bluuebunny/crossref_metadata_2025_split huggingface dataset:
Increasing nitrogenase catalytic efficiency for MgATP by changing serine 16 of its Fe protein to threonine: Use of Mn2+ to show interaction of serine 16 with Mg2+
Authors: Seefeldt Lance C. , Mortenson Leonard E. 
Published: July 2010

AbstractMgATP‐binding and hydrolysis are an integral part of the nitrogenase catalytic mechanism. We are exploring the function of MgATP hydrolysis in this reaction by analyzing the properties of the Fe protein (FeP) component of Azotobacter vinelandii nitrogenase altered by site‐directed mutagenesis. We have previously (Seefeldt, L.C., Morgan, T.V., Dean, D.R., & Mortenson, L.E., 1992, J. Biol. Chem. 267, 6680–6688) identified a region near the N‐terminus of FeP that is involved in interaction with MgATP. This region of FeP is homologous to the well‐known nucleotide‐binding motif GXXXXGKS/T. In the present work, we examined the function of the four hydroxyl‐containing amino acids immediately C‐terminal to the conserved lysine 15 that is involved in interaction with the β‐phosphate of MgATP. We have established, by altering independently Thr 17, Thr 18, and Thr 19 to alanine, that a hydroxyl‐containing residue is not needed at these positions for FeP to function. In contrast, an hydroxyl‐containing amino acid at position 16 was found to be critical for FeP function. When the strictly conserved Ser 16 was altered to Ala, Cys, Asp, or Gly, the FeP did not support N2 fixation when expressed in place of the wild‐type FeP in A. vinelandii. Altering Ser 16 to Thr (S16T), however, resulted in the expression of an FeP that was partially active. This S16T FeP was purified to homogeneity, and its biochemical examination allowed us to assign a catalytic function to this hydroxyl group in the nitrogenase mechanism. Of particular importance was the finding that the S16T FeP had a significantly higher affinity for MgATP than the wild‐type FeP, with a measured Km of 20 μM compared to the wild‐type FeP Km of 220 μM. This increased kinetic affinity for MgATP was reflected in a significantly stronger binding of the S16T FeP for MgATP. In contrast, the affinity for MgADP, which binds at the same site as MgATP, was unchanged. The catalytic efficiency (kcat/Km) of S16T FeP was found to be 5.3‐fold higher than for the wild‐type FeP, with the S16T FeP supporting up to 10 times greater nitrogenase activity at low MgATP concentrations. This indicates a role for the hydroxyl group at position 16 in interaction with MgATP but not MgADP. The site of interaction of this residue was further defined by examining the properties of wild‐type and S16T FePs in utilizing MnATP compared with MgATP. The S16T FeP was severely compromised in its interaction with MnATP, suggesting a mechanism where the hydroxyl group of amino acid 16 interacts with the Mg2+ of bound MgATP.
Probing the pyrophosphate‐binding site in potato tuber UDP‐glucose pyrophosphorylase with pyridoxal diphosphate
Authors: Kazuta Yasuaki , Tagaya Mitsuo , Tanizawa Katsuyuki , Fukui Toshio 
Published: July 2010

AbstractPotato tuber UDP‐glucose pyrophosphorylase (EC 2.7.7.9) catalyzes the reversible uridylyl transfer from UDP‐glucose to MgPPi forming glucose 1‐phosphate and MgUTP, according to an ordered bi‐bi mechanism in which UDP‐glucose and MgPPi bind in this order. To probe the active site of this enzyme, we have applied pyridoxal 5′‐diphosphate, a reactive PPi analogue. The enzyme was rapidly inactivated when incubated with the reagent in the presence of Mg2+ followed by sodium borohydride reduction. The degree of the inactivation was decreased by MgUTP, MgPPi, and glucose 1‐phosphate, but enhanced by UDP‐glucose. The enhancement was prevented by co‐addition of Pi, the competitive inhibitor with respect to PPi. The complete inactivation corresponded to the incorporation of 0.9–1.1 mol of reagent/mol of enzyme monomer. In the presence of UDP‐glucose, labels were almost exclusively incorporated into Lys‐329. Thus, this residue may be located near the bound MgPPi and its modification is promoted, probably through conformational changes, by the binding of UDP‐glucose to the enzyme. The results of the modification by the same reagent of the mutant enzymes in which Lys‐329 and Lys‐263 are individually replaced by Gln suggest the roles of these lysyl residues in the binding of MgPPi and in the UDP‐glucose‐induced conformational changes, respectively.
The binding domain structure of retinoblastoma‐binding proteins
Authors: Figge James , Breese Kimberly , Vajda Sandor , Zhu Qing‐Lin , Eisele Leslie , Andersen Thomas T. , Maccoll Robert , Friedrich Thomas , Smith Temple F. 
Published: July 2010

AbstractThe retinoblastoma gene product (Rb), a cellular growth suppressor, complexes with viral and cellular proteins that contain a specific binding domain incorporating three invariant residues: Leu‐X‐Cys‐X‐Glu, where X denotes a nonconserved residue. Hydrophobic and electrostatic properties are strongly conserved in this segment even though the nonconserved amino acids vary considerably from one Rb‐binding protein to another. In this report, we present a diagnostic computer pattern for a high‐affinity Rb‐binding domain featuring the three conserved residues as well as the conserved physico‐chemical properties. Although the pattern encompasses only 10 residues (with only 4 of these explicitly defined), it exhibits 100% sensitivity and 99.95% specificity in database searches. This implies that a certain pattern of structural and physico‐chemical properties encoded by this short sequence is sufficient to govern specific Rb binding. We also present evidence that the secondary structural conformation through this region is important for effective Rb binding.
Characterization and 2D NMR study of the stable [9–21, 15–27] 2 disulfide intermediate in the folding of the 3 disulfide trypsin inhibitor EETI II
Authors: Le‐Nguyen D. , Heitz A. , Chiche L. , Hajji M. El , Castro B. 
Published: July 2010

AbstractThe three disulfide Ecballium elaterium trypsin inhibitor II (EETI II) reduction with dithiothreitol (DTT) and re‐oxidation of the fully reduced derivative have been examined. A common stable intermediate has been observed for both processes. Isolation and sequencing of carboxymethylated material showed that the intermediate lacks the [2–19] bridge. The NMR study showed a very strong structural conservation as compared to the native EETI II, suggesting that the bridges are the [9–21] and [15–27] native ones. The differences occurred in sections 2–7 (containing the free cysteine 2 and the Arg 4‐Ile 5 active site) and 19–21 (containing the second free cysteine). Distance geometry calculations and restrained molecular dynamics refinements were also in favor of a [9–21, 15–27] arrangement and resulted in a well‐conserved (7–28) segment.
Identification of a nucleic acid‐binding region within the largest subunit ofDrosophila melanogasterRNA polymerase II
Authors: Kontermann Roland E. , Kobor Michael , Bautz Ekkehard K. F. 
Published: July 2010

AbstractThe largest and the second‐largest subunit of the multisubunit eukaryotic RNA polymerases are involved in interaction with the DNA template and the nascent RNA chain. Using Southwestern DNA‐binding techniques and nitrocellulose filter binding assays of bacterially expressed fusion proteins, we have identified a region of the largest, 215‐kDa, subunit ofDrosophilaRNA polymerase II that has the potential to bind nucleic acids nonspecifically. This nucleic acid‐binding region is located between amino acid residues 309–384 and is highly conserved within the largest subunits of eukaryotic and bacterial RNA polymerases. A homology to a region of the DNA‐binding cleft ofEscherichia coliDNA polymerase I involved in binding of the newly synthesized DNA duplex provides indirect evidence that the nucleic acid‐binding region of the largest subunit participates in interaction with double‐stranded nucleic acids during transcription. The nonspecific DNA‐binding behavior of the region is similar to that observed for the native enzyme in nitrocellulose filter binding assays and that of the separated largest subunit in Southwestern assays. A high content of basic amino acid residues is consistent with the electrostatic nature of nonspecific DNA binding by RNA polymerases.
Retroviral nucleocapsid proteins possess potent nucleic acid strand renaturation activity
Authors: Dib‐Hajj Fadia , Khan Raza , Giedroc David P. 
Published: July 2010

AbstractThe nucleocapsid protein (NC) is the major genomic RNA binding protein that plays integral roles in the structure and replication of all animal retroviruses. In this report, select biochemical properties of recombinant Mason‐Pfizer monkey virus (MPMV) and HIV‐1 NCs are compared. Evidence is presented that two types of saturated Zn2 NC‐polynucleotide complexes can be formed under conditions of low [NaCl] that differ in apparent site‐size (n = 8 vs. n = 14). The formation of one or the other complex appears dependent on the molar ratio of NC to RNA nucleotide with the putative low site‐size mode apparently predominating under conditions of protein excess. Both MPMV and HIV‐1 NCs kinetically facilitate the renaturation of two complementary DNA strands, suggesting that this is a general property of retroviral NCs. NC proteins increase the second‐order rate constant for renaturation of a 149‐bp DNA fragment by more than four orders of magnitude over that obtained in the absence of protein at 37°C. The protein‐assisted rate is 100–200‐fold faster than that obtained at 68°C, 1 M NaCl, solution conditions considered to be optimal for strand renaturation. Provided that sufficient NC is present to coat all strands, the presence of 400–1,000‐fold excess nonhomologous DNA does not greatly affect the reaction rate. The HIV‐1 NC‐mediated renaturation reaction functions stoichiometrically, requiring a saturated strand of DNA nucleotide:NC ratio of about 7–8, rather than 14. Under conditions of less protein, the rate acceleration is not realized. The finding of significant nucleic acid strand renaturation activity may have important implications for various events of reverse transcription particularly in initiation and cDNA strand transfer.
Structural analysis based on state‐space modeling
Authors: Stultz Collin M. , White James V. , Smith Temple F. 
Published: July 2010

AbstractA new method has been developed to compute the probability that each amino acid in a protein sequence is in a particular secondary structural element. Each of these probabilities is computed using the entire sequence and a set of predefined structural class models. This set of structural classes is patterned after Jane Richardson's taxonomy for the domains of globular proteins. For each structural class considered, a mathematical model is constructed to represent constraints on the pattern of secondary structural elements characteristic of that class. These are stochastic models having discrete state spaces (referred to as hidden Markov models by researchers in signal processing and automatic speech recognition). Each model is a mathematical generator of amino acid sequences; the sequence under consideration is modeled as having been generated by one model in the set of candidates. The probability that each model generated the given sequence is computed using a filtering algorithm. The protein is then classified as belonging to the structural class having the most probable model. The secondary structure of the sequence is then analyzed using a “smoothing” algorithm that is optimal for that structural class model. For each residue position in the sequence, the smoother computes the probability that the residue is contained within each of the defined secondary structural elements of the model. This method has two important advantages: (1) the probability of each residue being in each of the modeled secondary structural elements is computed using the totality of the amino acid sequence, and (2) these probabilities are consistent with prior knowledge of realizable domain folds as encoded in each model. As an example of the method's utility, we present its application to flavodoxin, a prototypical α/β protein having a central β‐sheet, and to thioredoxin, which belongs to a similar structural class but shares no significant sequence similarity.
Total synthesis and functional properties of the membrane‐intrinsic protein phospholamban
Authors: Vorherr Thomas , Carafoli Ernesto , Wrzosek Antoni , Chiesi Michele 
Published: July 2010

AbstractThe membrane‐intrinsic protein phospholamban (PLN), the regulatory protein of the sarcoplasmic reticulum (SR) Ca2+‐ATPase, was chemically synthesized. The synthesis was accomplished by double couplings and efficient capping procedures, thus eliminating hydrophobic failure sequences. The crude peptide was purified by high‐performance liquid chromatographic ion exchange and gel permeation chromatography in chloroform–methanol mixtures. Ion spray mass spectroscopy showed that the product had the correct molecular mass. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis runs produced the typical monomer–pentamer structural pattern. A predominantly helical CD spectrum was obtained in 0.075% C12E8 (67.9% helix, 1.8% beta, 12.2% turn, 18.1% random coil). Synthetic PLN was phosphorylated in detergent solutions by protein kinase A with a stoichiometry close to 1:1 (Pi to PLN monomer). Reconstitution of the isolated skeletal muscle SR Ca2+ ATPase in phosphatidylcholine membranes in the presence of PLN using the freezing and thawing technique yielded a preparation with lower Ca2+‐dependent ATPase activity. The inhibition was mainly due to a decrease in the affinity (Km(Ca)) of the ATPase for Ca2+ and was partially reversed by PLN phosphorylation with protein kinase A. By contrast, addition of PLN to diluted intact SR vesicles uncoupled the Ca2+‐transport reaction, suggesting an ionophoric effect of PLN. Because this effect was observed at very high PLN‐to‐SR vesicle ratios and was not influenced by PLN phosphorylation, its biological function is doubtful.
Unnatural amino acid packing mutants of Escherichia coli thioredoxin produced by combined mutagenesis/chemical modification techniques
Authors: Wynn R. , Richards F. M. 
Published: July 2010

AbstractWe have produced several mutants of Escherichia coli thioredoxin (Trx) using a combined mutagenesis/chemical modification technique. The protein C32S, C35S, L78C Trx was produced using standard mutagenesis procedures. After unfolding the protein with guanidine hydrochloride (GdmCl), the normally buried cysteine residue was modified with a series of straight chain aliphatic thiosulfonates, which produced cysteine disulfides to methane, ethane, 1‐n‐propane, 1‐n‐butane, and 1‐n‐pentane thiols. These mutants all show native‐like CD spectra and the ability to activate T7 gene 5 protein DNA polymerase activity. In addition, all mutants show normal unfolding transitions in GdmCl solutions. However, the midpoint of the transition, [GdmCl]1/2, and the free energy of unfolding at zero denaturant concentration, δG, give inverse orders of stability. This effect is due to changes in m, the dependence of δGunfolding0 on the GdmCl concentration. The method described here may be used to produce unnatural amino acids in the hydrophobic cores of proteins.
Crystal structure of human annexin I at 2.5 Å resolution
Authors: Weng Xiangwei , Luecke Hartmut , Song In Sung , Kang Doe Sun , Kim Sung‐Hou , Huber Robert 
Published: July 2010

AbstractcDNA coding for N‐terminally truncated human annexin I, a member of the family of Ca2+‐dependent phospholipid binding proteins, has been cloned and expressed in Escherichia coli. The expressed protein is biologically active, and has been purified and crystallized in space group P212121 with cell dimensions a = 139.36 Å, b = 67.50 Å, and c = 42.11 Å. The crystal structure has been determined by molecular replacement at 3.0 Å resolution using the annexin V core structure as the search model. The average backbone deviation between these two structures is 2.34 Å. The structure has been refined to an R‐factor of 17.7% at 2.5 Å resolution. Six calcium sites have been identified in the annexin I structure. Each is located in the loop region of the helix–loop–helix motif. Two of the six calcium sites in annexin I are not occupied in the annexin V structure. The superpositions of the corresponding loop regions in the four domains show that the calcium binding loops in annexin I can be divided into two classes: type II and type III. Both classes are different from the well‐known EF‐hand motif (type I).
The escherichia coli adenylyl cyclase complex: Stimulation by GTP and other nucleotides
Authors: Peterkofsky Alan , Gollop Natan 
Published: July 2010

AbstractEscherichia coli cells permeabilized by treatment with low concentrations of toluene contain an adenylyl cyclase activity that can be stimulated 3.6–7.6‐fold by GTP. The stimulatory effect of GTP is maximal at concentrations of the nucleotide in the physiological range (above 0.7 mM). Studies of the dependence of velocity on substrate (ATP) concentration indicate that the velocity vs. substrate plots are sigmoid in the absence of GTP but hyperbolic in the presence of GTP, suggesting an allosteric regulatory site that can be occupied by either ATP or GTP. Replacement of ATP by AMPPNP as substrate results in velocity vs. substrate plots that are hyperbolic in the absence or presence of GTP, although GTP increases the Vmax by a factor of 2.2; these findings indicate that AMPPNP specifically occupies the substrate site and GTP exclusively occupies the regulatory site. A test of the capacity of other guanine nucleotides to stimulate adenylyl cyclase activity showed that 2′‐deoxy‐GTP was almost as effective as GTP, but that GDP, GMP, ppGpp, and 3′, 5′‐cGMP were not stimulatory effectors; GTP‐γ‐S and GMPPNP stimulated adenylyl cyclase activity but to a lesser degree than did GTP. In addition to the previous indication that ATP can occupy the regulatory site on adenylyl cyclase, it was found that CTP and UTP were potent stimulators. Thus, all the naturally occurring RNA precursor nucleoside triphosphates are capable of stimulating adenylyl cyclase activity. In contrast, PPPi inhibits adenylyl cyclase activity. Additional studies showed that there is only one regulatory site for all the nucleotide stimulators and that the nucleotide occupying the substrate site can influence the properties of the regulatory site. These observations suggest a mechanism by which adenylyl cyclase activity can be regulated by the availability of nucleic acid precursors.
Determination of amide hydrogen exchange by mass spectrometry: A new tool for protein structure elucidation
Authors: Zhang Zhongqi , Smith David L. 
Published: July 2010

AbstractA new method based on protein fragmentation and directly coupled microbore high‐performance liquid chromatography–fast atom bombardment mass spectrometry (HPLC‐FABMS) is described for determining the rates at which peptide amide hydrogens in proteins undergo isotopic exchange. Horse heart cytochrome c was incubated in D2O as a function of time and temperature to effect isotopic exchange, transferred into slow exchange conditions (pH 2–3, 0 °C), and fragmented with pepsin. The number of peptide amide deuterons present in the proteolytic peptides was deduced from their molecular weights, which were determined following analysis of the digest by HPLC‐FABMS. The present results demonstrate that the exchange rates of amide hydrogens in cytochrome c range from very rapid (k > 140 h−1) to very slow (k < 0.002 h−1). The deuterium content of specific segments of the protein was determined as a function of incubation temperature and used to indicate participation of these segments in conformational changes associated with heating of cytochrome c. For the present HPLC‐FABMS system, approximately 5 nmol of protein were used for each determination. Results of this investigation indicate that the combination of protein fragmentation and HPLC‐FABMS is relatively free of constraints associated with other analytical methods used for this purpose and may be a general method for determining hydrogen exchange rates in specific segments of proteins.
Three‐dimensional structural model of the serine receptor ligand‐binding domain
Authors: Jeffery Constance J. , Koshland D.E. 
Published: July 2010

AbstractComputer‐based homology modeling techniques were used to construct a three‐dimensional model of the Escherichia coli serine receptor ligand‐binding domain based on the crystal structure of the Salmonella typhimurium aspartate receptor and the sequence homology between the two receptors. Residues that have been found in mutagenesis studies to be necessary for serine binding are located in a proposed serine‐binding site. Several other mutations that affect swimming behavior require relatively small shifts in α‐carbon positions in the model to give a minimized structure, suggesting that small changes in receptor conformation can affect the signaling state of the receptor.
Structural investigation of catalytically modified F120L and F120Y semisynthetic ribonucleases
Authors: Demel V. Srini J. , Doscher Marilynn S. , Glinn Michele A. , Martin Philip D. , Ram Michal L. , Edwards Brian F.P. 
Published: July 2010

AbstractThe structures of two catalytically modified semisynthetic RNases obtained by replacing phenylalanine 120 with leucine and tyrosine have been determined and refined at a resolution of 2.0 Å (R = 0.161 and 0.184, respectively). These structures have been compared with the refined 1.8‐Å structure (R = 0.204) of the fully active phenylalanine‐containing enzyme (Martin PD, Doscher MS, Edwards BFP, 1987, J Biol Chem 262:15930‐15938) and with the catalytically defective D121A (2.0 Å, R = 0.172) and D121N (2.0 Å, R = 0.186) analogs (deMel VSJ, Martin PD, Doscher MS, Edwards BFP, 1992, J Biol Chem 267:247‐256). The movement away from the active site of the loop containing residues 65‐72 is seen in all three catalytically defective analogs‐F120L, D121A, and D121N‐but not in the fully active (or hyperactive) F120Y. The insertion of the phenolic hydroxyl of Tyr 120 into a hydrogen‐bonding network involving the hydroxyl group of Ser 123 and a water molecule in F120Y is the likely basis for the hyperactivity toward uridine 2′,3′‐cyclic phosphate previously found for this analog (Hodges RS, Merrifield RB, 1974, Int J Pept Protein Res 6:397‐405) as well as the threefold increase in KM for cytidine 2′,3′‐cyclic phosphate found for this analog by ourselves.
Crystal structure of the unactivated ribulose 1, 5‐bisphosphate carboxylase/oxygenase complexed with a transition state analog, 2‐carboxy‐D‐arabinitol 1, 5‐bisphosphate
Authors: Zhang Kam Y.J. , Cascio Duilio , Eisenberg David 
Published: July 2010

AbstractThe crystal structure of unactivated ribulose 1, 5‐bisphosphate carboxylase/oxygenase from Nicotiana tabacum complexed with a transition state analog, 2‐carboxy‐D‐arabinitol 1, 5‐bisphosphate, was determined to 2.7 Å resolution by X‐ray crystallography. The transition state analog binds at the active site in an extended conformation. As compared to the binding of the same analog in the activated enzyme, the analog binds in a reverse orientation. The active site Lys 201 is within hydrogen bonding distance of the carboxyl oxygen of the analog. Loop 6 (residues 330‐339) remains open and flexible upon binding of the analog in the unactivated enzyme, in contrast to the closed and ordered loop 6 in the activated enzyme complex. The transition state analog is exposed to solvent due to the open conformation of loop 6.