bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation and characterization of native human renin derived from Chinese hamster ovary cells
Authors: Poorman Roger A. , Palermo Daniel P. , Post Leonard E. , Murakami Kazuo , Kinner John H. , Smith Clark W. , Reardon Ilene , Heinrikson Robert L. 
Published: May 2005

AbstractTransfection of Chinese hamster ovary (CHO) cells with a plasmid containing the cDNA for human preprorenin has provided cell lines that secrete 15 mg of native prorenin per liter of culture medium. Tryptic activation of the prorenin occurs by selective cleavage of the Arg66‐Leu67 bond (numbering as in preprorenin). The renin product, purified in a single step and in high yield by affinity chromatography, is fully stable for as long as 8 months when stored in solution at 4°C and pH 6.5. Purity of the renin was judged to be greater than 95% by gel electrophoresis, compositional and N‐terminal sequence analyses, and specific enzyme activity. An important aspect of the present work is the development of a direct assay for renin which permits accurate and reproducible evaluation of enzyme units and kinetic parameters. Application of methods described herein, combined with appropriate scale‐up fermentation capabilities, provides the means for generating gram quantities of human renin and its zymogen.
An algorithm for determining the conformation of polypeptide segments in proteins by systematic search
Authors: Moult J. , James M. N. G. 
Published: May 2005

AbstractThe feasibility of determining the conformation of segments of polypeptide chain up to six residues in length in globular proteins by means of systematic search through the possibles conformations has been investigated. Trial conformations are generated by using representative sets of ϕ, ψ, and χ angels that have been derived from an examination of the distributions of these angles in refined protein structures. A set of filters based on simple rules that protein structures obey is used to reduce the number of conformations to a manageable total. The most important filters are the maintenance of chain integrity and the avoidance of tooshort van der Waals contacts with the rest of the protein and with other portions of the segment under construction. The procedure is intended to be used with approximate models so that allowance is made throughout for errors in the rest of the structure. All possible main chains are first constructed and then all possible side‐chain conformations are built onto each of these. The electrostatic energy, including a solvent screening term, and the exposed hyrophobic area are evaluated for each accepted conformation. The method has been tested on two segments of chain in the trypsin like enzyme from Streptomyces griseus. It is found that there is a wide spread of energies among the accepted conformations, and the lowest energy ones have satisfactorily small root mean square deviations from the X‐ray structure.
The restoration of electron micrographs blurred by drift and rotation
Authors: Carragher Bridget , Bluemke David A. , Potel Michael J. , Josephs Robert 
Published: May 2005

AbstractWe have investigated the restoration of electron micrographs exhibiting blurring due to drift and rotation. Blurring due to drift arises in micrographs taken of a specimen which is moving relative to the image plane. A related problem is that of rotational blurring which arises in micrographs of thin sections of helical particles viewed in cross section. The twist of the particle within the finite thickness of the section causes the image to appear rotationally blurred about the helical axis.Restoration algorithms were evaluated by applying them to the restoration of blurred model images degraded by additive Gaussian noise. Model images were also used to investigate how an incorrect estimate of the point spread function function describing the blur would effect the restoration. Images were, if necessary, geometrically transformed to a space in which the point spread function of the blur can be considered as linear and space invariant as, under these conditions, the restoration algorithms are greatly simplified. In the case of the rotationally blurred images this procedur was accomplished by transforming the image to polar coordinates.The restoration techniques were successfully applied to blurred micrographs of bacteriophage T4 and crystals of catalase. The quality of the restoration was judged by comparisons of the restored images to undegraded images. Application to micrographs of rotationally blurred cross sections of helical macrofibers of sickle hemoglobin resulted in a reduction in the amount of rotational blurring.
Preliminary x‐ray diffraction analysis of HhaII endonuclease‐DNA cocrystals
Authors: Chandrasegaran Srinivasan , Smith Hamilton O. , Amzel Mario L. , Ysern Xavier 
Published: May 2005

AbstractHhaII restriction endonuclease purified from an overproducing recombinant E. coli clone has been cocrystallized with a heptanucleotide duplex, d‐GGAGTCC:GGACTCC. The cocrystals are monoclonic and belong to the space group C2. The unit cell dimensions are a = 199.0±1.0 Å, b = 100.0±0.5 Å, c = 80.3±0.4 Å, and β = 101.0±1.0°. There appear to be two dimers per asymmetric unit and the crystals diffract to 4‐Å resolution.
Predicting antibody hypervariable loop conformations II: Minimization and molecular dynamics studies of MCPC603 from many randomly generated loop conformations
Authors: Fine R. M. , Wang H. , Shenkin P. S. , Yarmush D. L. , Levinthal C. 
Published: May 2005

AbstractWe describe a method for predicting the conformations of loops in proteins and its application to four of the complementarity determining regions [CDRs] in the crystallographically determined structure of MCPC603. The method is based on the generation of a large number of randomly generated conformations for the backbone of the loop being studied, followed by either minimization or molecular dynamics followed by minimization starting from these random structures. The details of the algorithm for the generation of the loops are presented in the first paper in this series (Shenkin etal.[submitted]). The results of minimization and molecular dynamics applied to these loops is presented here. For the two shortest CDRs studied (H1 and L2, which are five and seven amino acids long), minimizations and dynamics simulations which ignore interactions of the loop amino acids beyond the carbon ture closely. This suggests that these loops fold independently of sequence variation. For the third CDR (L3, which is nine amino acids), those portions of the CDR near its base which are hydrogen bonded to framework are well replicated by our procedures, but the top of the loop shows singificant conformational variability. This variability persists when side chain interactions for the MCPC603 sequence are included. For a fourth CDR (H3, which is 11 amino acids long), new low‐energy backbone conformations are found; however, only those which are close to the crystal are compatible with the sequence when side chain interactions are taken into account. Results from minimuzation and dynamics on single CDRs with all other CDRs removed are presented. These allow us to explore the extent to which individual CDR conformations are determined by interactions with framework only.
Estimation of uncertainties in X‐ray refinement results by use of perturbed structures
Authors: Kuriyan John , Karplus Martin , Petsko Gregory A. 
Published: May 2005

AbstractThe uncertainties in the refined parameters for a 1.5‐Å X‐ray structure of carbon‐monoxy (FeII) myoglobin are estimated by combining energy minimization with least‐squares refinement against the X‐ray data. The energy minimization, done without reference to the X‐ray data, provide perturbed structures which are used to restart conventional X‐ray refinement. The resulting refined structures have the same, or better, R‐factor and stereochemical parameters as the original X‐ray structure, but deviate from it by 0.13 Å rms for the backbone atoms and 0.31 Å rms for the sidechain atoms. Atoms interacting with a disordered sidechain, Arg 45 CD3, are observed to have larger positional uncertainties. The uncertainty in the B‐factors, within the isotropic harmonic motion approximation, is estimated to be 15%. The resulting X‐ray structures are more consistent with the energy parameters used in simulations.
Subunit assembly and metabolic stability of E. coli RNA polymerase
Authors: Ishihama Akira , Fujita Nobuyuki , Glass Robert E. 
Published: May 2005

AbstractImmunological cross‐reaction was employed for identification of proteolytic fragments of E. coli RNA polymerase genered both in vitro and in vivo. Several species of partially denatured but assembled RNA polymerase were isolated, which were composed of fragments of the two large subunits, β and β′, and the two small and intact subunits, α and σ. Comparison of the rate and pathway of proteolytic cleavage in vitro of unassembled subunits, subassemblies, and intact enzymes indicated that the susceptibility of RNA polymerase subunits to proteolytic degradation was dependent on the assembly state.Using this method, degradation in vivo was found for some, but not all, of the amber fragments of β subunit in merodiploid cells carrying both wild‐type and mutant rpoB genes. Although the RNA polymerase is a metabolically stable component in exponentially growing cells of E. coli, degradation of the full‐sized subunits was found in two cases, i.e., several temperature‐sensitive E. coli mutants with a defect in the assembly of RNA polymerase and the stationary‐phase cells of a wild‐type E. coli. The in vivo degradation of RNA polymerase was indicated to be initiated by alteration of the enzyme structure.
Continuous compact protein domains
Authors: Zehfus Micheal H. 
Published: May 2005

AbstractThe coefficient of compactness was recently introduced and used to locate domains in lysozyme and ribonuclease (Zehfus and Rose: Bio‐chemistry 25:5759–5765, 1986). Nineteen additional proteins now have been analyzed by using this measure. Complete listings of compact units and plots showing their hierarchic organization are presented for all twenty‐one proteins. Large compact units correspond well to protein domains; however, many smaller compact structures of equal or better compactness are also found. Since small compact units could represent subdomains or protein‐folding intermediates, their structural composition is further examined.
Prediction of secondary structure by evolutionary comparison: Application to the α subunit of tryptophan synthase
Authors: Crawford Irving P. , Niermann Thomas , Kirschner Kasper 
Published: May 2005

AbstractThe amino acid sequences of the α subunits of tryptophan synthase form ten different microorganisms were alingned by standard procedures. The α helics, β strands and turns of each sequence were predicted separately by two standard prediction algorithms and averaged at homologous sequence positions. Additional evidence for conserved secondary structure was derived form profiles of average hydropathy and chain flexibility values, leading to a joint prediction. There is good agreement between (1) predicted β strands, maximal hydropathy and minimal flexibility, and (2) predicted loops, great chain flexibility, and protein segments that accept insertions of various lengths in individual sequences. The α subunit is predicted to have eight repeated β‐loop‐α‐loop motifs with an extra N‐terminal α helix and an intercalated segment of highly conserved residues. This pattern suggests that the teritiary structure of the α subunit is an eightfold α/β barrel. The distribution of conserved amino acid residues and publilshed data on limited proteolysis, chemical modification, and mutagenesis are consistent with the α/β barrel structure. Both the active site of the α subunit and the combining site for the β2 subunit are at the end of the barrel formed by the carboxyl‐termini of the β strands.
Prediction of the three‐dimensional structure of human growth hormone
Authors: Cohen F.E. , Kuntz I.D. 
Published: May 2005

AbstractIn recent years, the protein‐folding problem has attracted the attention of molecular biologists. Efforts have focused on developing heuristic and energy‐based algorithms to predict the three‐dimensional structure of a protein from its amino acid sequence. We have applied a series of heuristic algorithms to the sequence of human growth hormone. A family of five structures which are generically right‐handed fourfold α‐helical bundles are found from an investigaion of ∼108 structures. A plausible receptor binding site is suggested. Independent crystallographic analysis confirms some aspects of these predictions. These methods only deal with the “core” structure, and conformations of many residues are not defined. Further work is required to identify a unique set of coordinates and to clarify the topological alternatives available to α‐helical proteins.
Determination of three‐dimensional protein structures from nuclear magnetic resonance data using fragments of known structures
Authors: Krauli Per J. , Jones T. Alwyn 
Published: May 2005

AbstractA method to build a three‐dimensional protein model from nuclear magnetic resonance (NMR) data using fragments from a data base of crystallographically determined protein structures is presented. The interproton distances derived from the nuclear Overhauser effect (NOE) data are compared to the precalculated distances in the known protein structures. An efficient search algorithm is used, which arranges the distancs in matrices akin to a Cα diagonal distance plot, and compares the NOE distance matrices for short sequential zones of the protein to the data base matrices. After cluster analysis of the fragments found in this way, the structure is built by aligning fragments in overlapping zones. The sequentially long‐range NOEs cannot be used in the initial fragments search but are vital to discriminate between several possible combinations of different groups of fragments. The method has been tested on one simulated NOE data set derived from a crystal structure and one experimental NMR data set. The method produces models that have good local structure, but may contain larger global errors. These models can be used as the starting point for further refinement, e.g., by restrained molecular dynamics or interactive graphics.
Association of calmodulin and smooth muscle myosin light chain kinase: Application of a lable selection technique with trace acetylated calmodulin
Authors: Jackson Arthur E. , Carraway Kermit L. , Payne M. Elizabeth , Means Anthony R. , Puett David , Brew Keith 
Published: May 2005

AbstractA method is described for rapidly surveying the effects of modifying individual amino acid residues of a protein on its ability to interact specifically with another macromolecule. The procedure has been used to examine the individual roles of the seven lysyl residues of calmodulin in its ability to bind to smooth muscle myosin light chain kinase; previous studies by Jackson et al. (J. Biol. Chem. 261:1226–12232, 1986) have suggested that certain lysines may be located close to the interaction site. Trace [3H]‐acetylated calmodulin, consisting predominantly of molecules acetylated at single sites together with unmodified protein, was incubated in excess (five‐ to 20‐fold) with smooth muscle MLC kinase to allow the modified and unmodified molecules to compete for binding to the enzyme. Subsequently, the calmodulin‐enzyme complex was separated from unbound calmodulin, and the level of acetylation of each of the seven lysines of the bound fraction of calmodulin was determined and compared to that of each corresponding group of the starting preparation. Significant changes were found at only two of the lysines, 21 and 75, where the extent of acetylation in the bound fraction was three‐ and fivefold lower, respectively, than that in the original preparation. These results were reproducible in three separate selection experiments employing both chicken and turkey gizzard MLC kinase. It is concluded that acetylation of calmodulin at either lysine 21 or 75 markedly reduces its affinity for MLC kinase, but acetylation at any of the other lysines (13, 30, 77, 94, or 148) has only minor effects. This finding supports the proposal that the face of the central helix containing lysine 75 is involved in interaction with MLC kinase and suggests also that additional contact near Ca2‐binding site 1 occurs.
Identification of structural motifs from protein coordinate data: Secondary structure and first‐level supersecondary structure*
Authors: Richards Frederic M. , Kundrot Craig E. 
Published: May 2005

AbstractA computer program is described that produces a description of the secondary structure and supersecondary structure of a polypeptide chain using the list of alpha carbon coordinates as input. Restricting the term “secondary structure” to the conformation of contiguous segments of the chain, the program determines the initial and final residues in helices, extended strands, sharp turns, and omega loops. This is accomplished through the use of difference distance matrices. The distances in idealized models of the segments are compared with the actual structure, and the differences are evaluated for agreement within preset limits. The program assigns 90–95% of the residues in most proteins to at least one type of secondary elementIn a second step the now‐defined helices and strands are idealized as straight line segments, and the axial directions and locations are compiled from the input Cα coordinate list. These data are used to check for moderate curvature in strands and helices, and the secondary structure list is corrected where necessary. The geometric relations between these line segments are then calculated and output as the first level of supersecondary structure. A maximum of six parameters are required for a complete description of the relations between each pair. Frequently a less complete description will suffice, for example just the interaxial separation and angle. Both the secondary structure and one aspect of the supersecondary structure can be displayed in a character matrix analogous to the distance matrix format. This allows a quite accurate two‐dimensional display of the three‐dimensional structure, and several examples are presentedA procedure for searching for arbitrary substructures in proteins using distance matrices is also described. A search for the DNA binding helix‐turnhelix motif in the Protein Data Bank serves as an exampleA further abstraction of the above data can be made in the form of a metamatrix where each diagonal element represents an entire secondary segment rather than a single atom, and the off‐diagonal elements contain all the parameters describing their interrelations. Such matrices can be used in a straightforward search for higher levels of supersecondary structure or used in toto as a representation of the entire tertiary structure of the polypeptide chain.
Differences in crystal properties and ligand affinities of an antifluorescyl fab (4‐4‐20) in two solvent systems
Authors: Gibson A. L. , Herron J. N. , He X.‐M. , Patrick V. A. , Mason M. L. , Lin J.‐N. , Kranz D. M. , Voss E. W. , Edmundson A. B. 
Published: May 2005

AbstractAn antigen‐binding fragment (Fab) from a murine monoclonal antibody (4‐4‐20) with high affinity for fluorescein was cocrystallized with ligand in polyethylene glycol (PEG) and 2‐methl‐2,4‐pentanediol (MPD) in forms suitable for X‐ray analyses. In MPD the affinity of the intact antibody for fluorescein was 300 times lower than the value (3.4 × 1010 M−1) obtained in aqueous buffers. This decreased affinity was manifested by the partial release of bound fluorescein when MPD was added to solutions of liganded Feb during crystallization trials, In PEG, the ligand remained firmly bound to the protein. The liganded Feb crystallized in the monoclinic space group P21 in PEG, with a = 58.6, b = 97.2, c = 44.5 Å and β = 95.2°. In MPD the space group was triclinic P1, with a = 58.3, b = 43.4, c = 42.3 Å, α = 83.9°, β = 87.6°, and γ = 84.5°. X‐ray diffraction data were collected for both forms to 2.5‐Å resolution. Surprisingly, the triclinic form of the liganed antifluorescyl Feb had the same space group, closely similar cell dimensions, and practically the same orientation in the unit cell as an unliganded Fab (BV04‐01) with activity against single‐stranded DNA.
Combined procedure of distance geometry and restrained molecular dynamics techniques for protein structure determination from nuclear magnetic resonance data: Application to the DNA binding domain of lac repressor from Escherichia coli
Authors: de Vlieg J. , Scheek R. M. , van Gunsteren W. F. , Berendsen H. J. C. , Kaptein R. , Thomason J. 
Published: May 2005

AbstractThe technique of two‐dimensional nuclear magnetic resonance (2D‐NMR) has recently assumed an active role in obtaining information on structures of polypeptides, small proteins, sugars, and DNA fragments in solution. In order to generate spatial structures from the atom‐atom distance information obtained by the NMR method, different procedures have been developed. Here we introduce a combined procedure of distance geometry (DG) and molecular dynamics (MD) calculations for generating 3D structures that are consistent with the NMR data set and have reasonable internal energies. We report the application of the combined procedure on the lac repressor DNA binding domain (headpiece) using a set of 169 NOE and 17 “hydrogen bond” distance constraints. Eight of ten structures generated by the distance geometry algorithm were refined within 10 ps MD simulation time to structures with low internal energies that satisfied the distance constraints.Although the combination of DG and MD was designed to combine the good sampling properties of the DG algorithm with an efficient method of lowering the internal energy of the molecule, we found that the MD algorithm contributes significantly to the sampling as well.
Structural comparison of the prokaryotic ribosomal proteins L7/L12 and L30
Authors: Leijonmarck Marie , Appelt Krzysztof , Badger John , Liljas Anders , Wilson Keith S. , White Stephen W. 
Published: May 2005

AbstractThe structure of two prokaryotic ribosomal proteins, the carboxyterminal half of L7/L12 from Escherichia coli (L12CTF) and 1.30 from Bacilus Stearothermophilus display a remarkably similar fold in which alpaha‐helices pack onto one side of an antiparallel, three‐stranded, beta‐pleated sheet. A detailed comparison of the structures by least‐squares methods reveals that more than two‐thirds of the alpha carbons can be superimposed with a root mean square distance of 2.33 Å. The principal difference is an extra alpha‐helix in L12 CTF. The sequences of the proteins display a distinct conservation in regions which are crucial to the common fold, in particular the hydrophobic core. It is proposed that the similarity is a result of divergent evolution.
Photochemical crosslinking of bacteriophage T4 single‐stranded DNA‐binding protein (gp32) to oligo‐p(dT)8: Identification of phenylalanine‐183 as the site of crosslinking
Authors: Shamoo Yousif , Williams Kenneth R. , Konigsberg William H. 
Published: May 2005

AbstractUsing ultraviolet light, both the 33,000‐dalton single‐standard DNA‐binding protein from T4 bacteriophage (gp32)as well as a 25,000‐dalton limited trypsin cleavage product of gp32 (core gp32*) that retains high affinity for single‐stranded DNA can be crosslinked to an oligodeoxynucleotide, p(dT)8. After photolysis, a single tryptic peptide crosslinked to p(dT)8 was isolated by anion‐exchange high‐performance liquid chromatography. Gas‐phase sequencing of this modified peptide gave the following sequence: Gln‐Val‐Ser‐Gly‐(X)‐Ser‐Asn‐Tyr‐Asp‐Glu‐Ser‐Lys, which corresponds to residues 179–190 in gp32. Based on the absence of the expected phenylthiohydantoin derivative of phenylalanine 183 at cycle 5 (X) we infer that crosslinking has occurred at this position and that phenylalanine 183 is at the interface of the gp32:P(dT)8 complex in an orientation that allows covalent bond formation with the thymine radical produced by ultraviolet irradiation.