bluuebunny/crossref_metadata_2025_split huggingface dataset:
Crystallization of purified recombinant human interleukin‐1β
Authors: Carter D. B. , Curry K. A. , Tomich C‐S. C. , Yem A. W. , Deibel M. R. , Tracey D. E. , Paslay J. W. , Carter J. B. , Theriault N. Y. , Harris P. K. W. , Reardon I. M. , Zurcher‐Neely H. A. , Heinrikson R. L. , Clancy L. L. , Muchmore S. W. , Watenpaugh K. D. , Einspahr H. M. 
Published: May 2005

AbstractThe gene for human interleukin‐1β was cloned from SK‐hep‐1 hepatoma cellular RNA and expressed at high levels in Escherichia coli both as the naturally processed form (rIL‐1β) and as a variant with an additional sequence of three amino acids on the N‐terminus (rIL‐1β+). Expressed protein was purified to homogeneity by a sequence of steps, which included low pH incubation, adsorption and desorption from Procion Red Sepharose, sizing on a Superose 12 fast‐performance liquid chromatography (FPLC) column, and anion exchange chromatography on QAE Sepharose. The final step provided a biologically active protein that migrates on twodimensional (2‐D) gels as a single spot with a pI of 6.7 ± 0.2 and a molecular mass of 17,500 daltons. Concentrated solutions of rIL‐1β have produced crystals by ammonium sulfate precipitation. The crystals are tetragonal, show the symmetry of space group P41 or its enantiomer, have lattice constants of a = 58.46 (1) and c = 77.02 (3) A, and scatter to at least 2 Å resolution. A structure determination based on these crystals is under way.
Comparison of model and nuclear magnetic resonance structures for the human inflammatory protein C5a
Authors: Zuiderweg Erik R. P. , Henkin Jack , Mollison Karl W. , Carter George W. , Greer Jonathan 
Published: May 2005

AbstractThe model structure previously proposed for human C5a, based upon the crystal structure of the homologous protein human C3a, is compared to the solution structure of human C5a recently determined by nuclear magnetic resonance (NMR) methods in our laboratory. The general folding and helix topography of the C5a protein were modeled very well. The N‐terminus, which is disordered in teh C3a crystal, was correctly predicted in the C5a model both as to its being a helix and as to its docking site on the rest of the molecule. On the other hand, the NMR data show that the biologically important C‐terminal residues are disordered in solution, unlike the model and the C3a crystal structure where this region was helical.
Intermolecular localization of epitopes within an oligomeric protein by immunoelectron microscopy and image processing
Authors: Boisset Nicolas , Frank Joachim , Taveau Jean Christophe , Billiald Philippe , Motta Genevieve , Lamy Josette , Sizaret Pierre Yves , Lamy Jean 
Published: May 2005

AbstractThree epitopes have been localized by immunoelectron microscopy on subunits Aa6 of the 4 × 6‐meric hemocyanin of the scorpion Androctonus australis. Soluble immunocomplexes composed of monoclonal antibodies and of native hemocyanin were purified, negatively stained with uranyle acetate by the single‐layer technique, and examined under the electron microscope (EM). The molecule images were digitized, aligned, and submitted to correspondence analysis according to the method of Van Heel and Frank (Ultramicroscopy 6: 187‐194, 1981). A high‐precision localization of the attachment point of the Fab arm to the antigen was achieved through a careful analysis of the average images. This method easily allowed the discrimination of epitopes located in different domains (Mr 20 kDa) of the same subunit. Nonoverlapping epitopes located in the same structural domain of subunit Aa6 could be distinguished by the stain exclusion patterns of their Fab arms. The method is general and may be used for epitope mapping in any antigen producing definite EM views.
Lecithin: Cholesterol acyltransferase activation by synthetic amphipathic peptides
Authors: Subbarao Nanda K. , Fielding Christopher J. , Hamilton Robert L. , Szoka Francis C. 
Published: May 2005

AbstractThe amphipathic helical theory of Segrest and colleagues (FEBS Lett.: 38: 247–253, 1974) proposes that the lipid‐binding segments of serum apolipoproteins are in an alpha helical conformation. Furthermore the helices have a hydrophobic face and a hydrophilic face with a specific distribution of positively and negatively charged residues. The importance of the pattern of the charged residues in the lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation by the segments is still debated. We designed a 30‐residue peptide, GALA, which in the alpha helical conformation hs a hydrophilic face composed of glutamic acid residues (Sabbarao et al.: Biochemistry 26: 2964–2972, 1987). GALA behaves like the serum apolipoproteins in its interaction with dimyristoylphospatidylcholine (DMPC) at neutral pH; the amino terminal tryptophan of GALA undergoes a blue shift in its fluorescence emission spectrum, and the circular dichroism (CD) spectrum indicates that GALA acquires alpha helical structure in the presence of DMPC. A DMPC‐GALA:19/1 (molar ratio) complex can be isolated by gel‐permeation chromatography. This complex has a discoidal structure with the approximate dimensions of 44‐Å diameter. GALA edge thickness and a 170‐ to 350‐Å diameter. GALA activates LCAT with DMPC but not with unsaturated phospholipids as the substrate. The apparent partition coefficient of GALA into DMPC vesicles is 100‐fold larger than into egg phosphatidlylcholine vesicles. The interaction of GALA with unsaturated lipids at neutral pH is so weak that no detectable change in the spectroscopic properties of GALA or the structure of the liposomes can be detected under the conditions used here. The sequence of GALA differs from previously studied model Apo A1 peptides by the absence of positively charged residues on the hydrophilic face. This indicates that positive charges in Apo A1‐like peptides are not required in order to form discoidal structures with saturated phospholipids or to activate LCAT with such lipid substrates.
Preliminary X‐ray diffraction studies and biochemical characterization of the antitumor protein mitomalcin indicate close similarity to neocarzinostatin
Authors: Sieker L. C. , Gnanarajah G. G. 
Published: May 2005

AbstractThe antitumor antibiotic protein mitomalcin, from the microorganism Streptomyces malayensis, has been purified to apparent homogenity and crystallized. The crystals belong to space group P212121 and have the following cell parameters : a=27.2 Å, b=34.1 Å, c=101.7 Å, and alpha;=β=γ=90°. These crystal properties are extremely similar to crystals of the antitumor protein neocarzinostatin (11.7 kilodaltons [kDa]) from Streptomyces carzinostaticus in spite of differing pH conditions for crystallizing the two proteins and an apparent difference in molecular weight is similar to that of neocarzinostatin. An amino acid composition analysis of mitomalcin indicates that some differences may exist between the two molecules, but a preliminary amino acid sequence analysis of the first 37 residues found no difference in the N‐terminal region of the molecule.
Microfolding: Conformational probability map for the alanine dipeptide in water from molecular dynamics simulations
Authors: Anderson Amil G. , Hermans Jan 
Published: May 2005

AbstractA direct attack on the protein‐folding problem has been initiated with the free energy perturbation methods of molecular dynamics. The complete conformational probability map for the alanine dipeptide is presented. This work uses the SPC model for the explicit hydration of the dipeptide. Free energy differences for the four observed minima (β, αR, αL, C) are given, and the free energy barriers between minima are outlined.
Criteria that discriminate between native proteins and incorrectly folded models
Authors: Novotný Jiři , Rashin A. A. , Bruccoleri R. E. 
Published: May 2005

AbstractVarious theoretical concepts, such as free energy potentials, electrostatic interaction potentials, atomic packing, solvent‐exposed surface, and surface charge distribution between native proteins and misfolded protein models. Misfolded models were constructed by introducing incorrect side chains onto polypeptide backbones: side chains of the α‐helical hemerythrin were modeled on the β‐sheeted backbone of immunoglobulin VL domain, whereas those of the VL domain were similarly modeled on he hemerythrin backbone. CONGEN, a conformational space sampling program, was used to construct the side chains, in contrast to the previous work,1where incorrect side chains were modeled inall transconformations. Capability of the conformational search procedure to reproduce native conformations was gauged first by rebuilding (the correct) side chains in hemerythrin and the VL domain: constructs with r.m.s differences from the x‐ray side chains 2.2–2.4 Å were produced, and many calculated conformations matched the native ones quite well. Incorrectly folded models were then constructed by the same conformational protocol applied to incorrect amino acid sequences. All CONGEN constructs, both correctly and incorrectly folded, were characterized by exceptionally small molecular surfaces and low potential energies. Surface charge density, atomic packing, and Coulomb formula‐based electrostatic interactions of the misfolded structures and the correctly folded proteins were similar, and therefore of little criteria clearly favored the native structures over the misfolded ones: (1) solvent‐exposed side‐chain nonpolar surface, (2) number of buried ionizable groups, and (3) empirical free energy functions that incorporate solvent effects.
Structure‐function relationships in 3‐phosphoglycerate kinase: Role of the carboxy‐terminal peptide
Authors: Mas Maria T. , Resplandor Zenaida E. 
Published: May 2005

AbstractYeast 3‐phosphoglycerate kinase (PGK) is a monomeric enzyme (Mr ∼ 45,000) composed of two globular domains. Each domain corresponds approximately to the amino‐ and carboxyterminal halves of the polypeptide chain. The carboxy‐terminal end extends over the interdomain “hinge” region and packs against the amino‐terminal domain. It has been proposed that domain movement, resulting in closure of the active site left, is essential for the catalytic of PGK. Large‐scale conformational changes have also been postulated to explain activation of the enzyme by sulfate ions. Using site‐specific mutagenesis, we have removed a 15‐amino‐acid carboxy‐terminal fragment, in order to probe its role in the substrate‐ and sulfate‐induced conformational changes. The truncated enzyme exhibited approximately 1% of the activity of native PGK and lost the ability to undergo sulfateinduced activation. The Km for ATP was essentially unchanged (Km = 0.23mM), whereas the Km value for 3‐phosphoglycerate was increased about eightfold (Km = 3.85 mM and 0.50 mM, respectively). These results suggest that the carboxy‐terminal segment is important for the mechanism of substrate‐ and specific‐induced conformational transitions. CD spectra and sedimentation velocity measurements indicate that the carboxy‐terminal peptide is essential for structural integrity of PGK. The increased susceptibility of the truncated enzyme to thermal inactivation implies that the carboxy‐terminal peptide also contributes to the stability of PGK.
Crystallographic structure analysis of lamprey hemoglobin from anomalous dispersion of synchrotron radiation
Authors: Hendrickson Wayne A. , Smith Janet L. , Phizackerley R. Paul , Merritt Ethan A. 
Published: May 2005

AbstractThe molecular structure of lamprey hemoglobin was previously determined and refined by conventional crystallographic analysis. In this study, the structural analysis has been repeated in the course of developing the method of multiwavelength anomalous diffraction (MAD) for phase determination. New experimental and analytical procedures that were devised to perform this determination should have general applicability. These include an experimental design to optimize signal strength and reduce systematic errors, experimental evaluation of anomalous scattering factors, and a least‐squares procedure for analyzing the MAD data. MAD phases for the structure at 3Å resolution are as accurate overall as the multiple isomorphous replacement (MIR) phases determined previously.
Comparison of the dynamics of the membrane‐bound form of fd coat protein in micelles and in bilayers by solution and solid‐state nitrogen‐15 nuclear magnetic resonance spectroscopy
Authors: Bogusky M. J. , Leo G. C. , Opella S. J. 
Published: May 2005

AbstractSolid‐state and solution 15N nuclear magnetic resonance experiments on uniformly and specifically 15N labeled coat protein in phospholipid bilayers and in detergent micelles are used to describe the dynamics of the membrane‐bound form of the protein. The residues in the N‐ and C‐terminal portions of the coat protein in both phospholipid bilayers and in detergent micelles are mobile, while those in the hydrophobic midsection are immobile. There is evidence for a gradient of mobility in the C‐terminal region of the coat protein in micelles; at 25°C only the last two residues are mobile on the 109‐Hz timescale, while the last six to eight residues appear to be mobile on slower timescales and highly mobile at higher temperatures. Since all of the C‐terminal residues are immobile in the virus particles, the mobility of these residues in the membrane‐bound form of the protein may be important for the formation of protein‐DNA interactions in the assembly process.
Polar hydrogen positions in proteins: Empirical energy placement and neutron diffraction comparison
Authors: Brünger Axel T. , Karplus Martin 
Published: May 2005

AbstractA method for the prediction of hydrogen positions in proteins is presented. The method is based on the knowledge of the heavy atom positions obtained, for instance, from X‐ray crystallography. It employs an energy minimization limited to the environment of the hydrogen atoms bound to a common heavy atom or to a single water molecule. The method is not restricted to proteins and can be applied without modification to nonpolar hydrogens and to nucleic acids. The method has been applied to the neutron diffraction structures of trypsin ribonuclease A, and bovine pancreatic trypsin inhibitor. A comparison of the constructed and the observed hydrogen positions shows few deviations except in situations in which several energetically similar conformations are possible. Analysis of the potential energy of rotation of Lys amino and Ser, Thr, Tyr hydroxyl groups reveals that the conformations of lowest intrinsic torsion energies are statistically favored in both the crystal and the constructed structures.
Helix lap‐joints as ion‐binding sites: DNA‐binding motifs and Ca‐binding “EF hands” are related by charge and sequence reversal
Authors: Richardson Jane S. , Richardson David C. 
Published: May 2005

AbstractThe DNA‐binding helix pairs in gene repressor and activator were compared with other approximately perpendicular pairs of adjacent helices in the known protein structures. Two other examples of closely matching conformations were found in cytochrome c peroxidase (residues 153–174) and in ribosomal L7/L12 protein (residues 68–89). Another group of such offset “lap‐joints” are the Ca‐binding “EF hand” structures, which bind a positive rather than a negative ligand. The EF hands turn out to match the DNA‐binding motifs quite well (outside of the loop) if their sequence direction is reversed. This conformation is thus not as unusual as had been thought, but may have a more generalized role in ion binding and occasionally occur in a purely structural role.
Test of circular dichroism (CD) methods for crambin and CD‐assisted secondary structure prediction of its homologous toxins
Authors: Teeter M. M. , Whitlow Marc 
Published: May 2005

AbstractMethods that analyze protein circular dichroism (CD) spectra for fractions of secondary structure are evaluated for the plant protein crambin, which has a known high‐resolution crystal structure. In addition, a two‐step secondary structure prediction scheme is presented and used for the toxins homologous to crambin, shown by others to have secondary structures similar to crambin.The test of CD spectral analysis methods with the protein crambin employed two computer programs and several CD basis sets. Crambin's crystal structure, known to 0.945 Å resolution (Hendrickson, W.A., Teeter, M.M. Nature 290:107–113, 1981), allows accurate evaluation of results. Analysis with the protein spectra basis sets (Provencher, S. W., Glöckner, J. Biochemistry 20:33–37, 1981) as modified (Manavalan, P., Johnson, W. C., Jr. Anal. Biochem. 167:76–85, 1987) agreed most closely with crambin's crystal structure. This method was then applied to the CD spectra of the membrane‐active toxins homologous to crambin (α1‐ and β‐purothionin, phoratoxin A and B, an viscotoxin A3 and B).The new program SEQ (pronounced “seek”) was developed to assign the secondary structure along the protein chain in a hierarchical fashion and applied to the plant toxins. The method constrained the secondary structure fractions to those from CD analysis and combined standard statistical methods with amphipathic helix location.Both CD‐arrived secondary structure percentages and sequence assignment indicate that the viscotoxins are structurally most similar to crambin. Purothionin's secondary structure was predicted to be fundamentally similar to crambin's with a difference at the start of the first helix. This assignment agreed with Raman and NMR analyses of Purothionin and lends validity to the method presented here. Differences from the NMR in the CD secondary structure fraction analysis for phoratoxin suggest interference in the CD from tryptophan residues.
The construction of new proteins: V. A template‐assembled synthetic protein (TASP) containing both a 4‐helix bundle and β‐barrel‐like structure
Authors: Mutter Manfred , Hersperger René , Gubernator Klaus , Müller Klaus 
Published: May 2005

AbstractThe construction of a template‐assembled synthetic protein (TASP) designed to contain both a 4‐helix bundle and a β‐barrel as two folding “domains” is described. For the de novo design of proteins, amphiphilic helices (α) and β‐sheets (β) are covalently attached to a template peptide (T) carrying functional side chains suitably oriented to promote intarmolecular folding of the secondary structure blocks into a characteristic packing arrangement, i.e., T8‐(4α)(4β). The design of this new macromolecule was assisted by computer modeling, which suggested a low‐energy conformation with tight hydrophobic packing of the secondary structure subunits. Solid‐phase synthesis of the “two‐domain” TASP molecule was achieved using orthogonal protection techniques. The solution properties as well as circular dichroism (CD) and infrared spectroscopy (IR) data under various experimental conditions are consistent with the folded conformation suggested by modeling.
Electrostatic interactions in the assembly of Escherichia coli aspartate transcarbamylase
Authors: Glackin M. P. , McCarthy M. P. , Mallikarachchi D. , Matthew J. B. , Allewell N. M. 
Published: May 2005

AbstractAlthough ionizable groups are known to play important roles in the assembly, catalytic, and regulatory mechanisms of Escherichia coli aspartate transcarbamylase, these groups have not been characterized in detail. We report the application of static accessibility modified Tanford‐Kirkwood theory to model electrostatic effects associated with the assembly of pair of chains, subunits, and the holoenzyme. All of the interchain interfaces except R1–R6 are stabilized by electrostatic interactions by −2 to −4 kcal−m−1 at pH 8. The pH dependence of the electrostatic component of the free energy of stabilization of intrasubunit contacts (C1–C2 and R1–R6) is qualitatively different from that of intersubunit contacts (C1–C4, C1–R1, and C1–R4). This difference may allow the transmission of information across subunit interfaces to be selectively regulated. Groups whose calculated pK or charge changes as a result of protein‐protein interactions have been identified and the results correlated with available information about their function. Both the 240s loop of the c chain and the region near the Zn(II) ion of the r chain contain clusters of ionizable groups whose calculated pK values change by relatively large amounts upon assembly. These pK changes in turn extend to regions of the protein remote from the interface. The possibility that networks of ionizable groups are involved in transmitting information between binding sites is suggested.
Structural basis of hierarchical multiple substates of a protein. I: Introduction
Authors: Noguti Tosiyuki , Gō Nobuhiro 
Published: May 2005

AbstractA computer experiment of protein dynamics is carried out, which consists of two steps: (1) A Monte Carlo simulation of thermal fluctuations in the native state of a globular protein, bovinepancreatic trypsin inhibitor; and (2) a simulation of the quick freezingof fluctuating conformations into energy minima by minimization of the energy of a number of conformations sampled in the Monte Carlo simulations sampled in the Monte Carlo simulation. From the analysis of results of the computer experiment is obtained the following picture of protein dynamics:multiple energy minima exist in the native state, and they are distributedin clusters in the conformational space. The dynamics has a hierarchical structure which has at least two levels. In the first level, dynamics is restricted within one of the clusters of minima. In the second, transitions occur among the clusters. Local parts of a protein molecule, side chains and local main chain segments, can take multiple locally stable conformations in the native state. Many minima result from combinations of these multiple local conformations. The hierarchical structure in the dynamics comes from interactions among the local parts. Protein moleculeshave two types of flexibility, each associated with elastic and plastic deformations, respectively.
Structural basis of hierarchical multiple substates of a protein. II: Monte carlo simulation of native thermal fluctuations and energy minimization
Authors: Noguti Tosiyuki , Gō Nobuhiro 
Published: May 2005

AbstractConformational fluctuations in a globular protein, bovine pancreatic trypsin inhibitor, in the time range between picoseconds and nanoseconds are studied by a Monte Carlo simulation method. Multipleenergy minima are derived from sampled conformations by minimizing their energy. They are distributed in clusters in the conformational space. A hierarchical structure is observed in the simulated dynamics. In the time range between 10−14 and 10−10 seconds dynamics is well represented by a superposition of vibrational motions within an energy well with transitions among minima within each cluster. Transitions among clusters take place in the time range of nanoseconds or longer.
The pro‐peptide is not necessary for active renin secretion from transfected mammalian cells
Authors: Harrison T. M. , Chidgey M. A. J , Brammar W. J , Adams G. J 
Published: May 2005

AbstractCultured mouse myeloma cells were transfected with expression vectors encoding the aspartyl proteinase, human renin. The full construct, encoding the renin precursor prorenin, allows transfected cellsto secrete the enzymically inactive pro‐protein. Activity is detectable only following trypsin treatment which mimics the physiological activation step. Accordingly, it appears that myeloma cells do not contain detectable levels of an appropriate activating proteinase. However, when these cells are transfected with a construct from which the pro‐peptide coding sequence has been deleted, they secrete an apparently fully active enzyme which is indistinguishable from mature renin. We conclude that expression of the pro‐peptide is not necessary to allow correct folding of the molecule and its passage through the secretory pathway.