bluuebunny/crossref_metadata_2025_split huggingface dataset:
Simulations of CRP:(cAMP)2 in noncrystalline environments show a subunit transition from the open to the closed conformation
Authors: García Angel E. , Harman James G. 
Published: July 2010

AbstractThe CRP:cAMP complex functions as a transcription factor that facilitates RNA polymerase recognition of several bacterial promoters. Detailed crystal structure information is available for CRP:(cAMP)2 and for CRP:(cAMP)2 complexed with DNA. In the crystalline environment, CRP:(cAMP)2 subunits are asymmetrically related; one subunit has a closed conformation and the other has an open conformation. The CRP:(cAMP)2 complexed with DNA shows both subunits in a closed conformation. We have studied the molecular dynamics of CRP:(cAMP)2 in noncrystalline environments. CRP:(cAMP)2 was simulated for 625 ps in vacuo and for 140 ps in solution. The crystal structure of CRP:(cAMP)2 in the absence of DNA was used as the initial conformation. Molecule optimal dynamic coordinates (MODCs) (Garcia A, 1992, Phys Rev Lett 68:2696) were used to analyze protein conformations sampled during the course of the simulations. Two MODCs define a transition of the open subunit to a closed subunit conformation during the first 125 ps of simulation in vacuo; the resulting subunit conformation is similar to that observed in CRP:(cAMP)2:DNA crystals. Simulation of CRP:(cAMP)2 in solution showed that a transition from the open to the closed state also occurs when water is explicitly included in the calculations. These calculations suggest that the asymmetric conformation of CRP:(cAMP)2 is stabilized by crystal lattice interactions. The predicted solution conformation is more symmetric, with both subunits in a closed conformation.
Human protoporphyrinogen oxidase: Expression, purification, and characterization of the cloned enzyme
Authors: Dailey Tamara A. , Dailey Harry A. 
Published: July 2010

AbstractProtoporphyrinogen oxidase (E.C.1.3.3.4) catalyzes the oxygen‐dependent oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from human placenta has been cloned, sequenced, expressed in Escherichia coli, purified to homogeneity, and characterized. Northern blot analysis of eight different human tissues show evidence for only a single transcript in all tissue types and the size of this transcript is approximately 1.8 kb. The human cDNA has been inserted into an expression vector for E. coli and the protein produced at high levels in these cells. The protein is found in both membrane and cytoplasmic fractions. The enzyme was purified to homogeneity in the presence of detergents using a metal chelate affinity column. The purified protein is a homodimer composed of subunits of a molecular weight of 51,000. The enzyme contains one noncovalently bound FAD per dimer, has a monomer extinction coefficient of 48,000 at 270 nm and contains no detectable redox active metals. The apparent Km and Kcat for protoporphyrinogen IX are 1.7 μM and 10.5 min−1, respectively. The enzyme does not use coproporphyrinogen III as a substrate and is inhibited by micromolar concentrations of the herbicide acifluorfen. Protein database searches reveal significant homology between protoporphyrinogen oxidase and monoamine oxidase.
Two mutations in recombinant Hb β F41(C7)Y, K82 (EF6)D show additive effects in decreasing oxygen affinityxs
Authors: Dumoulin A. , Kiger L. , Griffon N. , Vasseur C. , Kister J. , Génin P. , Marden M.C. , Pagnier J. , Poyart C. 
Published: July 2010

AbstractBased on the properties of two low oxygen affinity mutated hemoglobins (Hb), we have engineered a double mutant Hb (rHb βYD) in which the βF41Y substitution is associated with K82D. Functional studies have shown that the Hb α2/β2(C7)F41Y exhibits a decreased oxygen affinity relative to Hb A, without a significantly increased auto‐oxidation rate. The oxygen affinity of the natural mutant βK82D (Hb Providence‐Asp) is decreased due to the replacement of two positive charges by two negative ones at the main DPG‐binding site. The functional properties of both single mutants are interesting in the view of obtaining an Hb‐based blood substitute, which requires: (1) cooperative oxygen binding with an overall affinity near 30 mm Hg at half saturation, at 37 °C, and in the absence of 2,3 diphosphoglycerate (DPG), and (2) a slow rate of autooxidation in order to limit metHb formation. It was expected that the two mutations were at a sufficient distance (20 Å) that their respective effects could combine to form low oxygen affinity tetramers. The double mutant does display additive effects resulting in a fourfold decrease in oxygen affinity; it can insure, in the absence of DPG, an oxygen delivery to the tissues similar to that of a red cell suspension in vivo at 37 °C. Nevertheless, the rate of autooxidation, 3.5‐fold larger than that of Hb A, remains a problem.
Pressure‐induced perturbation of ANS‐apomyoglobin complex: Frequency domain fluorescence studies on native and acidic compact states
Authors: Bismuto Ettore , Irace Gaetano , Sirangelo Ivana , Gratton Enrico 
Published: July 2010

AbstractThe pressure dependence of the flexibility of the 8‐anilino‐1‐naphthalene sulfonate (ANS)‐apomyoglobin complex was investigated in the range between atmospheric pressure and 2.4 kbar by frequency domain fluorometry. We examined two structural states: native and acidic compact. The conformational dynamics of the ANS‐apomyoglobin complex were deduced by studying the emission decay of ANS, which can form a noncovalent complex with the apoprotein in both the native and the acidic compact forms. Because the free fluorophore has a very short lifetime (less than 75 ps), its contribution can be separated from the long‐lived emission. The latter arises from ANS molecules bound to the protein and provides information on the structural and dynamic characteristics of the macromolecule. The fluorescence emission decay of the ANS‐apomyoglobin complex at neutral pH has a broad fluorescence lifetime distribution (width at half‐maximum = 4.1 ns). The small changes in the fluorescence distribution parameters that occur with changes in pressure indicate that the ANS‐apomyoglobin complex at neutral pH holds its compactness even at 2.4 kbar. A small contraction of molecular volume has been detected at low pressure, followed by a slight swelling with an increase in flexibility at higher pressures. The heterogeneity of ANS fluorescence in the acidic compact state of apomyoglobin is even greater than that in the native form (distribution width = 10 ns); moreover, the acidic compact state appears more expanded and accessible to solvent molecules than the native state, as suggested by the distribution center, which is 11 ns for the former and 19 ns for the latter. The lifetime distribution center remains constant with increasing pressure, which suggests that no other binding site is formed at high pressure.
Dynamics of the three methionyl side chains of Streptomyces subtilisin inhibitor. Deuterium NMR studies in solution and in the solid state
Authors: Tamura Atsuo , Matsushita Michio , Naito Akira , Kojima Shuichi , Miura Kin‐Ichiro , Akasaka Kazuyuki 
Published: July 2010

AbstractStreptomyces subtilisin inhibitor (SSI) contains three methionine residues in a subunit: two (at positions 73 and 70) in the crucial enzyme‐recognition sites P1 and P4, respectively, and one (Met 103) in the hydrophobic core. The motions of the side chains of these three Met residues and the changes in mobility on binding with subtilisin were studied by deuterium NMR spectroscopy in solution and in crystalline and powder solids. For this purpose, the wild‐type SSI was deuterium‐labeled at the methyl groups of all three Met residues, and three artificial mutant proteins were labeled at only one specific Met methyl group each. In solution, for methionines 73 and 70, the effective correlation times were only 0.8–1.0 × 10−10 sindicating that the two side chains on the surface fluctuate almost freely. On formation of a complex with subtilisin, however, these high mobilities were quenched, giving a correlation time of 1.1 ± 10−8 s for the side chains of methionines 70 and 73. The correlation time of Met 103, located in the hydrophobic core, was at least 1.0 ± 10−8 s in free SSI, showing that its side chain motion is highly restricted. The nature of the internal motions of the three Met side chains was examined in more detail by deuterium NMR spectroscopy of powder and crystalline samples. The spectral patterns of the powder samples depended critically on hydration: immediately after lyophilization, the side‐chain motions of the three Met residues were nearly quenched. With gradual hydration to 0.20 gram of water per gram protein‐water, the orientational fluctuation of the methyl axes of methionines 70 and 73 was selectively enhanced in both amplitude and frequency (to about 1 MHz) and, at nearly saturating hydration (0.60 gram of water per gram protein‐water), became extremely high in amplitude and frequency (>10 MHz). In contrast, the polycrystalline wild‐type SSI spectrum showed fine structures, reflecting characteristic motions of the Met side chains. The polycrystalline spectrum could be reproduced reasonably well by the same motion models and parameters used to simulate the powder spectrum at the final level of hydration, suggesting that the side‐chain motions are similar in the fully hydrated powder and in crystals. Spin‐lattice relaxation measurements gave evidence that, even in crystals, the methyl axes of all three Met residues undergo rapid motions with correlation times between 10−8 and 10−10 s, comparable to the correlation times in solution. Finally, in the hydrated stoichiometric complex of SSI with subtilisin BPN' in the solid state, large‐amplitude motions are absent, but the side chains of methionines 70 and/or 73 are likely to have small‐amplitude motions.
Identification of the residues responsible for the alkaline inhibition of Cu, Zn superoxide dismutase: A site‐directed mutagenesis approach
Authors: Polticelli Fabio , Desideri Alessandro , Battistoni Andrea , Rotilio Giuseppe , O'Neill Peter 
Published: July 2010

AbstractThe catalytic rate of wild type, two single (Lys 120 → Leu, Lys 134 → Thr), and one double (Lys 120 → Leu‐Lys 134 → Thr) mutants of Xenopus laevis B Cu, Zn superoxide dismutase has been studied by pulse radiolysis as a function of pH. The pH dependence curve of the activity of the wild‐type enzyme can be deconvoluted by two deprotonation equilibria, at pH 9.3 (pK1) and at pH 11.3 (pK2). Catalytic rate measurements on single and double mutants indicate that pK1 is mainly due to the deprotonation of Lys 120 and Lys 134, with only a minor contribution from other surface basic residues, whereas pK2 is due to titration of the invariant Arg 141, likely coupled to deprotonation of the copper‐bound water molecule. Accordingly, Brownian dynamics simulations carried out as a function of pH reproduce well the pH dependence of the catalytic rate, when the experimentally determined pKs are assigned to Lys 120, Lys 134, and Arg 141.
Equilibrium dissociation and unfolding of the dimeric human papillomavirus strain‐16 E2 DNA‐binding domain
Authors: Mok Yu‐Keung , Bycroft Mark , Gay Gonzalo De Prat , Jonathan Butler P. 
Published: July 2010

AbstractThe equilibrium unfolding reaction of the C‐terminal 80‐amino‐acid dimeric DNA‐binding domain of human papillomavirus (HPV) strain 16 E2 protein has been investigated using fluorescence, far‐UV CD, and equilibrium sedimentation. The stability of the HPV‐16 E2 DNA‐binding domain is concentration‐dependent, and the unfolding reaction is well described as a two‐state transition from folded dimer to unfolded monomer. The conformational stability of the protein, ΔGH2O, was found to be 9.8 kcal/mol at pH 5.6, with the corresponding equilibrium unfolding/dissociation constant, Ku, being 6.5 → 10−8 M. Equilibrium sedimentation experiments give a Kd of 3.0 → 10−8 M, showing an excellent agreement between the two different techniques. Denaturation by temperature followed by the change in ellipticity also shows a concomitant disappearance of secondary and tertiary structures. The Ku changes dramatically at physiologically relevant pH's: with a change in pH from 6.1 to 7.0, it goes from 5.5 → 10−8 M to 4.4 → 1010 M. Our results suggest that, at the very low concentration of protein where DNA binding is normally measured (e.g., 10−11 M), the protein is predominantly monomeric and unfolded. They also stress the importance of the coupling between folding and DNA binding.
Posttranslational processing of recombinant human interferon‐γ in animal expression systems
Authors: Freedman Robert B. , Jenkins Nigel , James David C. , Goldman Merlin H. , Hoare Michael , Oliver Ronald W. A. , Green Brian N. 
Published: July 2010

AbstractWe have characterized the heterogeneity of recombinant human interferon‐γ (IFN‐γ) produced by three expression systems: Chinese hamster ovary cells, the mammary gland of transgenic mice, and baculovirus‐infected Spodoptera frugiperda (Sf9) insect cells. Analyses of whole IFN‐γ proteins by electrospray ionization‐mass spectrometry (ESI‐MS) from each recombinant source revealed heterogeneous populations of IFN‐γ molecules resulting from variations in N‐glycosylation and C‐terminal polypeptide cleavages. A series of more specific analyses assisted interpretation of maximum entropy deconvoluted ESI‐mass spectra of whole IFN‐γ proteins; MALDI‐MS analyses of released, desialylated N‐glycans and of deglycosylated IFN‐γ polypeptides were combined with analyses of 2‐aminobenzamide labeled sialylated N‐glycans by cation‐exchange high‐performance liquid chromatography. These analyses enabled identification of specific polypeptide cleavage sites and characterization of associated N‐glycans. Production of recombinant IFN‐γ in the mammalian expression systems yielded polypeptides C‐terminally truncated at dibasic amino acid sites. Mammalian cell derived IFN‐γ molecules displayed oligosaccharides with monosaccharide compositions equivalent to complex, sialylated, or high‐mannose type N‐glycans. In contrast, IFN‐γ derived from baculovirus‐infected Sf9 insect cells was truncated further toward the C‐terminus and was associated with neutral (nonsialylated) N‐glycans. These data demonstrate the profound influence of host cell type on posttranslational processing of recombinant proteins produced in eukaryotic systems.
BSTI, a trypsin inhibitor from skin secretions of bombina bombina related to protease inhibitors of nematodes
Authors: Mignogna Giuseppina , Pascarella Stefano , Amiconi Gino , Barra Donatella , Wechselberger Christian , Hinterleitner Christa , Mollay Christa , Kreil Günther 
Published: July 2010

AbstractFrom skin secretions of the European frog Bombina bombina, a new peptide has been isolated that contains 60 amino acids, including 10 cysteine residues. Its sequence was determined by automated Edman degradation and confirmed by analysis of the cDNA encoding the precursor. A search in the databanks demonstrated that the pattern of cysteine residues in this skin peptide is similar to the ones found in protease inhibitors from Ascaris and in a segment of human von Willebrand factor. The 3D structure of the trypsin inhibitor from Ascaris suum could be used as a template to build a model of the amphibian peptide. In addition, we have demonstrated that this constituent of skin secretion is indeed an inhibitor of trypsin and thrombin, with Ki values in the range of 0.1 to 1 μM. The new peptide was thus named BSTI for Bombina skin trypsin/thrombin inhibitor.
Interlobe communication in multiple calcium‐binding site mutants of Drosophila calmodulin
Authors: Mukherjea Poushali , Maune John F. , Beckingham Kathy 
Published: July 2010

AbstractWe have generated mutants of Drosophila calmodulin in which pairs of calcium‐binding sites are mutated so as to prevent calcium binding. In all sites, the mutation involves replacement of the —Z position glutamate residue with glutamine. Mutants inactivated in both N‐terminal sites (B12Q) or both C‐terminal sites (B34Q), and two mutants with one N‐ and one C‐terminal site inactivated (B13Q and B24Q) were generated. The quadruple mutant with all four sites mutated was also studied. UV‐difference spectroscopy and near‐UV CD were used to examine the influence of these mutations upon the single tyrosine (Tyr‐138) of the protein. These studies uncovered four situations in which Tyr‐138 in the C‐terminal lobe responds to a change in the calcium‐binding properties of the N‐terminal lobe. Further, they suggest that N‐terminal calcium‐binding events contribute strongly to the aberrant behavior of Tyr‐138 seen in mutants with a single functional C‐terminal calcium‐binding site. The data also indicate that loss of calcium binding at site 1 adjusts the aberrant conformation of Tyr‐138 produced by mutation of site 3 toward the wild‐type structure. However, activation studies for skeletal muscle myosin light chain kinase (SK‐MLCK) established that all of the multiple binding site mutants are poor activators of SK‐MLCK. Thus, globally, the calcium‐induced conformation of B13Q is not closer to wild type than that of either the site 1 or the site 3 mutant. The positioning of Tyr‐138 within the crystal structure of calmodulin suggests that effects of the N‐terminal lobe on this residue may be mediated via changes to the central linker region of the protein.
A stable intermediate in the thermal unfolding process of a chimeric 3‐isopropylmalate dehydrogenase between a thermophilic and a mesophilic enzymes
Authors: Hayashi‐Iwasaki Yoko , Numata Koichi , Yamagishi Akihiko , Yutani Katsuhide , Sakurai Masahiro , Tanaka Nobuo , Oshima Tairo 
Published: July 2010

AbstractThe thermal unfolding process of a chimeric 3‐isopropylmalate dehydrogenase made of parts from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, enzymes was studied by CD spectrophotometry and differential scanning calorimetry (DSC). The enzyme is a homodimer with a subunit containing two structural domains. The DSC melting profile of the chimeric enzyme in 20 mM NaHCO3, pH 10.4, showed two endothermic peaks, whereas that of the T. thermophilus wild‐type enzyme had one peak. The CD melting profiles of the chimeric enzyme under the same conditions as the DSC measurement, also indicated biphasic unfolding transition. Concentration dependence of the unfolding profile revealed that the first phase was protein concentration‐independent, whereas the second transition was protein concentration‐dependent. When cooled after the first transition, the intermediate was isolated, which showed only the second transition upon heating. These results indicated the existence of a stable dimeric intermediate followed by the further unfolding and dissociation in the thermal unfolding of the chimeric enzyme at pH 10–11. Because the portion derived from the mesophilic isopropylmalate dehydrogenase in the chimeric enzyme is located in the hinge region between two domains of the enzyme, it is probably responsible for weakening of the interdomain interaction and causing the decooperativity of two domains. The dimeric form of the intermediate suggested that the first unfolding transition corresponds to the unfolding of domain 1 containing the N‐ and C‐termini of the enzyme, and the second to that of domain 2 containing the subunit interface.
Crystallization and preliminary diffraction studies of NodL, a rhizobial O‐acetyl‐transferase involved in the host‐specific nodulation of legume roots
Authors: Dunn Steven M. , Moody Peter C. E. , Downie J. Allan , Shaw William V. 
Published: July 2010

AbstractThe NodL specified O‐acetyltransferase from the microbial symbiont Rhizobium leguminosarum has been over‐expressed in Escherichia coli and purified using affinity‐elution dye chromatography as the key step. The protein has been crystallized at 20 °C in 18% PEG 600, 0.1 M Tris/HCl buffer, pH 8.5, containing 1% dioxane, 0.25% octyl‐β‐glucoside, and 5 mM coenzyme A using the hanging drop vapor diffusion method. Ambient temperature X‐ray diffraction studies reveal the space group to be hexagonal (P6322) with lattice constants a = b = 77.08 å, c = 160.6 å, and α = β = 90°, γ = 120°. Crystals that are flash‐frozen to 120 K diffract beyond 2.7 å.
Solution structure and lipid binding of a nonspecific lipid transfer protein extracted from maize seeds
Authors: Gomar Jérôme , Petit Marie‐Christine , Sodano Patrick , Sy Denise , Marion Didier , Kader Jean‐Claude , Vovelle Françoise , Ptak Marius 
Published: July 2010

AbstractThe three‐dimensional solution structure of a nonspecific lipid transfer protein extracted from maize seeds determined by 1H NMR spectroscopy is described. This cationic protein consists of 93 amino acid residues. Its structure was determined from 1, 091 NOE‐derived distance restraints, including 929 interresidue connectivities and 197 dihedral restraints (ϕ, ψ, χ1) derived from NOEs and 3J coupling constants. The global fold involving four helical fragments connected by three loops and a C‐terminal tail without regular secondary structures is stabilized by four disulfide bridges. The most striking feature of this structure is the existence of an internal hydrophobic cavity running through the whole molecule. The global fold of this protein, very similar to that of a previously described lipid transfer protein extracted from wheat seeds (Gincel E et al., 1994, Eur J Biochem 226:413–422) constitutes a new architecture for α‐class proteins. 1H NMR and fluorescence studies show that this protein forms well‐defined complexes in aqueous solution with lysophosphatidylcholine. Dissociation constants, Kd, of 1.9 ± 0.6 × 10−6 M and >10−3 M were obtained with lyso‐C16 and ‐C12, respectively. A structure model for a lipid—protein complex is proposed in which the aliphatic chain of the phospholipid is inserted in the internal cavity and the polar head interacts with the charged side chains located at one end of this cavity. Our model for the lipid—protein complex is qualitatively very similar to the recently published crystal structure (Shin DH et al., 1995, Structure 3:189–199).