. The method is based on inferring structural information from determinations of protection against enzymatic proteolysis, as governed by solvent accessibility and protein flexibility. This approach was applied to the study of the transcription factor Max —a member of the basic/helix‐loop‐helix/zipper family of DNA‐binding proteins. In the absence of DNA and at low ionic strengths, Max is rapidly digested by each of six endoproteases selected for the study, results consistent with an open and flexible structure of the protein. At physiological salt levels, the rates of digestion are moderately slowed; this and the patterns of cleavage are consistent with homodimerization of the protein through a predominantly hydrophobic interface. In the presence of Max‐specific DNA, the protein becomes dramatically protected against proteolysis, exhibiting up to a 100‐fold reduction in cleavage rates. Over a 2‐day period, both complete and partial proteolysis of the Max‐DNA complex is observed. The partial proteolytic fragmentation patterns reflect a very high degree of protection in the N‐terminal and helix‐loop‐helix regions of the protein, correlating with those expected of a stable dimer bound to DNA at its basic N‐terminals. Less protection is seen at the C‐terminal where a slow, sequential proteolytic cleavage occurs, correlating to the presence of a leucine zipper. The results also indicate a high affinity of Max for its target DNA that remains high even when the leucine zipper is proteolytically removed. In addition to the study of the helix‐loop‐helix protein Max, the present method appears well suited for a range of other structural biological applications.
Crystallization and preliminary X‐ray investigation of lipoxygenase‐3 from soybeans
Authors: Axelrod Bernard , Steczko Janusz , Minor Wladek , Stojanoff Vivian
Published: June 2010

Soybean lipoxygenase‐3 has been crystallized by the vapor diffusion method in 16–20% polyethylene glycol (average Mr, 3,400), 0.2 M sodium acetate buffer, pH 5.7, at 21 °C, at a protein concentration of 8–15 mg/mL. The crystals, which diffract to 3‐Å spacings, belong to the monoclinic space group C2. Cell constants are a = 111.9, b= 136.4, and c = 61.6 Å and β = 95.7°. The calculated value of Matthews's constant, Vm = 2.48 Å3 /kDa, is consistent with the presence of one molecule of lipoxygenase per crystallographic asymmetric unit (Z = 4).
Disulfide bond tethering of extracellular loops does not affect the closure of OmpF porin at acidic pH
Authors: Wager Beau , Baslé Arnaud , Delcour Anne H.
Published: June 2010

The permeability of the outer membrane of gram‐negative bacteria is essentially controlled by pore‐forming proteins of the porin family. The trimeric E. coli porin OmpF is assembled as a triple β‐barrel, where each monomer contains a central pore and extracellular loops. Electrophysiological analysis of the behavior of OmpF at acidic pH reveals that the protein undergoes a conformational change leading to the sequential step‐wise closure of the three monomers. A previous atomic force microscopy study suggested that the conformational change might be due to a bending of extracellular loops over the pore opening, and loop deletion experiments suggested that loops L1, L7, and L8 are involved. In order to test the hypothesis for loop movement, we engineered a series of double cysteine mutants in loops L1, L6, L7 and L8 in order to create disulfide bonds linking two loops to each other, or the two branches of a loop, or a loop to the β‐barrel. Five out of the six mutants showed the formation of the disulfide bond. However, none of these had an altered response to acidic pH relative to the wildtype channel. Although we cannot dismiss the possibility that the mobility restriction introduced by each disulfide bond was too localized to impact a more global conformational change of the three loops, the fact that all of the different types of disulfide bond tethering were similarly ineffective suggests that the extracellular loops L1, L7, and L8 may not undergo a major acidic‐pH induced conformational change leading to channel closure. Proteins 2010. © 2010 Wiley‐Liss, Inc.
A structural dissection of amino acid substitutions in helical transmembrane proteins
Authors: Mokrab Younes , Stevens Tim J. , Mizuguchi Kenji
Published: June 2010

The evolution of protein folds is under strong constraints from their surrounding environment. Although folding in water‐soluble proteins is driven primarily by hydrophobic forces, the nature of the forces that determine the folding and stability of transmembrane proteins are still not fully understood. Furthermore, the chemically heterogeneous lipid bilayer has a non‐uniform effect on protein structure. In this article, we attempt to get an insight into the nature of this effect by examining the impact of various types of local structure environment on amino acid substitution, based on alignments of high‐resolution structures of polytopic helical transmembrane proteins combined with sequences of close homologs. Compared to globular proteins, burying amino acid sidechains, especially hydrophilic ones, led to a lower increase in conservation in both the lipid‐water interface region and the hydrocarbon core region. This observation is due to surface residues in HTM proteins especially in the HC region being relatively highly conserved, suggesting higher evolutionary constraints from their specific interactions with the surrounding lipid molecules. Polar and small residues, particularly Pro and Gly, show a noticeable increase in conservation as they are positioned more towards the centre of the membrane, which is consistent with their recognized key roles in structural stability. In addition, the examination of hydrogen bonds in the membrane environment identified some exposed hydrophilic residues being better conserved when not hydrogen‐bonded to other residues, supporting the importance of lipid‐protein sidechain interactions. The conclusions presented in this study highlight the distinct features of substitution matrices that take into account the membrane environment, and their potential role in improving sequence‐structure alignments of transmembrane proteins. Proteins 2010; © 2010 Wiley‐Liss, Inc.
ReplicOpter: A replicate optimizer for flexible docking
Authors: Demerdash Omar N. A. , Buyan Amanda , Mitchell Julie C
Published: June 2010

We present a computationally efficient method for flexible refinement of docking predictions that reflects observed motions within a protein's structural class. Using structural homologs, we derive deformation models that capture likely motions. The models or “replicates” typically align along a rigid core, with a handful of flexible loops, linkers and tails. A few replicates can generate a much larger number of conformers, by exchanging each flexible region independently of the others. In this way, 10 replicates of a protein having 6 flexible regions can be used to generate a million conformations of a molecule. While this has obvious advantages in terms of sampling, the cost of assessing energies at every conformer is prohibitive, particularly when both molecules are flexible. Our approach addresses this combinatorial explosion, using key assumptions to compress the sampling by many orders of magnitude. ReplicOpter can perform hierarchical clustering from a list of rigid docking predictions and find nearby structures to any promising cluster representatives. These predicted complexes can then be refined and rescored. ReplicOpter's scoring function includes a Lennard‐Jones potential softened using the Anderson‐Chandler‐Weeks decomposition, a desolvation term derived from the Atomic Contact Energy function, Coulombic electrostatics, hydrogen bonding, and terms to model pi‐pi and pi‐cation interactions. ReplicOpter has performed well on several recent CAPRI systems. We are presently benchmarking ReplicOpter on the complete docking benchmark set to fully establish its utility in refining rigid docking predictions and identifying near‐native solutions. © 2010 Wiley‐Liss, Inc.
Docking by structural similarity at protein‐protein interfaces
Authors: Sinha Rohita , Kundrotas Petras J. , Vakser Ilya A.
Published: June 2010

Rapid accumulation of experimental data on protein‐protein complexes drives the paradigm shift in protein docking from “traditional,” template free approaches to template based techniques. Homology docking algorithms based on sequence similarity between target and template complexes can account for up to 20% of known protein‐protein interactions. When highly homologous templates for the target complex are not available, but the structure of the target monomers is known, docking by local structural alignment may provide an adequate solution. Such an algorithm was developed based on the structural comparison of monomers to cocrystallized interfaces. A library of the interfaces was generated from cocrystallized protein‐protein complexes in PDB. The partial structure alignment algorithm was validated on the DOCKGROUND benchmark sets. The optimal performance of the partial (interface) structure alignment was achieved with the interface residues defined by 12 Å distance across the interface. Overall, the partial structure alignment yielded more accurate models than the full structure alignment. Most templates identified by the partial structure alignment had low sequence identity to the target, which makes them hard to detect by sequence‐based methods. The results indicate that the structure alignment techniques provide a much needed addition to the docking arsenal, with the combined structure alignment and template free docking success rate significantly surpassing that of the free docking alone. Proteins 2010. © 2010 Wiley‐Liss, Inc.
Correlation of anatase particle size with photocatalytic properties
Authors: Lee Sangwook , Cho In‐Sun , Noh Jun‐Hong , Hong Kug Sun , Han Gill Sang , Jung Hyun Suk , Jeong Seonghoon , Lee Chanjong , Shin Hyunho
Published: June 2010

The influence of the anatase TiO2 particle size on the photocatalytic properties was systematically investigated on the nanometer‐size scale. The anatase TiO2 crystallite size estimated using the Scherrer equation was varied from 8.0 to 16.4 nm, and the photocatalytic properties were significantly deteriorated with decreasing crystallites size. The photocurrent measurements showed that the charge generation diminished with decreasing TiO2 particle size. The bandgap energies were measured as a function of the particle size using UV–Vis spectroscopy in order to investigate the origin of the lowered charge‐generation characteristics. The absorption band edge shift was found in the anatase nanocrystallites, which was attributed to the quantum size effect. Therefore, the increased bandgap energy induced the low light absorption capability, which was responsible for the low photoactivity of the very tiny anatase nanoparticles. These findings demonstrated that the particle‐size reduction was not the best way to maximize the photocatalytic properties of TiO2 because of the diminished light absorption.