bluuebunny/crossref_metadata_2025_split huggingface dataset:
Crystallization and preliminary X‐ray analysis of the monomeric Cu, Zn superoxide dismutase from Escherichia coli
Authors: Battistoni Andrea , Folcarelli Silvia , Rotilio Giuseppe , Desideri Alessandro , Capasso Clemente , Pesce Alessandra , Bolognesi Martino 
Published: February 2009

AbstractThe Cu, Zn superoxide dismutase (Cu, Zn SOD) originally isolated from the periplasmic space of Escherichia coli has been cloned and overexpressed in the E. coli strain BMH 71/18. The protein has been purified as a single component of 17,000 Da, corresponding to one subunit of the common dimeric eukaryotic Cu, Zn SODs. Large crystals of the purified protein have been grown in the presence of polyethylene glycol 4,000 at pH 8.5; the crystals belong to the monoclinic space group P21, with unit cell constants α = 33.1 Å, b = 52.6 Å, c = 43.3 Å, β = 111.4 degrees. One SOD subunit is contained in the asymmetric unit, yielding a Vm value of 2.1 Å3/Da; the crystals diffract X‐rays beyond 2.0 Å resolution.
Crystallization and preliminary X‐ray analysis of aldehyde dehydrogenase from Vibrio harveyi
Authors: Croteau Nathalie , Vedadi Masoud , Delarge Marc , Meighen Edward , Vrielink Alice , Abu‐Abed Mona , Howell P. Lynne 
Published: February 2009

AbstractAldehyde dehydrogenase from Vibrio harveyi catalyzes the oxidation of long‐chain aliphatic aldehydes to acids. The enzyme is unique among the family of aldehyde dehydrogenases in that it exhibits much higher specificity for the cofactor NADP+ than for NAD+. The sequence of this form of the enzyme varies significantly from the NAD+ dependent forms, suggesting differences in the three‐dimensional structure that may be correlated to cofactor specificity. Crystals of the enzyme have been grown both in the presence and absence of NADP+ using the hanging drop vapor diffusion technique. In order to improve crystal size and quality, iterative seeding techniques were employed. The crystals belong to space group P21, with unit cell dimensions a = 79.4 Å, b = 131.1 Å, c = 92.2 Å, and β = 92.4°. Freezing the crystal to 100K has enabled a complete set of data to be collected using a rotating anode source (λ. = 1.5418 Å). The crystals diffract to a minimum d‐spacing of 2.6 Å resolution. Based on density calculations, two homodimers of molecular weight 110 kDa are estimated to be present in the asymmetric unit. Self‐rotation functions show the presence of 3 noncrystallographic twofold symmetry axes.
High‐resolution X‐ray structure of UDP‐galactose 4‐epimerase complexed with UDP‐phenol
Authors: Thoden James B. , Frey Perry A. , Holden Hazel M. 
Published: February 2009

AbstractUDP‐galactose 4‐epimerase from Escherichia coli catalyzes the interconversion of UDP‐glucose and UDP‐galactose. In recent years, the enzyme has been the subject of intensive investigation due in part to its ability to facilitate nonstereospecific hydride transfer between β‐NADH and a 4‐keto hexopyranose intermediate. The first molecular model of the epimerase from E. coli was solved to 2.5 Å resolution with crystals grown in the presence of a substrate analogue. UDP‐phenol (Bauer AJ, Rayment I, Frey PA, Holden HM, 1992, Proteins Struct Funct Genet 12:372–381). There were concerns at the time that the inhibitor did not adequately mimic the sugar moiety of a true substrate. Here we describe the high‐resolution X‐ray crystal structure of the ternary complex of UDP‐galactose 4‐epimerase with NADH and UDP‐phenol. The model was refined to 1.8 Å resolution with a final overall R‐factor of 18.6%. This high‐resolution structural analysis demonstrates that the original concerns were unfounded and that, in fact, UDP‐phenol and UDP‐glucose bind similarly. The carboxamide groups of the dinucleotides, in both subunits, are displaced significantly from the planes of the nicotinamide rings by hydrogen bonding interactions with Ser 124 and Tyr 149. UDP‐galactose 4‐epimerase belongs to a family of enzymes known as the short‐chain dehydrogenases, which contain a characteristic Tyr‐Lys couple thought to be important for catalysis. The epimerase/NADH/UDP‐phenol model presented here represents a well‐defined ternary complex for this family of proteins and, as such, provides important information regarding the possible role of the Tyr‐Lys couple in the reaction mechanism.
A missing link in cupredoxins: Crystal structure of cucumber stellacyanin at 1.6 Å resolution
Authors: Hart P. John , Eisenberg David , Nersissian Aram M. , Valentine Joan Selverstone , Herrmann Reinhold G. , Nalbandyan Robert M. 
Published: February 2009

AbstractStellacyanins are blue (type I) copper glycoproteins that differ from other members of the cupredoxin family in their spectroscopic and electron transfer properties. Until now, stellacyanins have eluded structure determination. Here we report the three‐dimensional crystal structure of the 109 amino acid, non‐glycosylated copper binding domain of recombinant cucumber stellacyanin refined to 1.6 Å resolution. The crystallographic R‐value for all 18,488 reflections (σ > 0) between 50–1.6 Å is 0.195. The overall fold is organized in two β‐sheets, both with four β‐strands. Two α‐helices are found in loop regions between β‐strands. The β‐sheets form a β‐sandwich similar to those found in other cupredoxins, but some features differ from proteins such as plastocyanin and azurin in that the β‐barrel is more flattened, there is an extra N‐terminal α‐helix, and the copper binding site is much more solvent accessible. The presence of a disulfide bond at the copper binding end of the protein confirms that cucumber stellacyanin has a phytocyanin‐like fold. The ligands to copper are two histidines, one cysteine, and one glutamine, the latter replacing the methionine typically found in mononuclear blue copper proteins. The Cu‐Gln bond is one of the shortest axial ligand bond distances observed to date in structurally characterized type I copper proteins. The characteristic spectroscopic properties and electron transfer reactivity of stellacyanin, which differ significantly from those of other well‐characterized cupredoxins, can be explained by its more exposed copper site, its distinctive amino acid ligand composition, and its nearly tetrahedral ligand geometry. Surface features on the cucumber stellacyanin molecule that could be involved in interactions with putative redox partners are discussed.
Cloning, expression, and spectroscopic characterization of Cucumis sativus stellacyanin in its nonglycosylated form
Authors: Nersissian Aram M. , Valentine Joan Selverstone , Mehrabian Zara B. , Nalbandyan Robert M. , Hart P. John , Fraczkiewicz Grazyna , Czernuszewicz Roman S. , Bender Christopher J. , Peisach Jack , Herrmann Reinhold G. 
Published: February 2009

AbstractThe cDNA encoding the 182 amino acid long precursor stellacyanin from Cucumis sativus was isolated and characterized. The protein precursor consists of four sequence domains: I, a 23 amino acid hydrophobic N‐terminal signal peptide with features characteristic of secretory proteins; II, a 109 amino acid copper‐binding domain; III, a 26 amino acid hydroxyproline‐ and serine‐rich peptide characteristic of motifs found in the extensin family, extracellular structural glycoproteins found in plant cell walls; and IV, a 22 amino acid hydrophobic extension. Maturation of the protein involves posttranslational processing of domains I and IV. The copper‐binding domain (domain II), which shares high sequence identity with other stellacyanins, has been expressed without its carbohydrate attachment sites, refolded from the Escherichia coli inclusion bodies, purified, and characterized by electronic absorption, EPR, ESEEM, and RR spectroscopy. Its spectroscopic properties are nearly identical to those of stellacyanin from the Japanese lacquer tree Rhus vernicifera, the most extensively studied and best characterized stellacyanin, indicating that this domain folds correctly, even in the absence of its carbohydrate moiety. The presence of a hydroxyproline‐ and serine‐rich domain III suggests that stellacyanin may have a function other than that of a diffusible electron transfer protein, conceivably participating in redox reactions localized at the plant cell wall, which are known to occur in response to wounding or infection of the plant.
Probing the structure and mobility of Pseudomonas aeruginosa azurin by circular dichroism and dynamic fluorescence anisotropy
Authors: Mei G. , Agró A. Finazzi , Rosato N. , Gilardi G. , Venanzi M. , Canters G.W. 
Published: February 2009

AbstractThe UV dynamic fluorescence and CD of several Pseudomonas aeruginosa azurins bearing single amino acid mutation have been studied. Two classes of mutants were examined. In the first class, two hydrophobic residues in the core of the protein, Ile 7 and Phe 110, nearest to the azurin single tryptophan Trp 48, were substituted by a serine (mutants I7S and F110S). In the second class, two residues in the outer sphere of the copper ligand field were changed, obtaining the following mutants: M44K, H35F, H35L, and H35Q. All these proteins showed two fluorescence lifetimes in the copper‐containing form, but only one in the copper‐free form. The lifetime of the latter derivatives was different from either those of the metal‐bound samples, definitely ruling out the presence of apo‐like species in the holo protein. Copper‐free I7S and F110S showed a more complex fluorescence decay profile requiring a distribution of lifetimes rather than a single lifetime. Holo F110S was also better fitted, in the limit of confidence, with two distributions rather than a pair of lifetimes. Time‐resolved anisotropy of these two mutants as well as of wild‐type (wt) protein showed two components (rotational times for wt ≤ 200 ps and 7 ns, respectively). These components were not affected significantly by copper removal in the case of wt protein. Instead, the short rotational component of the mutants dropped dramatically to values near zero, indicating a much greater mobility of the tryptophanyl residue in the mutant apo azurins. These data were supported by CD measurements showing a small effect of the copper presence in the region below 250 nm, i.e., in the secondary structure, but almost a collapse of the aromatic asymmetry at 270–295 nm related to a relaxation of the structural constraint around the tryptophan. Altogether these data show that copper does not play a structural role in wt azurin, whereas it is crucial in the stabilization of I7S and F110S mutants. Furthermore, although the metal site geometry is rigidly kept in wt apo‐azurin, it regains the native form only in the presence of the metal in the “core” mutants. This finding is important for the theory of entatic states in metalloproteins (Williams RJP, 1995, Eur J Biochem 234:363–381).
The allosteric activator ATP induces a substrate‐dependent alteration of the quaternary structure of a mutant aspartate transcarbamoylase impaired in active site closure
Authors: Baker Darren P. , Kantrowitz Evan R. , Vachette Patrice , Fetler Luc 
Published: February 2009

AbstractAspartate transcarbamoylase from Escherichia coli shows homotropic cooperativity for aspartate as well as hetero‐tropic regulation by nucleotides. Structurally, it consists of two trimeric catalytic subunits and three dimeric regulatory subunits, each chain being comprised of two domains. Glu‐50 and Ser‐171 are involved in stabilizing the closed conformation of the catalytic chain. Replacement of Glu‐50 or Ser‐171 by Ala in the holoenzyme has been shown previously to result in marked decreases in the maximal observed specific activity, homotropic cooperativity, and affinity for aspartate (Dembowski NJ, Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:3716–3723; Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:1444–1451). We have constructed a double mutant enzyme combining both mutations. The resulting Glu‐50/Ser‐171 &RR; Ala enzyme is 9‐fold less active than the Ser‐171 &RR; Ala enzyme, 69‐fold less active than the Glu‐50 &RR; Ala enzyme, and shows 1.3‐fold and 1.6‐fold increases in the [S]0.5Asp as compared to the Ser‐171 &RR; Ala and Glu‐50 &RR; Ala enzymes, respectively. However, the double mutant enzyme exhibits some enhancement of homotropic cooperativity with respect to aspartate, relative to the single mutant enzymes. At subsaturating concentrations of aspartate, the Glu‐50/Ser‐171 &RR; Ala enzyme is activated less by ATP than either the Glu‐50 &RR; Ala or Ser‐171 &RR; Ala enzyme, whereas CTP inhibition is intermediate between that of the two single mutants. As opposed to the wild‐type enzyme, the Glu‐50/Ser‐171 &RR; Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate. Structural analysis of the Ser‐171 &RR; Ala and Glu‐50/Ser‐171 &RR; Ala enzymes by solution X‐ray scattering indicates that both mutants exist in the same T quaternary structure as the wild‐type enzyme in the absence of ligands, and in the same R quaternary structure in the presence of saturating N‐(phosphonoacetyl)‐L‐aspartate. However, saturating concentrations of carbamoyl phosphate and succinate are unable to convert a significant fraction of either mutant enzyme population to the R quaternary structure, as has been observed previously for the Glu‐50 &RR; Ala enzyme. The curves for both the Ser‐171 &RR; Ala and Glu‐50/Ser‐171 &RR; Ala enzymes obtained in the presence of substoichiometric amounts of PALA are linear combinations of the two extreme T and R states. The structural consequences of nucleotide binding to these two enzymes were also investigated. Most surprisingly, the direction and amplitude of the effect of ATP upon the double mutant enzyme were shown to vary depending upon the substrate analogue used.
Evidence for an active T‐state pig kidney fructose 1,6‐bisphosphatase: Interface residue Lys‐42 is important for allosteric inhibition and AMP cooperativity
Authors: Lu Guqiang , Stec Boguslaw , Giroux Eugene L. , Kantrowitz Evan R. 
Published: February 2009

AbstractDuring the R &RR; T transition in the tetrameric pig kidney fructose‐1,6‐bisphosphatase (Fru‐1,6‐P2ase, EC 3.1.3.11) a major change in the quaternary structure of the enzyme occurs that is induced by the binding of the allosteric inhibitor AMP (Ke HM, Liang JY, Zhang Y, Lipscomb WN, 1991, Biochemistry 30:4412–4420). The change in quaternary structure involving the rotation of the upper dimer by 17° relative to the lower dimer is coupled to a series of structural changes on the secondary and tertiary levels. The structural data indicate that Lys‐42 is involved in a complex set of intersubunit interactions across the dimer‐dimer interface with residues of the 190′s loop, a loop located at the pivot of the allosteric rotation. In order to test the function of Lys‐42, we have replaced it with alanine using site‐specific mutagenesis. The kcat and Km values for Lys‐42 &RR; Ala Fru‐1,6‐P2ase were 11 s−1 and 3.3 μM, respectively, resulting in a mutant enzyme that was slightly less efficient catalytically than the normal pig kidney enzyme. Although the Lys‐42 &RR; Ala Fru‐1,6‐P2ase was similar kinetically in terms of Km and kcat the response to inhibition by AMP was significantly different than that of the normal pig kidney enzyme. Not only was AMP inhibition no longer cooperative, but also it occurred in two stages, corresponding to high‐ and low‐affinity binding sites. Saturation of the high‐affinity sites only reduced the activity by 30%, compared to 100% for the wild‐type enzyme. In order to determine in what structural state the enzyme was after saturation of the high‐affinity sites, the Lys‐42 &RR; Ala enzyme was crystallized in the presence of Mn2+, fructose‐6‐phosphate (Fru‐6‐P), and 100 μM AMP and the data collected to 2.3 Å resolution. The X‐ray structure showed the T state with AMP binding with full occupancy to the four regulatory sites and the inhibitor Fru‐6‐P bound at the active sites. The results reported here suggest that, in the normal pig kidney enzyme, the interactions between Lys‐42 and residues of the 190′s loop, are important for propagation of AMP cooperativity to the adjacent subunit across the dimer‐dimer interface as opposed to the monomer‐monomer interface, and suggest that AMP cooperativity is necessary for full allosteric inhibition by AMP.
Perturbations of the denatured state ensemble: Modeling their effects on protein stability and folding kinetics
Authors: Wrabl James O. , Shortle David 
Published: February 2009

AbstractBy considering the denatured state of a protein as an ensemble of conformations with varying numbers of sequence‐specific interactions, the effects on stability, folding kinetics, and aggregation of perturbing these interactions can be predicted from changes in the molecular partition function. From general considerations, the following conclusions are drawn: (1) A perturbation that enhances a native interaction in denatured state conformations always increases the stability of the native state. (2) A perturbation that promotes a non‐native interaction in the denatured state always decreases the stability of the native state. (3) A change in the denatured state ensemble can alter the kinetics of aggregation and folding. (4) The loss (or increase) in stability accompanying two mutations, each of which lowers (or raises) the free energy of the denatured state, will be less than the sum of the effects of the single mutations, except in cases where both mutations affect the same set of partially folded conformations. By modeling the denatured state as the ensemble of all non‐native conformations of hydrophobic‐polar (HP) chains configured on a square lattice, it can be shown that the stabilization obtained from enhancement of native interactions derives in large measure from the avoidance of non‐native interactions in the D state. In addition, the kinetic effects of fixing single native contacts in the denatured state or imposing linear gradients in the HH contact probabilities are found, for some sequences, to significantly enhance the efficiency of folding by a simple hydrophobic zippering algorithm. Again, the dominant mechanism appears to be avoidance of non‐native interactions. These results suggest stabilization of native interactions and imposition of gradients in the stability of local structure are two plausible mechanisms involving the denatured state that could play a role in the evolution of protein folding and stability.
Novel domains in NADPH oxidase subunits, sorting nexins, and PtdIns 3‐kinases: Binding partners of SH3 domains?
Authors: Ponting Christopher P. 
Published: February 2009

AbstractTwo SH3 domain‐containing cytosolic components of the NADPH oxidase, p47phox and p40phox, are shown by analyses of their sequences to contain single copies of a novel class of domain, the PX (phox) domain. Homologous domains are demonstrated to be present in the Cpk class of phosphatidylinositol 3‐kinase, S. cerevisiae Bem1p, and S. pombe Scd2, and a large family of human sorting nexin 1 (SNX1) homologues. The majority of these domains contains a polyproline motif, typical of SH3 domain‐binding proteins. Two further findings are reported. A third NADPH oxidase subunit, p67phox, is shown to contain four tetratricopeptide repeats (TPRs) within its N‐terminal RaclGTP‐binding region, and a 28 residue motif in p40phox is demonstrated to be present in protein kinase C isoforms τ/λ and ζ, and in three ZZ domain‐containing proteins.
Kinetic analysis of ligand binding to interleukin‐2 receptor complexes created on an optical biosensor surface
Authors: Myszka David G. , Arulanantham Peter R. , Sana Theodore , Wu Zining , Morton Thomas A. , Ciardelli Thomas L. 
Published: February 2009

AbstractThe interleukin‐2 receptor (IL‐2R) is composed of at least three cell surface subunits, IL‐2Rα, IL‐2Rβ, and IL‐2Rγc. On activated T‐cells, the α‐ and β‐subunits exist as a preformed heterodimer that simultaneously captures the IL‐2 ligand as the initial event in formation of the signaling complex. We used BIAcore to compare the binding of IL‐2 to biosensor surfaces containing either the α‐subunit, the β‐subunit, or both subunits together. The receptor ectodomains were immobilized in an oriented fashion on the dextran matrix through unique solvent‐exposed thiols. Equilibrium analysis of the binding data established IL‐2 dissociation constants for the individual α‐ and β‐subunits of 37 and 480 nM, respectively. Surfaces with both subunits immobilized, however, contained a receptor site of much higher affinity, suggesting the ligand was bound in a ternary complex with the α‐ and β‐subunits, similar to that reported for the pseudo‐high‐affinity receptor on cells. Because the binding responses had the additional complexity of being mass transport limited, obtaining accurate estimates for the kinetic rate constants required global fitting of the data sets from multiple surface densities of the receptors. A detailed kinetic analysis indicated that the higher‐affinity binding sites detected on surfaces containing both α‐ and β‐subunits resulted from capture of IL‐2 by a preformed complex of these subunits. Therefore, the biosensor analysis closely mimicked the recognition properties reported for these subunits on the cell surface, providing a convenient and powerful tool to assess the structure‐function relationships of this and other multiple subunit receptor systems.
Crystal structure of the coat protein from the GA bacteriophage: Model of the unassembled dimer
Authors: Ni Chao‐Zhou , White Carol A. , Mitchell Richard S. , Wickersham John , Kodandapani Ramadurgam , Peabody David S. , Ely Kathryn R. 
Published: February 2009

AbstractThere are four groups of RNA bacteriophages with distinct antigenic and physicochemical properties due to differences in surface residues of the viral coat proteins. Coat proteins also play a role as translational repressor during the viral life cycle, binding an RNA hairpin within the genome. In this study, the first crystal structure of the coat protein from a Group II phage GA is reported and compared to the Group I MS2 coat protein. The structure of the GA dimer was determined at 2.8 Å resolution (R‐factor = 0.20). The overall folding pattern of the coat protein is similar to the Group I MS2 coat protein in the intact virus (Golmohammadi R, Valegård K, Fridborg K, Liljas L, 1993, J Mol Biol 234:620–639) or as an unassembled dimer (Ni CZ, Syed R, Kodandapani R, Wickersham J, Peabody DS, Ely KR, 1995, Structure 3:255–263). The structures differ in the FG loops and in the first turn of the αA helix. GA and MS2 coat proteins differ in sequence at 49 of 129 amino acid residues. Sequence differences that contribute to distinct immunological and physical properties of the proteins are found at the surface of the intact virus in the AB and FG loops. There are six differences in potential RNA contact residues within the RNA‐binding site located in an antiparallel β‐sheet across the dimer interface. Three differences involve residues in the center of this concave site; Lys/Arg 83, Ser/Asn 87, and Asp/Glu 89. Residue 87 was shown by molecular genetics to define RNA‐binding specificity by GA or MS2 coat protein (Lim F, Spingola M, Peabody DS, 1994, J Biol Chem 269:9006–9010). This sequence difference reflects recognition of the nucleotide at position ‐5 in the unpaired loop of the translational operators bound by these coat proteins. In GA, the nucleotide at this position is a purine whereas in MS2, it is a pyrimidine.