bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structure and sequence relationships in the lipocalins and related proteins
Authors: Flower Darren R. , North ANTHONY C.T. , Attwood Teresa K. 
Published: February 2009

AbstractThe lipocalins and fatty acid‐binding proteins (FABPs) are two recently identified protein families that both function by binding small hydrophobic molecules. We have sought to clarify relationships within and between these two groups through an analysis of both structure and sequence. Within a similar overall folding pattern, we find large parts of the lipocalin and FABP structures to be quantitatively equivalent. The three largest structurally conserved regions within the lipocalin common core correspond to characteristic sequence motifs that we have used to determine the constitution of this family using an iterative sequence analysis procedure. This afforded a new interpretation of the family, which highlighted the difficulties of determining a comprehensive and coherent classification of the lipocalins. The first of the three conserved sequence motifs is also common to the FABPs and corresponds to a conserved structural element characteristic of both families. Similarities of structure and sequence within the two families suggests that they form part of a larger “structural superfamily”; we have christened this overall group the calycins to reflect the cup‐shaped structure of its members.
Reduced representation model of protein structure prediction: Statistical potential and genetic algorithms
Authors: Sun Shaojian 
Published: February 2009

AbstractA reduced representation model, which has been described in previous reports, was used to predict the folded structures of proteins from their primary sequences and random starting conformations. The molecular structure of each protein has been reduced to its backbone atoms (with ideal fixed bond lengths and valence angles) and each side chain approximated by a single virtual united‐atom. The coordinate variables were the backbone dihedral angles ϕ and Ψ. A statistical potential function, which included local and nonlocal interactions and was computed from known protein structures, was used in the structure minimization. A novel approach, employing the concepts of genetic algorithms, has been developed to simultaneously optimize a population of conformations. With the information of primary sequence and the radius of gyration of the crystal structure only, and starting from randomly generated initial conformations, I have been able to fold melittin, a protein of 26 residues, with high computational convergence. The computed structures have a root mean square error of 1.66 Å (distance matrix error = 0.99 Å) on average to the crystal structure. Similar results for avian pancreatic polypeptide inhibitor, a protein of 36 residues, are obtained. Application of the method to apamin, an 18‐residue polypeptide with two disulfide bonds, shows that it folds apamin to native‐like conformations with the correct disulfide bonds formed.
Energy coupling between DNA binding and subunit association is responsible for the specificity of DNA‐Arc interaction
Authors: Silva Jerson L. , Silveira Cristina F. 
Published: February 2009

AbstractThe effects of several DNA molecules on the free energy of subunit association of Arc repressor were measured. The association studies under equilibrium conditions were performed by the dissociating perturbation of hydrostatic pressure. The magnitude of stabilization of the subunit interaction was determined by the specificity of the protein‐DNA interaction. Operator DNA stabilized the free energy of association by about 2.2 kcal/mol of monomeric unit, whereas poly(dG‐dC) stabilized the subunit interaction by only 0.26 kcal. Measurements of the stabilizing free energy at different DNA concentrations revealed a stoichiometry of two dimers per 21 bp for the operator DNA sequence and for the nonspecific DNA poly(dA‐dT). However, the maximum stabilization was much larger for operator sequence (Δp = 1, 750 bar) as compared for poly(dA‐dT) (Δp = 750 bar). The importance of the free‐energy linkage for the recognition process was corroborated by its absence in a mutant Arc protein (PL8) that binds to operator and nonspecific DNA sequences with equal, low affinity. We conclude that the coupling accounts for the high specificity of the Arc‐operator DNA interaction. We hypothesize a mutual coupling between the protein subunits and the two DNA strands, in which the much higher persistency of the associated form when Arc is bound to operator would stabilize the interactions between the two DNA strands.
Reconstitution of active catalytic trimer of aspartate transcarbamoylase from proteolytically cleaved polypeptide chains
Authors: Powers Vincent M. , Yang Ying R. , Fogli Michael J. , Schachman H. K. 
Published: February 2009

AbstractTreatment of the catalytic (C) trimer of Escherichia coli aspartate transcarbamoylase (ATCase) with α‐chymotrypsin by a procedure similar to that used by Chan and Enns (1978, Can. J. Biochem. 56, 654‐658) has been shown to yield an intact, active, proteolytically cleaved trimer containing polypeptide fragments of 26, 000 and 8, 000 MW. Vmax of the proteolytically cleaved trimer (CPC) is 75% that of the wild‐type C trimer, whereas Km for aspartate and Kd for the bisubstrate analog, N‐(phosphonacetyl)‐l‐aspartate, are increased about 7‐ and 15‐fold, respectively. CPC trimer is very stable to heat denaturation as shown by differential scanning microcalorimetry. Amino‐terminal sequence analyses as well as results from electrospray ionization mass spectrometry indicate that the limited chymotryptic digestion involves the rupture of only a single peptide bond leading to the production of two fragments corresponding to residues 1‐240 and 241‐310. This cleavage site involving the bond between Tyr 240 and Ala 241 is in a surface loop known to be involved in intersubunit contacts between the upper and lower C trimers in ATCase when it is in the T conformation. Reconstituted holoenzyme comprising two CPC trimers and three wild‐type regulatory (R) dimers was shown by enzyme assays to be devoid of the homotropic and heterotropic allosteric properties characteristic of wild‐type ATCase. Moreover, sedimentation velocity experiments demonstrate that the holoenzyme reconstituted from CPC trimers is in the R conformation. These results indicate that the intact flexible loop containing Tyr 240 is essential for stabilizing the T conformation of ATCase. Following denaturation of the CPC trimer in 4.7 M urea and dilution of the solution, the separate proteolytic fragments re‐associate to form active trimers in about 60% yield. How this refolding of the fragments, docking, and association to form trimers are achieved is not known.
Purification and characterization of Klebsiella aerogenes UreE protein: A nickel‐binding protein that functions in urease metallocenter assembly
Authors: Lee Mann Hyung , Pankratz H. Stuart , Wang Shengke , Scott Robert A. , Finnegan Michael G. , Johnson Michael K. , Ippolito Joseph A. , Christianson David W. , Hausinger Robert P. 
Published: February 2009

AbstractThe Klebsiella aerogenes ureE gene product was previously shown to facilitate assembly of the urease metallocenter (Lee, M.H., et al., 1992, J. Bacteriol. 174, 4324‐4330). UreE protein has now been purified and characterized. Although it behaves as a soluble protein, UreE is predicted to possess an amphipathic β‐strand and exhibits unusually tight binding to phenyl‐Sepharose resin. Immunogold electron microscopic studies confirm that UreE is a cytoplasmic protein. Each dimeric UreE molecule (Mr = 35, 000) binds 6.05 + 0.25 nickel ions (Kd of 9.6 + 1.3 μM) with high specificity according to equilibrium dialysis measurements. The nickel site in UreE was probed by X‐ray absorption and variable‐temperature magnetic circular dichroism spectroscopies. The data are most consistent with the presence of Ni(II) in pseudo‐octahedral geometry with 3‐5 histidyl imidazole ligands. The remaining ligands are nitrogen or oxygen donors. UreE apoprotein has been crystallized and analyzed by X‐ray diffraction methods. Addition of nickel ion to apoprotein crystals leads to the development of fractures, consistent with a conformational change upon binding nickel ion. We hypothesize that UreE binds intracellular nickel ion and functions as a nickel donor during metallocenter assembly into the urease apoprotein.
Structure, function, and regulation of adrenergic receptors
Authors: Strosberg A. D. 
Published: February 2009

AbstractAdrenergic receptors for adrenaline and noradrenaline belong to the large multigenic family of receptors coupled to GTP‐binding proteins. Three pharmacologic types have been identified: α1‐, α2‐, and β‐adrenergic receptors. Each of these has three subtypes, characterized by both structural and functional differences. The α2and β receptors are coupled negatively and positively, respectively, to adenylyl cyclase via Gior Gsregulatory proteins, and the α1receptors modulate phospholipase C via the Goprotein. Subtype expression is regulated at the level of the gene, the mRNA, and the protein through various transcriptional and postsynthetic mechanisms. Adrenergic receptors constitute, after rhodopsin, one of the best studied models for the other receptors coupled to G proteins that are likely to display similar structural and functional properties.
Cloning, characterization, and expression of the β subunit of pig heart succinyl‐CoA synthetase
Authors: Bailey Darrin L. , Wolodko William T. , Bridger William A. 
Published: February 2009

AbstractThe form of succinyl‐CoA synthetase found in mammalian mitochondria is known to be an αβ dimer. Both GTP‐and ATP‐specific isozymes are present in various tissues. We have isolated essentially identical complementary DNA clones encoding the β subunit of pig heart succinyl‐CoA synthetase from both newborn and adult tissues. These cDNAs include a 1.4‐kb sequence encoding the cytoplasmic precursor to the β subunit comprised of 417 amino acid residues including a 22‐residue mitochondrial targeting sequence. The cDNA encoding the 395‐amino acid, 42,502‐Da mature protein was confirmed to be the succinyl‐CoA synthetase β subunit by agreement with the N‐terminal protein sequence and by high homology to prokaryotic forms of the β subunit that were previously cloned (about 45% identical to β from Escherichia coli). In contrast to a previous report (Nishimura, J.S., Ybarra, J., Mitchell, T., & Horowitz, P.M., 1988, Biochem. J. 250, 429–434), we found no tryptophan residue to be encoded in the sequence for the mature β subunit, and this finding is corroborated by the fact that highly purified pig heart succinyl‐CoA synthetase shows no tryptophan fluorescence or tryptophan content in amino acid compositional analysis. The cDNA clones encoding the mature pig heart β subunit and its counterpart α subunit were coexpressed in a deletion mutant strain of E. coli. Recovery of succinyl‐CoA synthetase activity demonstrated that this combination of subunits forms a productive enzymatic complex having GTP specificity.
Conformational intermediates in the folding of a coiled‐coil model peptide of the N‐terminus of tropomyosin and αα‐tropomyosin
Authors: Greenfield Norma J. , Hitchcock‐Degregori Sarah E. 
Published: February 2009

AbstractCircular dichroism was used to study the folding of αα‐tropomyosin and AcTM43, a 43‐residue peptide designed to serve as a model for the N‐terminal domain of tropomyosin. The sequence of the peptide is AcMDAIKKKMQMLKLDVENLLDRLEQLEADLKALEDRYKQLEGGC. The peptide appeared to form a coiled coil at low temperatures (<25 °C) in buffers with physiological ionic strength and pH. The folding and unfolding of the peptide, however, were noncooperative. When CD spectra were examined as a function of temperature, the apparent degree of folding differed when the ellipticity was followed at 222, 208, and 280 nm. Deconvolution of the spectra suggested that at least three component curves contributed to the CD in the far UV. One component curve was similar to the CD spectrum of the coiled‐coil α‐helix of native αα‐tropomyosin. The second curve resembled the spectrum of single‐stranded short α‐helical segments found in globular proteins. The third was similar to that of polypeptides in the random coil conformation. These results suggested that as the peptide folded, the α‐helical content increased before most of the coiled coil was formed.When the CD spectrum of striated muscle αα‐tropomyosin was examined as a function of temperature, the unfolding was also not totally cooperative. As the temperature was raised from 0 to 25 °C, there was a decrease in the coiled coil and an increase in the conventional α‐helix type spectrum without formation of random coil. The major transition, occurring at 40 °C, was a cooperative transition characterized by the loss of all of the remaining coiled coil and a concomitant increase in random coil.
Refinement of the structure of human basic fibroblast growth factor at 1.6 Å resolution and analysis of presumed heparin binding sites by selenate substitution
Authors: Eriksson A. Elisabeth , Matthews Brian W. , Cousens Lawrence S. 
Published: February 2009

AbstractThe three‐dimensional structure of human basic fibroblast growth factor has been refined to a crystallographic residual of 16.1% at 1.6 Å resolution. The structure has a Kunitz‐type fold and is composed of 12 antiparallel β‐strands, 6 of which form a β‐barrel. One bound sulfate ion has been identified in the model, hydrogen bonded to the side chains of Asn 27, Arg 120, and Lys 125. The side chain of Arg 120 has two conformations, both of which permit hydrogen bonds to the sulfate. This sulfate binding site has been suggested as the binding site for heparin (Eriksson, A.E., Cousens, L.S., Weaver, L.H., & Matthews, B.W., 1991, Proc. Natl. Acad. Sci. USA 88, 3441–3445). Two β‐mercaptoethanol (BME) molecules are also included in the model, each forming a disulfide bond to the Sγ atoms of Cys 69 and Cys 92, respectively. The side chain of Cys 92 has two conformations of which only one can bind BME. Therefore the BME molecule is half occupied at this site.The locations of possible sulfate binding sites on the protein were examined by replacing the ammonium sulfate in the crystallization medium with ammonium selenate. Diffraction data were measured to 2.2 Å resolution and the structure refined to an R‐factor of 13.8%. The binding of the more electron‐dense selenate ion was identified at two positions. One position was identical to the sulfate binding site identified previously. The second selenate binding site, which is of lower occupancy, is situated 5.6 Å from the first. This ion is hydrogen bonded by the side chain of Lys 135 and Arg 120. Thus the side chain of Arg 120 binds two selenate ions simultaneously. It is suggested that the observed second selenate binding site should also be considered as a possible binding site for heparin, or that both selenate binding sites might simultaneously contribute to the binding of heparin.
Backbone assignments and secondary structure of the Escherichia coli enzyme‐II mannitol a domain determined by heteronuclear three‐dimensional NMR spectroscopy
Authors: Kroon Gerard J. A. , Dijkstra Klaas , Scheek Ruud M. , Robillard George T. , Grötzinger Joachim 
Published: February 2009

AbstractThis report presents the backbone assignments and the secondary structure determination of the A domain of the Escherichia coli mannitol transport protein, enzyme‐IImtl.The backbone resonances were partially assigned using three‐dimensional heteronuclear 1H NOE 1H‐15N single‐quantum coherence (15N NOESY‐HSQC) spectroscopy and three‐dimensional heteronuclear 1H total correlation 1H‐15N single‐quantum coherence (15N TOCSY‐HSQC) spectroscopy on uniformly 15N enriched protein. Triple‐resonance experiments on uniformly 15N/13C enriched protein were necessary to complete the backbone assignments, due to overlapping 1H and 15N frequencies. Data obtained from three‐dimensional 1H‐15N‐13Cα correlation experiments (HNCA and HN(CO)CA), a three‐dimensional 1H‐15N‐13CO correlation experiment (HNCO), and a three‐dimensional 1Hα‐13Cα‐13CO correlation experiment (COCAH) were combined using SNARF software, and yielded the assignments of virtually all observed backbone resonances.Determination of the secondary structure of IIAmtl is based upon NOE information from the 15N NOESY‐HSQC and the 1Hα and 13Cα secondary chemical shifts. The resulting secondary structure is considerably different from that reported for IIAglc of E. coli and Bacillus subtilis determined by NMR and X‐ray.
A study of the effects of altering the sites for N‐glycosylation in α‐1‐proteinase inhibitor variants M and S
Authors: Samandari Taraz , Brown Jerry L. 
Published: February 2009

Abstractα‐1‐Proteinase inhibitor (A1Pi) is a monomeric secreted protein glycosylated at asparagines 46, 83, and 247. For this study cDNAs for M (normal) and S (Glu264 → Val) variants of A1Pi were altered by site‐directed mutagenesis to produce the combinations of single, double, and triple mutants that can be generated by changing the codons normally specifying these Asn residues to encode Gln. The fates of the mutant proteins were followed in transiently transfected COS‐1 cells. All variants with altered glycosylation sites are secreted at reduced rates, are partially degraded, accumulate intracellularly, and some form Nonidet P‐40‐insoluble aggregates. The carbohydrate attached at Asn83 seems to be of particular importance to the export of both A1PiM and A1PiS from the endoplasmic reticulum. All mutations affecting glycosylation of A1PiS notably reduce secretion, cause formation of insoluble aggregates, and influence degradation of the altered proteins. The variant of A1PiS missing all three glycosylation sites is poorly secreted, is incompletely degraded, and accumulates in unusual perinuclear vesicles. These studies show that N‐linked oligosaccharides in A1Pi are vital to its efficient export from the endoplasmic reticulum and that the consequences of changing the normal pattern of glycosylation vary depending upon the sites altered and the variant of A1Pi bearing these alterations.
Recombinant human erythropoietin (rHuEPO): Cross‐linking with disuccinimidyl esters and identification of the interfacing domains in EPO
Authors: Haniu Mitsuru , Narhi Linda O. , Arakawa Tsutomu , Elliott Steve , Rohde Michael F. 
Published: February 2009

AbstractSeveral amino groups of recombinant human erythropoietin are selectively cross‐linked by specific cross‐linkers including disuccinimidyl suberate or dithiobis(succinimidyl propionate). Intramolecular cross‐linkings are obtained without significant change of the protein conformation using appropriate concentrations (0.2 mM) of the cross‐linkers, which possess an 11–12‐Å length of a spacer between two reacting groups. Intramolecularly cross‐linked peptides obtained suggest that several amino groups in erythropoietin (EPO) are positioned at a distance of near 12 Å in the solution state. These interfacing amino groups include Lys 20–Lys 154, Lys 45–Lys 140, Lys 52–Lys 154, Lys 52–Lys 140, and Ala 1–Lys 116. A comparison of the cross‐linking results between nonglycosylated EPO and glycosylated EPO suggests that both proteins retain high similarity regarding protein conformation. These results fit a structural model similar to that of human growth hormone, in which four α‐helical bundles and a long stretch of β‐sheet structure are involved in the active protein.
Verification of protein structures: Patterns of nonbonded atomic interactions
Authors: Colovos Chris , Yeates Todd O. 
Published: February 2009

AbstractA novel method for differentiating between correctly and incorrectly determined regions of protein structures based on characteristic atomic interactions is described. Different types of atoms are distributed nonrandomly with respect to each other in proteins. Errors in model building lead to more randomized distributions of the different atom types, which can be distinguished from correct distributions by statistical methods.Atoms are classified in one of three categories: carbon (C), nitrogen (N), and oxygen (O). This leads to six different combinations of pairwise noncovalently bonded interactions (CC, CN, CO, NN, NO, and OO). A quadratic error function is used to characterize the set of pairwise interactions from nine‐residue sliding windows in a database of 96 reliable protein structures. Regions of candidate protein structures that are mistraced or misregistered can then be identified by analysis of the pattern of nonbonded interactions from each window.
Rapid protein separation and diffusion coefficient measurement by frit inlet flow field‐flow fractionation
Authors: Liu Min‐Kuang , Li Ping , Giddings J. Calvin 
Published: February 2009

AbstractIn this study three flow field‐flow fractionation (flow FFF) channels are utilized for the separation of proteins and for the simultaneous measurement of their translational diffusion coefficients, D. One channel has a traditional sample inlet, whereas the other two incorporate a frit inlet design that permits more convenient and rapid sample introduction. The dependence of retention time on D, which leads to differential elution and the opportunity to measure D for protein peaks purified by the flow FFF process, is described theoretically and examined experimentally. Factors affecting band broadening, resolution, and optimization are also examined. The separation of proteins is achieved in the time range 4–20 min. Partial resolution is achieved in multiple runs requiring 2 min each. Values of D calculated from retention times are reported for 15 proteins. These include two protein dimers (bovine serum albumin and γ‐globulin) not ordinarily accessible to measurement. The D values from the three channels are compared with one another and with literature data. Reasonable consistency (within 3–4%) is found. High‐speed repetitive runs can be used to acquire multiple values of D in time intervals as short as 1 min.
Disulfide bonds and the stability of globular proteins
Authors: Betz Stephen F. 
Published: February 2009

AbstractAn understanding of the forces that contribute to stability is pivotal in solving the protein‐folding problem. Classical theory suggests that disulfide bonds stabilize proteins by reducing the entropy of the denatured state. More recent theories have attempted to expand this idea, suggesting that in addition to configurational entropic effects, enthalpic and native‐state effects occur and cannot be neglected. Experimental thermodynamic evidence is examined from two sources: (1) the disruption of naturally occurring disulfides, and (2) the insertion of novel disulfides. The data confirm that enthalpic and native‐state effects are often significant. The experimental changes in free energy are compared to those predicted by different theories. The differences between theory and experiment are large near 300 K and do not lend support to any of the current theories regarding the stabilization of proteins by disulfide bonds. This observation is a result of not only deficiencies in the theoretical models but also from difficulties in determining the effects of disulfide bonds on protein stability against the backdrop of numerous subtle stabilizing factors (in both the native and denatured states), which they may also affect.