bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractThe 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

AbstractThe 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

AbstractTwo 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

AbstractThe 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

AbstractPhenylalanine 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

AbstractMost 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

AbstractSites 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

AbstractThe 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

AbstractThe 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

AbstractPeptides 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

AbstractThe 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

AbstractHolo 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

AbstractA 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

AbstractThe 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.