bluuebunny/crossref_metadata_2025_split huggingface dataset:
A test case for structure‐based functional assignment: The 1.2 Å crystal structure of the yjgF gene product from Escherichia coli
Authors: Volz Karl 
Published: February 2009

AbstractThe YER057c/YIL051c/YjgF protein family is a set of 24 full‐length homologs, each ∼130 residues in length, and each with no known function or relationship to proteins of known structure. To determine the function of this family, the structure of one member–the YjgF protein from Escherichia coli–was solved and refined at a resolution of 1.2 Å. The YjgF molecule is a homotrimer with exact threefold symmetry. Its tertiary and quaternary structures are related to that of Bacillus subtilis chorismate mutase, although their active sites are completely different. The YjgF protein has an active site curiously similar to protein tyrosine phosphatases, including a covalently modified cysteine, but it is unlikely to be functionally related. The lessons learned from this attempt to deduce function from structure may be useful to future projects in structural genomics.
Probing the location and function of the conserved histidine residue of phosphoglucose isomerase by using an active site directed inhibitor N‐bromoacetylethanolamine phosphate
Authors: Meng Menghsiao , Chane Tze‐Lieh , Sun Yuh‐Ju , Hsiao Chwan‐Deng 
Published: February 2009

AbstractPhosphoglucose isomerase (EC 5.3.1.9) catalyzes the interconversion of D‐glucopyranose‐6‐phosphate and D‐fructofuranose‐6‐phosphate by promoting an intrahydrogen transfer between C1 and C2. A conserved histidine exists throughout all phosphoglucose isomerases and was hypothesized to be the base catalyzing the isomerization reaction. In the present study, this conserved histidine, His311, of the enzyme from Bacillus stearothermophilus was subjected to mutational analysis, and the mutational effect on the inactivation kinetics by N‐bromoacetylethanolamine phosphate was investigated. The substitution of His311 with alanine, asparagine, or glutamine resulted in the decrease of activity, in kcat/KM, by a factor of 103, indicating the importance of this residue. N‐bromoacetylethanolamine phosphate inactivated irreversibly the activity of wild‐type phosphoglucose isomerase; however, His311 → Ala became resistant to this inhibitor, indicating that His311 is located in the active site and is responsible for the inactivation of the enzyme by this active site‐directed inhibitor. The pKa of His311 was estimated to be 6.31 according to the pH dependence of the inactivation. The proximity of this value with the pKa value of 6.35, determined from the pH dependence of kcat/KM supports a role of His311 as a general base in the catalysis.
Conformational and metal‐binding properties of androcam, a testis‐specific, calmodulin‐related protein from drosophila
Authors: Martin Stephen R. , Kleinjung Jens , Bayley Peter M. , Lu Alan Q. , Xiao Jie , Beckingham Kathy 
Published: February 2009

AbstractAndrocam is a testis‐specific protein of Drosophila melanogaster, with 67% sequence identity to calmodulin and four potential EF‐hand calcium‐binding sites. Spectroscopic monitoring of the thermal unfolding of recombinant calciumfree androcam shows a biphasic process characteristic of a two‐domain protein, with the apo‐N‐domain less stable than the apo‐C‐domain. The two EF hands of the C‐domain of androcam bind calcium cooperatively with 40‐fold higher average affinity than the corresponding calmodulin sites. Magnesium competes with calcium binding [Ka(Mg) ˜ 3 × 103 M−1]. Weak calcium binding is also detected at one or more N‐domain sites. Compared to apo‐calmodulin, apo‐androcam has a smaller conformational response to calcium and a lower α‐helical content over a range of experimental conditions. Unlike calmodulin, a tryptic cleavage site in the N‐domain of apo‐androcam remains trypsin sensitive in the presence of calcium, suggesting an altered calcium‐dependent conformational change in this domain. The affinity of model target peptides for androcam is 103‐105 times lower than for calmodulin, and interaction of the N‐domain of androcam with these peptides is significantly reduced. Thus, androcam shows calcium‐induced conformational responses typical of a calcium sensor, but its properties indicate calcium sensitivity and target interactions significantly different from those of calmodulin. From the sequence differences and the altered calcium‐binding properties it is likely that androcam differs from calmodulin in the conformation of residues in the second calcium‐binding loop. Molecular modeling supports the deduction that there are significant conformational differences in the N‐domain of androcam compared to calmodulin, and that these could affect the surface, conferring a different specificity on androcam in target interactions related to testis‐specific calcium signaling functions.
Real‐time measurements of dark substrate catalysis
Authors: Xie Dong , Suvorov Leonid , Erickson John W. , Gulnik Sergei 
Published: February 2009

AbstractWe have developed a novel procedure to monitor the real‐time cleavage of natural unmodified peptides (dark substrates). In the competition‐based assay, the initial cleavage rate of a fluorogenic peptide substrate is measured in the presence of a second substrate that is not required to exhibit any optical property change upon cleavage. Using a unique experimental design and steady‐state enzyme kinetics for a two‐substrate system, we were able to determine both Km and kcat values for cleavage of the dark substrate. The method was applied to HIV‐1 protease and to the V82F/I84V drug resistant mutant enzyme. Using two different substrates, we showed that the kinetic parameters derived from the competition assay are in good agreement with those determined independently using standard direct assay. This method can be applied to other enzyme systems as long as they have one substrate for which catalysis can be conveniently monitored in real time.
Purification and characterization of a cobalt‐activated carboxypeptidase from the hyperthermophilic archaeon Pyrococcus furiosus
Authors: Cheng Timothy C. , Ramakrishnan Vijay , Chan Sunney I. 
Published: February 2009

AbstractA novel metallocarboxypeptidase (PfuCP) has been purified to homogeneity from the hyperthermophilic archaeon, Pyrococcus furiosus, with its intended use in C‐terminal ladder sequencing of proteins and peptides at elevated temperatures. PfuCP was purified in its inactive state by the addition of ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) to purification buffers, and the activity was restored by the addition of divalent cobalt (Kd = 24 ± 4 μM at 80 °C). The serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) had no effect on the activity. The molecular mass of monomeric PfuCP is 59 kDa as determined by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and 58 kDa by SDS‐PAGE analysis. In solution, PfuCP exists as a homodimer of ˜ 128 kDa as determined by gel filtration chromatography. The activity of PfuCP exhibits a temperature optimum exceeding 90°C under ambient pressure, and a narrow pH optimum of 6.2‐6.6. Addition of Co2+ to the apoPfuCP at room temperature does not alter its far‐UV circular dichroism (CD) or its intrinsic fluorescence spectrum. Even when the CoPfuCP is heated to 80 °C, its far‐UV CD shows a minimal change in the global conformation and the intrinsic fluorescence of aromatic residues shows only a partial quenching. Changes in the intrinsic fluorescence appear essentially reversible with temperature. Finally, the far‐UV CD and intrinsic fluorescence data suggest that the overall structure of the holoenzyme is extremely thermostable. However, the activities of both the apo and holo enzyme exhibit a similar second‐order decay over time, with 50% activity remaining after ˜ 40 min at 80 °C. The N‐blocked synthetic dipeptide, N‐carbobenzoxy‐Ala‐Arg (ZAR), was used in the purification assay. The kinetic parameters at 80°C with 0.4 mM CoCl2 were: Km, 0.9 ± 0.1 mM; Vmax 2,300 ± 70 U mg−1; and turn over number, 600 ± 20 s−1. Activity against other ZAX substrates (X = V, L, I, M, W, Y, F, N, A, S, H, K) revealed a broad specificity for neutral, aromatic, polar, and basic C‐terminal residues. This broad specificity was confirmed by the C‐terminal ladder sequencing of several synthetic and natural peptides, including porcine N‐acetyl‐renin substrate, for which we have observed (by MALDI‐TOF MS) stepwise hydrolysis by PfuCP of up to seven residues from the C‐terminus: Ac‐Asp‐Arg‐Val‐Tyr‐Ile‐His‐Pro‐z Phe‐His‐Leu‐Leu‐Val‐Tyr‐Ser.
Locating and identifying posttranslational modifications by in‐source decay during MALDI‐TOF mass spectrometry
Authors: Lennon John J. , Walsh Kenneth A. 
Published: February 2009

AbstractA technique is described for identifying and locating posttranslational modifications (PTMs) in peptides and proteins of known sequence by interpretation of cn ion signals generated by in‐source decay during delayed ion extraction in matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. Sites of phosphorylation in seven synthetic peptides were determined, as was the location of both the heme group and N, N, N‐trimethyllysine in yeast cytochrome c. A semi‐automated data analysis process facilitates the identification of segments of the sequence on each side of the PTM, permitting its placement at the junction of the segments and definition of the added mass. A graphical display facilitates illustration of both the location and mass of the PTM.
NikR is a ribbon‐helix‐helix DNA‐binding protein
Authors: Chivers Peter T. , Sauer Robert T. 
Published: February 2009

AbstractEscherichia coli NikR, a repressor with homologs in other bacteria and archaea, was identified as a potential new member of the ribbon‐helix‐helix (β‐α‐α) family of transcription factors in profile based sequence searches and in structure prediction experiments. Biophysical and biochemical characterization of the N‐terminal domain of NikR show that it has many features expected of a β‐α‐α protein including α‐helical content, dimeric solution form, concentration dependent thermal stability, and ability to bind DNA in sequence‐specific manner. Mutation of a residue predicted to be important for DNA‐binding reduces operator affinity but does not affect the secondary structure or stability of the protein.
The relationship between effector binding and inhibition of activity in D‐3‐phosphoglycerate dehydrogenase
Authors: Grant Gregory A. , Xu Xiao Lan , Hu Zhiqin 
Published: February 2009

AbstractThe binding of L‐serine to phosphoglycerate dehydrogenase from Escherichia coli displays elements of both positive and negative cooperativity. At pH 7.5, ˜ 2 mol of serine are bound per mole of tetrameric enzyme. A substantial degree of positive cooperativity is seen for the binding of the second ligand, but the binding of the third and fourth ligand display substantial negative cooperativity. The data indicate a state of ˜ 50% inhibition when only one serine is bound and ˜ 80‐90% inhibition when two serines are bound. This is consistent with the tethered domain hypothesis that has been presented previously. Comparison of the data derived directly from binding stoichiometry to the binding constants determined from the best fit to the Adair equation, produce a close agreement, and reinforce the general validity of the derived binding constants. The data also support the conclusion that the positive cooperativity between the binding to the first and second site involves binding sites at opposite interfaces over 110 Å apart. Thus, an order of binding can be envisioned where the binding of the first ligand initiates a conformational transition that allows the second ligand to bind with much higher affinity at the opposite interface. This is followed by the third ligand, which binds with lesser affinity to one of the two already occupied interfaces, and in so doing, completes a global conformational transition that produces maximum inhibition of activity and an even lower affinity for the fourth ligand, excluding it completely. Thus, maximal inhibition is accomplished with less than maximal occupancy of effector sites through a mechanism that displays strong elements of both positive and negative cooperativity.
Methionine sulfoxidation of the chloroplast small heat shock protein and conformational changes in the oligomer
Authors: Gustavsson Niklas , Härndahl Ulrika , Sundby Cecilia , Emanuelsson Anna , Roepstorff Peter 
Published: February 2009

AbstractThe small heat shock proteins (sHsps), which counteract heat and oxidative stress in an unknown way, belong to a protein family of sHsps and α‐crystallins whose members form large oligomeric complexes. The chloroplast‐localized sHsp, Hsp21, contains a conserved methionine‐rich sequence, predicted to form an amphipatic helix with the methionines situated along one of its sides. Here, we report how methionine sulfoxidation was detected by mass spectrometry in proteolytically cleaved peptides that were produced from recombinant Arabidopsis thaliana Hsp21, which had been treated with varying concentrations of hydrogen peroxide. Sulfoxidation of the methionine residues in the conserved amphipatic helix coincided with a significant conformational change in the Hsp21 protein oligomer.
2′Halo‐ATP and ‐GTP analogues: Rational phasing tools for protein crystallography
Authors: Gruen Mathias , Becker Christian , Beste Andrea , Reinstein Jochen , Scheidig Axel J. , Goody Roger S. 
Published: February 2009

AbstractThe solution of the crystallographic macromolecular phase problem requires incorporation of heavy atoms into protein crystals. Several 2′‐halogenated nucleotides have been reported as potential universal phasing tools for nucleotide binding proteins. However, only limited data are available dealing with the effect of 2′‐substitution on recognition by the protein. We have determined equilibrium dissociation constants of 2′‐halogenated ATP analogues for the ATP binding proteins UMP/CMP kinase and the molecular chaperone DnaK. Whereas the affinities to UMP/CMP kinase are of the same order of magnitude as for unsubstituted ATP, the affinities to DnaK are drastically decreased to undetectable levels. For 2′‐halogenated GTP analogues, the kinetics of interaction were determined for the small GTPases p21ras (Y32W) (fluorescent mutant) and Rab5. The rates of association were found to be within about one order of magnitude of those for the nonsubstituted nucleotides, whereas the rates of dissociation were accelerated by factors of ˜100 (p21ras) or ˜105 (Rab5), and the resulting equilibrium dissociation constants are in the nm or μM range, respectively. The data demonstrate that 2′halo‐ATP and ‐GTP are substrates or ligands for all proteins tested except the chaperone DnaK. Due to the very high affinities of a large number of GTP binding proteins to guanine nucleotides, even a 105‐fold decrease in affinity as observed for Rab5 places the equilibrium dissociation constant in the μM range, so that they are still well suited for crystallization of the G‐protein:nucleotide complex.
Common protein architecture and binding sites in proteases utilizing a Ser/Lys dyad mechanism
Authors: Paetzel Mark , J. Strynadka Natalie C. 
Published: February 2009

AbstractEscherichia coli signal peptidase (SPase) and E. coli UmuD protease are members of an evolutionary clan of serine proteases that apparently utilize a serine‐lysine catalytic dyad mechanism. Recently, the crystallographic structure of a SPase inhibitor complex was solved elucidating the catalytic residues and the substrate binding subsites. Here we show a detailed comparison of the E. coli SPase structure to the native E. coli UmuD′ structure. The comparison reveals that despite a very low sequence identity these functionally diverse enzymes share the same protein fold within their catalytic core and allows by analogy for the assignment of the cleavage‐site orientation and substrate binding subsites in the UmuD(D′) protease. The structural alignment of SPase and UmuD′ predicts important mechanistic and structural similarities and differences within these newly characterized families of serine proteases.
Solution structure and dynamics of bovine β‐lactoglobulin A
Authors: Kuwata Kazuo , Era Seiichi , Hoshino Masaru , Forge Vincent , Goto Yuji , Batt Carl A. 
Published: February 2009

AbstractUsing heteronuclear NMR spectroscopy, we studied the solution structure and dynamics of bovine β‐lactoglobulin A at pH 2.0 and 45 °C, where the protein exists as a monomeric native state. The monomeric NMR structure, comprising an eight‐stranded continuous antiparallel β‐barrel and one major α‐helix, is similar to the X‐ray dimeric structure obtained at pH 6.2, including βIstrand that forms the dimer interface and loop EF that serves as a lid of the interior hydrophobic hole. {1H}‐15N NOE revealed that βf, βg, and βH strands buried under the major α‐helix are rigid on a pico‐ to nanosecond time scale and also emphasized rapid fluctuations of loops and the N‐ and C‐terminal regions.
The extracellular regions of PSMA and the transferrin receptor contain an aminopeptidase domain: Implications for drug design
Authors: Mahadevan Daruka , Saldanha José W. 
Published: February 2009

AbstractThe Aeromonas proteolytica aminopeptidase (AMP), Pseudomonas sp. (RS‐16) carboxypeptidase G2 (CPG2), and Streptomyces griseus aminopeptidase (SGAP) are zinc dependent proteolytic enzymes with cocatalytic zinc ion centers and a conserved aminopeptidase fold. A BLAST search with the sequence of the solved AMP structure indicated that a similar domain could be found in prostate‐specific membrane antigen (PSMA) and the transferrin receptor (TfR). When the PSMA or TfR sequence was input into the THREADER program, the top structural matches were SGAP and AMP confirming that these are structurally conserved domains. Optimal sequence alignment of PSMA and TfR using the known three‐dimensional structures of AMP, CPG2, and SGAP shows that the critical amino acids involved in forming the catalytic pocket are conserved in PSMA but absent in the TfR. The specificity pocket in AMP is formed from four aromatic side chains and the equivalent region in CPG2/PSMA has a changed sequence pattern. Since CPG2 and PSMA are folate hydrolases, the changed specificity pocket leaves space to accommodate the large pteroate moiety of folic acid. In contrast, no enzyme function has been ascribed to the TfR.
Acetylcholinesterase of schistosoma mansoni—Functional correlates: Contributed in honor of Professor Hans Neurath's 90th birthday
Authors: Arnon Ruth , Tarrab‐Hazdai Rebeca , Silman Israel 
Published: February 2009

AbstractAcetylcholinesterase (AChE) is an enzyme broadly distributed in many species, including parasites. It occurs in multiple molecular forms that differ in their quaternary structure and mode of anchoring to the cell surface. This review summarizes biochemical and immunological investigations carried out in our laboratories on AChE of the helmint, Schistosoma mansoni. AChE appears in S. mansoni in two principal molecular forms, both globular, with sedimentation coefficients of ∼6.5 and 8 S. On the basis of their substrate specificity and sensitivity to inhibitors, both are “true” acetylcholinesterases. Approximately half of the AChE activity of S. mansoni is located on the outer surface of the parasite, attached to the tegumental membrane via a covalently attached glycosylphosphatidylinositol anchor. The remainder is located within the parasite, mainly associated with muscle tissue. Whereas the internal enzyme is most likely involved in termination of neurotransmission at cholinergic synapses, the role of the surface enzyme remains to be established; there are, however, indications that it is involved in signal transduction. The two forms of AChE differ in their heparin‐binding properties, only the internal 8 S form of the AChE being retained on a heparin column. The two forms differ also in their immunological specificity, since they are selectively recognized by different monoclonal antibodies. Polyclonal antibodies raised against S. mansoni AChE purified by affinity chromatography are specific for the parasite AChE, reacting with both molecular forms, but do not recognize AChE from other species. They interact with the surface‐localized enzyme on the intact organism, and produce almost total complement‐dependent killing of the parasite. S. mansoni AChE is thus demonstrated to be a functional protein, involved in multifaceted activities, which can serve as a suitable candidate for diagnostic purposes, vaccine development, and drug design.
NMR assignments, secondary structure, and global fold of calerythrin, an EF‐hand calcium‐binding protein fromSaccharopolyspora erythraea
Authors: Aitio Helena , Heikkinen Sami , Äinen Ilkka Kilpel , Annila Arto , Thulin Eva , Drakenberg Torbjörn 
Published: February 2009

AbstractCalerythrin is a 20 kDa calcium‐binding protein isolated from gram‐positive bacteriumSaccharopolyspora erythraea.Based on amino acid sequence homology, it has been suggested that calerythrin belongs to the family of invertebrate sarcoplasmic EF‐hand calcium‐binding proteins (SCPs), and therefore it is expected to function as a calcium buffer. NMR spectroscopy was used to obtain structural information on the protein in solution. Backbone and side chain1H,13C, and15N assignments were obtained from triple resonance experiments HNCACB, HN(CO)CACB, HNCO, CC(CO)NH, and [15N]‐edited TOCSY, and HCCH‐TOCSY. Secondary structure was determined by using secondary chemical shifts and characteristic NOEs. In addition, backbone N‐H residual dipolar couplings were measured from a spin‐state selective [1H,15N] correlation spectrum acquired from a sample dissolved in a dilute liquid crystal. Four EF‐hand motifs with characteristic helix‐loop‐helix patterns were observed. Three of these are typical calcium‐binding EF‐hands, whereas site 2 is an atypical nonbinding site. The global fold of calerythrin was assessed by dipolar couplings. Measured dipolar couplings were compared with values calculated from four crystal structures of proteins with sequence homology to calerythrin. These data allowed us to recognize an overall similarity between the folds of calerythrin and sarcoplasmic calcium‐binding proteins from the sandwormNereis diversicolorand the amphioxusBranchiostoma lanceolatum.
Rotamer strain as a determinant of protein structural specificity
Authors: Lazar Greg A. , Handel Tracy M. , Johnson Eric C. , Desjarlais John R. 
Published: February 2009

AbstractWe present direct evidence for a change in protein structural specificity due to hydrophobic core packing. High resolution structural analysis of a designed core variant of ubiquitin reveals that the protein is in slow exchange between two conformations. Examination of side‐chain rotamers indicates that this dynamic response and the lower stability of the protein are coupled to greater strain and mobility in the core. The results suggest that manipulating the level of side‐chain strain may be one way of fine tuning the stability and specificity of proteins.