bluuebunny/crossref_metadata_2025_split huggingface dataset:
NMR analysis of regioselectivity in dephosphorylation of a triphosphotyrosyl dodecapeptide autophosphorylation site of the insulin receptor by a catalytic fragment of LAR phosphotyrosine phosphatase
Authors: Lee Jonathan P. , Cho Hyeongjin , Bannwarth Willi , Kitas Eric A. , Walsh Christopher T. 
Published: February 2009

AbstractAn autophosphorylation site in the activated insulin receptor tyrosine kinase domain has three tyrosines phosphorylated when fully activated. To begin to examine recognition of triphosphotyrosyl sites by protein tyrosine phosphatases in possible control of signal transduction a triphosphotyrosyl dodecapeptide TRDIpYETDpYpYRK corresponding to residues 1,142–1,153 of the insulin receptor was prepared and incubated with the 40‐kDa catalytic domain of the human PTPase LAR. To assess regioselectivity of recognition, the three diphosphotyrosyl regioisomers, and the three monophosphotyrosyl regioisomers were prepared and assayed. All seven peptides were PTPase substrates. To identify any preferences in dephosphorylation at pY5, pY9, or pY10, 1H‐NMR analyses were conducted during enzyme incubations and distinguishing fingerprint regions determined for each of the seven phosphotyrosyl peptides. LAR PTPase shows strong preference for dephosphorylation first at pY5 (at tri‐, di‐, and monophosphotyrosyl levels). Initially this regioselectivity gives the Y5(pY9)(pY10) diphospho regioisomer, followed by equal dephosphorylation at pY9 or pY10 to give the corresponding monophosphoryl species on the way to fully dephosphorylated product. The NMR methodology is applicable to other peptides with multiple sites of phosphorylation that undergo attack by any phosphatase.
Structure of the oxidized long‐chain flavodoxin from anabaena 7120 at 2 å resolution
Authors: Rao S. T. , Shaffie Fathieh , Yu Carlos , Satyshur Kenneth A. , Stockman Brian J. , Markley John L. , Sundaralingam M. 
Published: February 2009

AbstractThe structure of the long‐chain flavodoxin from the photosynthetic cyanobacterium Anabaena 7120 has been determined at 2 å resolution by the molecular replacement method using the atomic coordinates of the long‐chain flavodoxin from Anacystis nidulans. The structure of a third long‐chain flavodoxin from Chondrus crispus has recently been reported. Crystals of oxidized A. 7120 flavodoxin belong to the monoclinic space group P21 with a = 48.0, b = 32.0, c = 51.6 å, and β = 92°, and one molecule in the asymmetric unit. The 2 å intensity data were collected with oscillation films at the CHESS synchrotron source and processed to yield 9,795 independent intensities with Rmerg of 0.07. Of these, 8,493 reflections had I > 2σ and were used in the analysis. The model obtained by molecular replacement was initially refined by simulated annealing using the XPLOR program. Repeated refitting into omit maps and several rounds of conjugate gradient refinement led to an R‐value of 0.185 for a model containing atoms for protein residues 2–169, flavin mononucleotide (FMN), and 104 solvent molecules. The FMN shows many interactions with the protein with the isoalloxazine ring, ribityl sugar, and the 5′‐phosphate. The flavin ring has its pyrimidine end buried into the protein, and the functional dimethyl benzene edge is accessible to solvent. The FMN interactions in all three long‐chain structures are similar except for the O4′ of the ribityl chain, which interacts with the hydroxyl group of Thr 88 side chain in A. 7120, while with a water molecule in the other two. The phosphate group interacts with the atoms of the 9–15 loop as well as with NE1 of Trp 57. The N5 atom of flavin interacts with the amide NH of Ile 59 in A. 7120, whereas in A. nidulans it interacts with the amide NH of Val 59 in a similar manner. In C. crispus flavodoxin, N5 forms a hydrogen bond with the side chain hydroxyl group of the equivalent Thr 58. The hydrogen bond distances to the backbone NH groups in the first two flavodoxins are 3.6 å and 3.5 å, respectively, whereas in the third flavodoxin the distance is 3.1 å, close to the normal value. Even though the hydrogen bond distances are long in the first two cases, still they might have significant energy because their microenvironment in the protein is not accessible to solvent. In all three long‐chain flavodoxins, a water molecule bridges the ends of the inserted loop in the β5 strand and minimally perturbs its hydrogen bonding with β4. Many of the water molecules in these proteins interact with the flavin binding loops. The conserved β‐core of the three long‐chain and two short‐chain flavodoxins superpose with root mean square deviations ranging from 0.48 å to 0.97 å.
Arginine 54 in the active site of escherichia coli aspartate transcarbamoylase is critical for catalysis: A site‐specific mutagenesis, NMR, and X‐ray crystallographic study
Authors: Stebbins Jeffrey W. , Robertson Diane E. , Roberts Mary F. , Stevens Raymond C. , Lipscomb William N. , Kantrowitz Evan R. 
Published: February 2009

AbstractThe replacement of Arg‐54 by Ala in the active site of Escherichia coli aspartate transcarbamoylase causes a 17,000‐fold loss of activity but does not significantly influence the binding of substrates or substrate analogs (Stebbins, J.W., Xu, W., & Kantrowitz, E.R., 1989, Biochemistry 28, 2592–2600). In the X‐ray structure of the wild‐type enzyme, Arg‐54 interacts with both the anhydride oxygen and a phosphate oxygen of carbamoyl phosphate (CP) (Gouaux, J.E. & Lipscomb, W.N., 1988, Proc. Natl. Acad. Sci. USA 85, 4205–4208). The Arg‐54 → Ala enzyme was crystallized in the presence of the transition state analog N‐phosphonoacetyl‐l‐aspartate (PALA), data were collected to a resolution limit of 2.8 å, and the structure was solved by molecular replacement. The analysis of the refined structure (R factor = 0.18) indicates that the substitution did not cause any significant alterations to the active site, except that the side chain of the arginine was replaced by two water molecules. 31P‐NMR studies indicate that the binding of CP to the wild‐type catalytic subunit produces an upfield chemical shift that cannot reflect a significant change in the ionization state of the CP but rather indicates that there are perturbations in the electronic environment around the phosphate moiety when CP binds to the enzyme. The pH dependence of this upfield shift for bound CP indicates that the catalytic subunit undergoes a conformational change with a pKa ∼ 7.7 upon CP binding. Furthermore, the linewidth of the 31P signal of CP bound to the Arg‐54 → Ala enzyme is significantly narrower than that of CP bound to the wild‐type catalytic subunit at any pH, although the change in chemical shift for the CP bound to the mutant enzyme is unaltered. 31P‐NMR studies of PALA complexed to the wild‐type catalytic subunit indicate that the phosphonate group of the bound PALA exists as the dianion at pH 7.0 and 8.8, whereas in the Arg‐54 → Ala catalytic subunit the phosphonate group of the bound PALA exists as the monoanion at pH 7.0 and 8.8. Thus, the side chain of Arg‐54 is essential for the proper ionization of the phosphonate group of PALA and by analogy the phosphate group in the transition state. These data support the previously proposed proton transfer mechanism, in which a fully ionized phosphate group in the transition state accepts a proton during catalysis.
X‐ray structure of glu 53 human lysozyme
Authors: Harata Kazuaki , Muraki Michiro , Hayashi Yasuhiro , Jigami Yoshifumi 
Published: February 2009

AbstractThe three‐dimensional structure of a modified human lysozyme (HL), Glu 53 HL, in which Asp 53 was replaced by Glu, has been determined at 1.77 å resolution by X‐ray analysis. The backbone structure of Glu 53 HL is essentially the same as the structure of wild‐type HL. The root mean square difference for the superposition of equivalent Cα atoms is 0.141 å. Except for the Glu 53 residue, the structure of the active site region is largely conserved between Glu 53 HL and wild‐type HL. However, the hydrogen bond network differs because of the small shift or rotation of side chain groups. The carboxyl group of Glu 53 points to the carboxyl group of Glu 35 with a distance of 4.7 å between the nearest carboxyl oxygen atoms. A water molecule links these carboxyl groups by a hydrogen bond bridge. The active site structure explains well the fact that the binding ability for substrates does not significantly differ between Glu 53 HL and wild‐type HL. On the other hand, the positional and orientational change of the carboxyl group of the residue 53 caused by the mutation is considered to be responsible for the low catalytic activity (ca. 1%) of Glu 53 HL. The requirement of precise positioning for the carboxyl group suggests the possibility that the Glu 53 residue contributes more than a simple electrostatic stabilization of the intermediate in the catalysis reaction.
Internal water molecules and H‐bonding in biological macromolecules: A review of structural features with functional implications
Authors: Meyer Edgar 
Published: February 2009

AbstractConserved structural patterns of internal water molecules and/or H‐bond chains were observed and are here correlated in this review, which then describes two functional properties: equilibration of hydrostatic pressure and proton transport. Available evidence in support of these hypotheses is presented, together with suggested experiments to test them. High‐resolution crystal structures of a variety of proteins were studied with interactive computer graphics. Conserved H‐bonding linkages may be used as a paradigm for a rationalization of proton transport in membranes. The concept of the “proton wire,” which links buried active‐site amino acids with the surface of the protein raises the more general question of the functional role of the various molecular components
Crystal structure of the Y52F/Y73F double mutant of phospholipase A2: Increased hydrophobic interactions of the phenyl groups compensate for the disrupted hydrogen bonds of the tyrosines
Authors: Sekharudu Chandra , Ramakrishnan Boopathy , Huang Baohua , Jiang Ru‐Tai , Dupureur Cynthia M. , Tsai Ming‐Daw , Sundaralingam Muttaiya 
Published: February 2009

AbstractThe enzyme phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn‐2 ester bond of membrane phospholipids. The highly conserved Tyr residues 52 and 73 in the enzyme form hydrogen bonds to the carboxylate group of the catalytic Asp‐99. These hydrogen bonds were initially regarded as essential for the interfacial recognition and the stability of the overall catalytic network. The elimination of the hydrogen bonds involving the phenolic hydroxyl groups of the Tyr‐52 and ‐73 by changing them to Phe lowered the stability but did not significantly affect the catalytic activity of the enzyme. The X‐ray crystal structure of the double mutant Y52F/Y73F has been determined at 1.93 å resolution to study the effect of the mutation on the structure. The crystals are trigonal, space group P3121, with cell parameters α = b = 46.3 å and c = 102.95 å. Intensity data were collected on a Siemens area detector, 8,024 reflections were unique with an R(sym) of 4.5% out of a total of 27,203. The structure was refined using all the unique reflections by XPLOR to a final R‐factor of 18.6% for 955 protein atoms, 91 water molecules, and 1 calcium ion. The root mean square deviation for the α‐carbon atoms between the double mutant and wild type was 0.56 å. The crystal structure revealed that four hydrogen bonds were lost in the catalytic network; three involving the tyrosines and one involving Pro‐68. However, the hydrogen bonds of the catalytic triad, His‐48, Asp‐99, and the catalytic water, are retained. There is no additional solvent molecule at the active site to replace the missing hydroxyl groups; instead, the replacement of the phenolic OH groups by H atoms draws the Phe residues closer to the neighboring residues compared to wild type; Phe‐52 moves toward His‐48 and Asp‐99 of the catalytic diad, and Phe‐73 moves toward Met‐8, both by about 0.5 å. The closing of the voids left by the OH groups increases the hydrophobic interactions compensating for the lost hydrogen bonds. The conservation of the triad hydrogen bonds and the stabilization of the active site by the increased hydrophobic interactions could explain why the double mutant has activity similar to wild type. The results indicate that the aspartyl carboxylate group of the catalytic triad can function alone without additional support from the hydrogen bonds of the two Tyr residues.
Effects of calcium on recombinant bovine chromogranin A
Authors: Angeletti Ruth Hogue , Ali Ghazala , Shen Ning , Gee Patricia , Nieves Edward 
Published: February 2009

AbstractBovine chromogranin A, the acidic calcium‐binding protein characteristic of endocrine secretory vesicles, has been expressed inEscherichia coliusing the pET3a vector system under T7 polymerase control. The expressed protein is located in the bacterial cytosol and can be purified from bacterial proteins by a heat treatment step, followed by gel filtration, anion‐exchange, and reversed‐phase chromatography. The purified recombinant chromogranin A has an apparentMTof ca. 72,000 by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, in spite of its 432‐amino acid polypeptide chain, consistent with observations on natural chromogranin A. The primary structure has been confirmed by mass spectral analysis of tryptic peptides, by Edman degradation of the intact protein, and by immunoreactivity with sequence‐specific antibodies. Analysis by circular dichroism spectroscopy shows pH‐ and concentration‐dependent spectra. The spectra are Ca2+‐dependent from 5 to 40 μM.
Automated carboxy‐terminal sequence analysis of peptides and proteins using diphenyl phosphoroisothiocyanatidate
Authors: Bailey Jerome M. , Nikfarjam Firoozeh , Shenoy Narmada R. , Shively John E. 
Published: February 2009

AbstractProteins and peptides can be sequenced from the carboxy‐terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on the automation of the thiocyanate chemistry using acetic anhydride and trimethylsilylisothiocyanate (TMS‐ITC) to derivatize the C‐terminal amino acid to a thiohydantoin and sodium trimethylsilanolate for specific hydrolysis of the derivatized C‐terminal amino acid (Bailey, J.M., Shenoy, N.R., Ronk, M., & Shively, J.E., 1992, Protein Sci. 1, 68–80). A major limitation of this approach was the need to activate the C‐terminus with acetic anhydride. We now describe the use of a new reagent, diphenyl phosphoroisothiocyanatidate (DPP‐ITC) and pyridine, which combines the activation and derivatization steps to produce peptidylthiohydantoins. Previous work by Kenner et al. (Kenner, G.W., Khorana, H.G., & Stedman, R.J., 1953, Chem. Soc. J., 673–678) with this reagent demonstrated slow kinetics. Several days were required for complete reaction. We show here that the inclusion of pyridine was found to promote the formation of C‐terminal thiohydantoins by DPP‐ITC resulting in complete conversion of the C‐terminal amino acid to a thiohydantoin in less than 1 h. Reagents such as imidazole, triazine, and tetrazole were also found to promote the reaction with DPP‐ITC as effectively as pyridine. General base catalysts, such as triethylamine, do not promote the reaction, but are required to convert the C‐terminal carboxylic acid to a salt prior to the reaction with DPP‐ITC and pyridine. By introducing the DPP‐ITC reagent and pyridine in separate steps in an automated sequencer, we observed improved sequencing yields for amino acids normally found difficult to derivatize with acetic anhydride/TMS‐ITC. This was particularly true for aspartic acid, which now can be sequenced in yields comparable to most of the other amino acids. Automated programs are described for the C‐terminal sequencing of peptides covalently attached to carboxylic acid‐modified polyethylene and proteins (200 pmol to 5 nmol) noncovalently applied to Zitex (porous Teflon). The generality of our automated C‐terminal sequencing methodology was examined by sequencing model peptides containing all 20 of the common amino acids. All of the amino acids tested were found to sequence in good yield except for proline, which was found not to be capable of derivatization. In spite of this limitation, the methodology should be a valuable tool for the C‐terminal sequence analysis of peptides and proteins. This work represents the first time an automated, sequential, chemical method for C‐terminal sequence analysis has been applied to subnanomolar amounts of protein samples.
Escherichia coli K12 arabinose‐binding protein mutants with altered transport properties
Authors: Kehres D.G. , Hogg R.W. 
Published: February 2009

AbstractThe arabinose‐binding protein (ABP) of Escherichia coli binds l‐arabinose in the periplasm and delivers it to a cytoplasmic membrane complex consisting of the AraG and AraH proteins, for uptake into the cell. To study the interaction between the soluble and membrane components of this periplasmic transport system, regions of the ABP surface containing the opening of the arabinose‐binding cleft were subjected to site‐directed mutagenesis. Thirty‐eight ABP variants containing one to three amino acid substitutions were recovered. ABP variants were expressed with wild‐type AraG and AraH from a plasmid, in a strain lacking the chromosomal araFGH operon, and the whole cell uptake parameters, Ven (maximum initial velocity of arabinose entry) and Ken (concentration of arabinose yielding half‐maximal entry) were determined. Twenty‐four mutants had normal Ven values, 3 mutants had Ven and Ken values twice wild type, and 11 mutants had Ven and Ken values 20–50% of wild type. Binding proteins that had altered uptake properties were each expressed, processed, and localized to the periplasm at levels equivalent to wild type. The mutant binding proteins behaved the same as wild type during purification, and each had a Kd (dissociation constant for bound arabinose) comparable to that of wild‐type ABP. Mutations that resulted in altered uptake identified nine amino acids surrounding the arabinose‐binding cleft, all of which are charged in the wild‐type protein, and all of whose side chains project outward from the cleft. The evidence suggests that this surface of the binding protein and these nine charged loci play a major role in ABP interactions with the membrane complex.
A kinetic model for binding protein‐mediated arabinose transport
Authors: Kehres D.G. 
Published: February 2009

AbstractA kinetic model is presented based on the simplest plausible mechanism for bacterial binding protein‐dependent transport. The transport phenotypes of the 18 variant arabinose‐binding proteins analyzed by Kehres and Hogg (1992, Protein Sci. 1, 1652–1660) (wild type and 17 mutants) are interpreted to mean that in wild‐type arabinose uptake the forward transport rate (kfor) greatly exceeds the dissociation rate (kund) of a binding protein docked with the AraG:AraH membrane complex, and that kfor dominance is preserved in all of the binding protein surface mutants. The assumptions and predictions of the model are consistent with existing data from other periplasmic transport systems.
Stability and reconstitution of pyruvate oxidase from lactobacillus plantarum: Dissection of the stabilizing effects of coenzyme binding and subunit interaction
Authors: Risse Bernhard , Stempfer Günter , Rudolph Rainer , Möllering Hans , Jaenicke Rainer 
Published: February 2009

AbstractPyruvate oxidase from Lactobacillus plantarum is a homotetrameric flavoprotein with strong binding sites for FAD, TPP, and a divalent cation. Treatment with acid ammonium sulfate in the presence of 1.5 M KBr leads to the release of the cofactors, yielding the stable apoenzyme. In the present study, the effects of FAD, TPP, and Mn2+ on the structural properties of the apoenzyme and the reconstitution of the active holoenzyme from its constituents have been investigated.As shown by circular dichroism and fluorescence emission, as well as by Nile red binding, the secondary and tertiary structures of the apoenzyme and the holoenzyme do not exhibit marked differences. The quaternary structure is stabilized significantly in the presence of the cofactors. Size‐exclusion high‐performance liquid chromatography and analytical ultracentrifugation demonstrate that the holoenzyme retains its tetrameric state down to 20 μg/mL, whereas the apoenzyme shows stepwise tetramer‐dimer‐monomer dissociation, with the monomer as the major component, at a protein concentration of <20 μg/mL.In the presence of divalent cations, the coenzymes FAD and TPP bind to the apoenzyme, forming the inactive binary FAD or TPP complexes. Both FAD and TPP affect the quaternary structure by shifting the equilibrium of association toward the dimer or tetramer. High FAD concentrations exert significant stabilization against urea and heat denaturation, whereas excess TPP has no effect.Reconstitution of the holoenzyme from its components yields full reactivation. The kinetic analysis reveals a compulsory sequential mechanism of cofactor binding and quaternary structure formation, with TPP binding as the first step. The binary TPP complex (in the presence of 1 mM Mn2+/TPP) is characterized by a dimer‐tetramer equilibrium transition with an association constant of Ka = 2 × 107 M−1. The apoenzyme TPP complex dimer associates with the tetrameric holoenzyme in the presence of 10 μM FAD. This association step obeys second‐order kinetics with an association rate constant k = 7.4 × 103 M−1 s−1 at 20 °C. FAD binding to the tetrameric binary TPP complex is too fast to be resolved by manual mixing.
In vitro aging of calmodulin generates isoaspartate at multiple Asn–Gly and Asp–Gly sites in calcium‐binding domains II, III, and IV
Authors: Potter Steven M. , Henzel William J. , Aswad Dana W. 
Published: February 2009

AbstractWe have determined the major sites responsible for isoaspartate formation during in vitro aging of bovine brain calmodulin under mild conditions. Protein l‐isoaspartyl methyltransferase (EC 2.1.1.77) was used to quantify isoaspartate by the transfer of methyl‐3H from S‐adenosyl‐l‐[methyl‐3H]methionine to the isoaspartyl (α‐carboxyl) side chain. More than 1.2 mol of methyl‐acceptor sites per mol of calmodulin accumulated during a 2‐week incubation without calcium at pH 7.4, 37°C. Analysis of proteolytic peptides of aged calmodulin revealed that >95% of the methylation capacity is restricted to residues in the four calcium‐binding domains, which are predicted to be highly flexible in the absence of calcium. We estimate that domains III, IV, and II accumulated 0.72, 0.60, and 0.13 mol of isoaspartate per mol of calmodulin, respectively. The Asn‐97‐Gly‐98 sequence (domain III) is the greatest contributor to isoaspartate formation. Other major sites of isoaspartate formation are Asp‐131‐Gly‐132 and Asp‐133‐Gly‐134 in domain IV, and Asn‐60‐Gly‐61 in domain II. Significant isoaspartate formation was also localized to Asp‐20, Asp‐22, and/or Asp‐24 in domain I, to Asp‐56 and/or Asp‐58 in domain II, and to Asp‐93 and/or Asp‐95 in domain III. All of these residues are calcium ligands in the highly conserved EF‐hand calcium‐binding motif. Thus, other EF‐hand proteins may also be subject to isoaspartate formation at these ligands. The results support the idea that isoaspartate formation in structured proteins is strongly influenced by both the C‐flanking residue and by local flexibility.