bluuebunny/crossref_metadata_2025_split huggingface dataset:
The observation of chaperone‐ligand noncovalent complexes with electrospray ionization mass spectrometry
Authors: Bruce James E. , Liu Chuanliang , Smith Richard D. , Randall Linda L. , Smith Virginia F. 
Published: February 2009

AbstractFourier transform ion cyclotron resonance mass spectrometry (FTICR‐MS) was applied for the study of noncovalent chaperone SecB‐ligand complexes produced in solution and examined in the gas phase with the aid of electrospray ionization (ESI). Since chaperone proteins are believed to recognize and bind only with ligands with nonnative tertiary structure, this work required careful unfolding of the ligand and subsequent reaction with the intact chaperone (the noncovalent tetrameric protein, SecB). A high denaturant concentration was employed to produce nonnative structures of the OppA, and microdialysis of the resulting solutions containing the chaperone‐ligand complexes was carried out to rapidly remove the denaturant prior to analysis. Multistage mass spectrometry was essential to the successful study of these complexes since the initial mass spectra indicated extensive adduction that precluded mass measurements, even after microdialysis. However, low energy collisional activation of the ions in the FTICR trap proved useful for adduct removal, and careful control of excitation level preserved the intact complexes of interest, revealing a 1:1 SecB:OppA stoichiometry. To our knowledge, these results present the first direct observation of chaperone‐ligand noncovalent complexes and the highest molecular weight heterogeneous noncovalent complex observed to date by mass spectrometry. Furthermore, these results highlight the capabilities of FTICR for the study of such complex systems, and the development of a greater understanding of chaperone interactions in protein export.
Isolation and characterization of a DnaJ‐like protein in rats: The C‐terminal 10‐kDa domain of hsc70 is not essential for stimulating the ATP‐hydrolytic activity of hsc70 by a DnaJ‐like protein
Authors: Leng Chih‐Hsiang , Wang Chung , Brodsky Jeffrey L. 
Published: February 2009

AbstractA DnaJ‐like protein, RDJ1, was isolated from a rat brain cDNA library. The protein is predicted to have 397 amino acid residues and shares 99% identity to that of HDJ2, a human DnaJ‐like protein. RDJ1 was also shown to rescue the temperature‐sensitive lethality of a strain containing a mutated cytosolic DnaJ in yeast, ydj1‐151. Fragments containing the J‐domain of RDJl either with or without the G/F motif were expressed in Escherichia coli. The purified proteins stimulated the ATPase activity of hsc70 and of the 60‐kDa N‐terminal fragment of hsc70. These results imply that RDJl can interact with the N‐terminal 60‐kDa fragment of hsc70 to activate ATP hydrolysis by hsc70.
Calorimetric analyses of the interaction between SecB and its ligands
Authors: Randall Linda L. , Topping Traci B. , Suciu Dominic , Hardy Simon J. S. 
Published: February 2009

AbstractSecB is a chaperone in Escherichia coli dedicated to export of proteins from the cytoplasm to the periplasm and outer membrane. It functions to bind and deliver precursors of exported proteins to the translocation apparatus before they fold into their native structures, thus maintaining them in a competent state for translocation across the membrane. The natural ligands of SecB are precursor proteins containing leader sequences. There are numerous reports in the literature indicating that SecB does not specifically recognize the leader peptides. However, two published investigations have concluded that the leader peptide is the recognition element (Watanabe M, Blobel G. 1989. Cell 58:685‐705; Watanabe M, Blobel G. 1995. Proc Natl Acad Sci USA 92:10133‐10136). In this work we use titration calorimetry to show that SecB binds two physiological ligands, which contain leader sequences, with no higher affinity than the same molecules lacking their leader sequences. Indeed, for one ligand the presence of the leader sequence reduces the affinity. Therefore, it can be concluded that the leader sequence provides no positive contribution to the binding energy.
What should the Z‐score of native protein structures be?
Authors: Zhang Li , Skolnick Jeffrey 
Published: February 2009

AbstractThe Z‐score of a protein is defined as the energy separation between the native fold and the average of an ensemble of misfolds in the units of the standard deviation of the ensemble. The Z‐score is often used as a way of testing the knowledge‐based potentials for their ability to recognize the native fold from other alternatives. However, it is not known what range of values the Z‐scores should have if one had a correct potential. Here, we offer an estimate of Z‐scores extracted from calorimetric measurements of proteins. The energies obtained from these experimental data are compared with those from computer simulations of a lattice model protein. It is suggested that the Z‐scores calculated from different knowledge‐based potentials are generally too small in comparison with the experimental values.
The role of tyrosine 121 in cofactor binding of 5‐aminolevulinate synthase
Authors: Tan Dongwei , Barber Michael J. , Ferreira Gloria C. 
Published: February 2009

Abstract5‐Aminolevulinate synthase (EC 2.3.1.37) is the first enzyme in the heme biosynthesis in nonplant eukaryotes and some prokaryotes. It functions as a homodimer and requires pyridoxal 5′‐phosphate as an essential cofactor. Tyr‐121 is a conserved residue in all known sequences of 5‐aminolevulinate synthases. Further, it corresponds to Tyr‐70 of Escherichia coli aspartate aminotransferase, which has been shown to interact with the cofactor and prevent the dissociation of the cofactor from the enzyme. To test whether Tyr‐121 is involved in cofactor binding in murine erythroid 5‐aminolevulinate synthase, Tyr‐121 of murine erythroid 5‐aminolevulinate synthase was substituted by Phe and His using site‐directed mutagenesis. The Y121F mutant retained 36% of the wild‐type activity and the Km value for substrate glycine increased 34‐fold, while the activity of the Y121H mutant decreased to 5% of the wild‐type activity and the Km value for glycine increased fivefold. The pKal values in the pH‐activity profiles of the wild‐type and mutant enzymes were 6.41, 6.54, and 6.65 for wild‐type, Y121F, and Y121H, respectively. The UV‐visible and CD spectra of Y121F and Y121H mutants were similar to those of the wild‐type with the exception of an absorption maximum shift (420 ∼ 395 nm) for the Y121F mutant in the visible spectrum region, suggesting that the cofactor binds the Y121F mutant enzyme in a more unrestrained manner. Y121F and Y121H mutant enzymes also exhibited lower affinity than the wild‐type for the cofactor, reflected in the Kd values for pyridoxal 5′‐phosphate (26.5, 6.75, and 1.78 μ. for Y121F, Y121H, and the wild‐type, respectively). Further, Y121F and Y121H proved less thermostable than the wild type. Taken together, these findings indicate that Tyr‐121 plays a critical role in cofactor binding of murine erythroid 5‐aminolevulinate synthase.
Mutational analysis of the active site of indoleglycerol phosphate synthase fromEscherichia coli
Authors: Darimont Beatrice , Stehlin Catherine , Szadkoski Halina , Kirscner Kasper 
Published: February 2009

AbstractIndoleglycerol phosphate synthase catalyzes the ring closure of 1‐(2‐carboxyphenylamino)‐1‐deoxyribulose 5′‐phosphate to indoleglycerol phosphate, the fifth step in the pathway of tryptophan biosynthesis from chorismate. Because chemical synthesis of indole derivatives from arylamino ketones requires drastic solvent conditions, it is interesting by what mechanism the enzyme catalyzes the same condensation reaction. Seven invariant polar residues in the active site of the enzyme fromEscherichia colihave been mutated directly or randomly, to identify the catalytically essential ones. A strain ofE. colisuitable for selecting and classifying active mutants by functional complementation was constructed by precise deletion of thetrpC gene from the genome. Judged by growth rates of transformants on selective media, mutants with either S58 or S60 replaced by alanine were indistinguishable from the wild‐type, but R186 replaced by alanine was still partially active. Saturation random mutagenesis of individual codons showed that E53 was partially replaceable by aspartate and cysteine, whereas K114, E163, and N184 could not be replaced by any other residue. Partially active mutant proteins were purified and their steady‐state kinetic and inhibitor binding constants determined. Their relative catalytic efficiencies paralleled their relative complementation efficiencies. These results are compatible with the location of the essential residues in the active site of the enzyme and support a chemically plausible catalytic mechanism. It involves two enzyme‐bound intermediates and general acid‐base catalysis by K114 and E163 with the support of E53 and N184.
Characterization of Escherichia coli thioredoxin variants mimicking the active‐sites of other thiol/disulfide oxidoreductases
Authors: Mössner Ekkehard , Huber‐Wunderlich Martina , Glockshuber Rudi 
Published: February 2009

AbstractThiol/disulfide oxidoreductases like thioredoxin, glutaredoxin, DsbA, or protein disulfide isomerase (PDI) share the thioredoxin fold and a catalytic disulfide bond with the sequence Cys‐Xaa‐Xaa‐Cys (Xaa corresponds to any amino acid). Despite their structural similarities, the enzymes have very different redox properties, which is reflected by a 100,000‐fold difference in the equilibrium constant (Keq) with glutathione between the most oxidizing member, DsbA, and the most reducing member, thioredoxin. Here we present a systematic study on a series of variants of thioredoxin from Escherichia coli, in which the Xaa‐Xaa dipeptide was exchanged by that of glutaredoxin, PDI, and DsbA. Like the corresponding natural enzymes, all thioredoxin variants proved to be stronger oxidants than the wild‐type, with the order wild‐type < PDI‐type < DsbA‐type < glutaredoxin‐type. The most oxidizing, glutaredoxin‐like variant has a 420‐fold decreased value of Keq, corresponding to an increase in redox potential by 75 mV. While oxidized wild‐type thioredoxin is more stable than the reduced form (ΔΔGox/red = 16.9 kJ/mol), both redox forms have almost the same stability in the variants. The pH‐dependence of the reactivity with the alkylating agent iodoacetamide proved to be the best method to determine the pKa value of thioredoxin's nucleophilic active‐site thiol (Cys32). A pKa of 7.1 was measured for Cys32 in the reduced wild‐type. All variants showed a lowered pKa of Cys32, with the lowest value of 5.9 for the glutaredoxin‐like variant. A correlation of redox potential and the Cys32 pKa value could be established on a quantitative level. However, the predicted correlation between the measured ΔΔGox/red values and Cys32 pKa values was only qualitative.
The crystal structure of lactococcus lactis dihydroorotate dehydrogenase A complexed with the enzyme reaction product throws light on its enzymatic function
Authors: Rowland Paul , Larsen Sine , Björnberg Olof , Nielsen Finn S. , Jensen Kaj Frank 
Published: February 2009

AbstractDihydroorotate dehydrogenases (DHODs) catalyze the oxidation of (S)‐dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. A description is given of the crystal structure of Lactococcus lactis dihydroorotate dehydrogenase A (DHODA) complexed with the product of the enzyme reaction orotate. The structure of the complex to 2.0 Å resolution has been compared with the structure of the native enzyme. The active site of DHODA is known to contain a water filled cavity buried beneath a highly conserved and flexible loop. In the complex the orotate displaces the water molecules from the active site and stacks above the DHODA flavin isoalloxazine ring, causing only small movements of the surrounding protein residues. The orotate is completely buried beneath the protein surface, and the orotate binding causes a significant reduction in the mobility of the active site loop. The orotate is bound by four conserved asparagine side chains (Asn 67, Asn 127, Asn 132, and Asn 193), the side chains of Lys 43 and Ser 194, and the main chain NH groups of Met 69, Gly 70, and Leu 71. Of these the Lys 43 side chain makes hydrogen bonds to both the flavin isoalloxazine ring and the carboxylate group of the orotate. Potential interactions with bound dihydroorotate are considered using the orotate complex as a basis for molecular modeling. The role of Cys 130 as the active site base is discussed, and the sequence conservation of the active site residues across the different families of DHODs is reviewed, along with implications for differences in substrate binding and in the catalytic mechanisms between these families.
Circular permutation of βB2‐crystallin changes the hierarchy of domain assembly
Authors: Wright G. , Basak A.K. , Wieligmann K. , Slingsby C. , Mayr E.‐M. 
Published: February 2009

AbstractThe βγ‐crystallins form a superfamily of eye lens proteins comprised of multiple Greek motifs that are symmetrically organized into domains and higher assemblies. In the βB2‐crystallin dimer each polypeptide folds into two similar domains that are related to monomeric γ‐crystallin by domain swapping. The crystal structure of the circularly permuted two‐domain βB2 polypeptide shows that permutation converts intermolecular domain pairing into intramolecular pairing. However, the dimeric permuted protein is, in fact, half a native tetramer. This result shows how the sequential order of domains in multi‐domain proteins can affect quaternary domain assembly.
Crystal structure of human cathepsin S
Authors: Mcgrath Mary E. , Palmer James T. , Brömme Ddeter , Somoza John R. 
Published: February 2009

AbstractWe have determined the 2.5 Å structure (Rcryst= 20.5%, Rfree = 28.5%) of a complex between human cathepsin S and the potent, irreversible inhibitor 4‐morpholinecarbonyl‐Phe‐hPhe‐vinyl sulfone‐phenyl. Noncrystallographic symmetry averaging and other density modification techniques were used to improve electron density maps which were nonoptimal due to systematically incomplete data. Methods that reduce the number of parameters were implemented for refinement. The refined structure shows cathepsin S to be similar to related cysteine proteases such as papain and cathepsins K and L. As expected, the covalently‐bound inhibitor is attached to the enzyme at Cys 25, and enzyme binding subsites S3‐S1' are occupied by the respective inhibitor substituents. A somewhat larger S2 pocket than what is found in similar enzymes is consistent with the broader specificity of cathepsin S at this site, while Lys 61 in the S3 site may offer opportunities for selective inhibition of this enzyme. The presence of Arg 137 in the S1' pocket, and proximal to Cys 25 may have implications not only for substrate specificity C‐terminal to the scissile bond, but also for catalysis.
Crystal structure of apo‐glycine N‐methyltransferase (GNMT)
Authors: Pattanayek R. , Newcomer M.E. , Wagner C. 
Published: February 2009

AbstractThe crystal structure of the recombinant apo‐form of glycine N‐methyltransferase (GNMT) has been determined at 2.5 Å resolution. GNMT is a tetrameric enzyme (monomer Mr = 32,423Da, 292 amino acids) that catalyzes the transfer of a methyl group from S‐adenosylmethionine (AdoMet) to glycine with the formation of S‐adenosylmethionine (AdoHcy) and sarcosine (N‐methylglycine). GNMT is a regulatory enzyme, which is inhibited by 5‐methyltetrahydrofolate pentaglutamate and believed to control the ratio of AdoMet to AdoHcy in tissues. The crystals belong to the orthorhombic space group P21212 (a = 85.39, b = 174.21, c = 44.71 Å) and contain one dimer per asymmetric unit. The AdoMet‐GNMT structure served as the starting model. The structure was refined to an R‐factor of 21.9%. Each monomer is a three‐domain structure with a large cavity enclosed by the three domains. The tetramer resembles a Square with a central Channel about which N‐terminal domains are intertwined. Only localized changes of the residues involved in the binding pocket are observed for the apo‐GNMT structure when compared to that determined in the presence of Substrate and Substrate analog.
The three‐dimensional structure of a helix‐less variant of intestinal fatty acid‐binding protein
Authors: Steele Ruth A. , Emmert Daniel A. , Kao Jeff , Hodson Michaele E. , Frieden Carl , Cistola David P. 
Published: February 2009

AbstractIntestinal fatty acid‐binding protein (I‐FABP) is a cytosolic 15.1‐kDa protein that appears to function in the intracellular transport and metabolic trafficking of fatty acids. It binds a Single molecule of long‐chain fatty acid in an enclosed cavity surrounded by two five‐stranded antiparallel β‐sheets and a helix‐turn‐helix domain. To investigate the role of the helical domain, we engineered a variant of I‐FABP by deleting 17 contiguous residues and inserting a Ser‐Gly linker (Kim K et al., 1996, Biochemistry 35:7553‐7558). This variant, termed Δ17‐SG, was remarkably stable, exhibited a high β‐sheet content and was able to bind fatty acids with some features characteristic of the wild‐type protein. In the present study, we determined the structure of the Δ17‐SG/palmitate complex at atomic resolution using triple‐resonance 3D NMR methods. Sequence‐specific 1H, 13C, and 15N resonance assignments were established at pH 7.2 and 25 °C and used to define the consensus 1H/13C chemical shift‐derived secondary structure. Subsequently, an iterative protocol was used to identify 2,544 NOE‐derived interproton distance restraints and to calculate its tertiary structure using a unique distance geometry/simulated annealing algorithm. In spite of the sizable deletion, the Δ17‐SG structure exhibits a backbone conformation that is nearly superimposable with the β‐sheet domain of the wild‐type protein. The selective deletion of the α‐helical domain creates a very large opening that connects the interior ligand‐binding cavity with exterior solvent. Unlike wild‐type I‐FABP, fatty acid dissociation from Δ17‐SG is structurally and kinetically un‐impeded, and a protein conformational transition is not required. The Δ17‐SG variant of I‐FABP is the only wild‐type or engineered member of the intracellular lipid‐binding protein family whose structure lacks α‐helices. Thus, Δ17‐SG I‐FABP constitutes a unique model System for investigating the role of the helical domain in ligand‐protein recognition, protein stability and folding, lipid transfer mechanisms, and cellular function.
Comparative analysis of the noncollagenous NC1 domain of type IV collagen: Identification of structural features important for assembly, function, and pathogenesis
Authors: Netzer Kai‐Olaf , Suzuki Ko , Itoh Yoshifumi , Hudson Bolly G. , Khalifah Raja G. 
Published: February 2009

AbstractType IV collagen α1‐α6 chains have important roles in the assembly of basement membranes and are implicated in the pathogenesis of Goodpasture Syndrome, an autoimmune disorder, and Alport Syndrome, a hereditary renal disease. We report comparative sequence analyses and structural predictions of the noncollagenous C‐terminal globular NC1 domain (28 sequences). The inferred tree verified that type IV collagen sequences fall into two groups, α‐1‐like and α2‐like, and suggested that vertebrate α3/α4 sequences evolved before α1/α2 and α5/α6. About one fifth of NC1 residues were identified to confer either the α1 or α2 group‐specificity. These residues accumulate opposite Charge in subdomain B of α1 (positive) and α2 (negative) sequences and may play a role in the stoichiometric chain selection upon type IV collagen assembly. Neural network secondary structure prediction on multiple aligned sequences revealed a subdomain core structure consisting of six hydrophobic β‐strands and one short α‐helix with a significant hydrophobic moment. The existence of opposite charges in the a‐helices may carry implications for intersubdomain interactions. The results provide a rationale for defining the epitope that binds Goodpasture autoantibodies and a framework for understanding how certain NC1 mutations may lead to Alport Syndrome. A search algorithm, based entirely on amino acid properties, yielded a possible similarity of NC1 to tissue inhibitor of metalloproteinases (TIMP) and prompted an investigation of a possible functional relationship. The results indicate that NC1 preparations decrease the activity of matrix metalloproteinases 2 and 3 (MMP‐2, MMP‐3) toward a peptide Substrate, though not to [14C]‐gelatin. We suggest that an ancestral NC1 may have been incorporated into type IV collagen as an evolutionarily mobile domain carrying Proteinase inhibitor function.
Deciphering the structural code for proteins: Helical propensities in domain classes and statistical multiresidue information in α‐helices
Authors: Negrete Josée A. , Viñuales Yolanda , Palau Jaume 
Published: February 2009

AbstractWe made several statistical analyses in a large sample of nearly 4,000 helices (from 546 redundancy‐controlled PDB protein subunits), which give new insights into the helical properties of globular proteins. In a first experiment, the amino acid composition of the whole sample was compared with the composition of two helical sample subgroups (the “mainly‐α” and the “(α/β)8 barrel” domain classes); we reached the conclusion that composition‐based helical propensities for secondary structure prediction do not depend on the structural class.Running a five‐residue window through the whole sample, the positional composition revealed that positive and negative residues are located throughout the helices and tend to neutralize the macrodipole effect. On this basis, we analyzed charged triplets using a running five‐residue window. The conclusion was that only mixed charged residues [positive (+) and negative (‐)] located at positions 1‐2‐5 and 1‐4‐5 are clearly favored. In these locations the most abundant are (–.+) and (‐.++), and this shows the existence of side chain microdipoles, which neutralize the large macrodipole of the helix.We made a systematic statistical analysis of charged, dipolar, and hydrophobic + aromatic residues, which enabled us to work out rules that should be useful for modeling and design purposes.Finally, we analyzed the relative abundance of all the different amphipathic double‐arcs that are present in helices formed by octapeptides (8) and nonapeptides (18). All of the double‐arcs that make up Schiffer and Edmundson's classical helical wheel are found in abundance in the sample.