bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of ligand effector binding sites in transmembrane regions of the human G protein‐coupled C3a receptor
Authors: Sun Jianzhong , Ember Julia A. , Chao Ta‐Hsiang , Hugli Tony E. , Fukuoka Yoshihiro , Ye Richard D. 
Published: February 2009

AbstractThe human C3a anaphylatoxin receptor (C3aR) is a G protein‐coupled receptor (GPCR) composed of seven transmem‐brane α‐helices connected by hydrophilic loops. Previous studies of chimeric C3aR/C5aR and loop deletions in C3aR demonstrated that the large extracellular loop2 plays an important role in noneffector ligand binding; however, the effector binding site for C3a has not been identified. In this study, selected charged residues in the transmembrane regions of C3aR were replaced by Ala using site‐directed mutagenesis, and mutant receptors were stably expressed in the RBL‐2H3 cell line. Ligand binding studies demonstrated that R161A (helix IV), R340A (helix V), and D417A (helix VII) showed no binding activity, although full expression of these receptors was established by flow cytometric analysis. C3a induced very weak intracellular calcium flux in cells expressing these three mutant receptors. H81A (helix II) and K96A (helix III) showed decreased ligand binding activity. The calcium flux induced by C3a in H81A and K96A cells was also consistently reduced. These findings suggest that the charged transmembrane residues Arg161, Arg340, and Asp417 in C3aR are essential for ligand effector binding and/or signal coupling, and that residues His81 and Lys96 may contribute less directly to the overall free energy of ligand binding. These transmembrane residues in C3aR identify specific molecular contacts for ligand interactions that account for C3a‐induced receptor activation.
Arginine 197 of the cholecystokinin‐A receptor binding site interacts with the sulfate of the peptide agonist cholecystokinin
Authors: Gigouj Véronique , Escrieut Chantal , Silvente‐Poirot Sandrine , Bouisson Michèle , Vaysse Nicole , Fourmy Daniel , Maigret Bernard , Fehrentz Jean‐Alain , Martinez Jean , Moroder Luis , Gully Danielle 
Published: February 2009

AbstractThe knowledge of the binding sites of G protein‐coupled cholecystokinin receptors represents important insights that may serve to understand their activation processes and to design or optimize ligands. Our aim was to identify the amino acid of the cholecystokinin‐A receptor (CCK‐AR) binding site in an interaction with the sulfate of CCK, which is crucial for CCK binding and activity. A three‐dimensional model of the [CCK‐AR‐CCK] complex was built. In this model, Arg197 was the best candidate residue for a ionic interaction with the sulfate of CCK. Arg197 was exchanged for a methionine by site‐directed mutagenesis. Wild‐type and mutated CCK‐AR were transiently expressed in COS‐7 cells for pharmacological and functional analysis. The mutated receptor on Arg197 did not bind the agonist radioligand 125I‐BH‐[Thr, Nle]‐CCK‐9; however, it bound the nonpeptide antagonist [3H]‐SR27,897 as the wild‐type receptor. The mutant was ≅1,470‐ and 3,200‐fold less potent than the wild‐type CCK‐AR to activate G proteins and to induce inositol phosphate production, respectively. This is consistent with the 500‐fold lower potency and 800‐fold lower affinity of nonsulfated CCK relative to sulfated CCK on the wild‐type receptor. These data, together with those showing that the mutated receptor failed to discriminate nonsulfated and sulfated CCK while it retained other pharmacological features of the CCK‐AR, strongly support an interaction between Arg 197 of the CCK‐AR binding site and the sulfate of CCK. In addition, the mutated CCK‐AR resembled the low affinity state of the wild‐type CCK‐AR, suggesting that Arg 197‐sulfate interaction regulates conformational changes of the CCK‐AR that are required for its physiological activation.
Mapping of ATP binding regions in poly (A) polymerases by photoaffinity labeling and by mutational analysis identifies a domain conserved in many nucleotidyltransferases
Authors: Jenö Paul , Keller Walter , Martin Georges 
Published: February 2009

AbstractWe have identified regions in poly(A) polymerases that interact with ATP. Conditions were established for efficient cross‐linking of recombinant bovine and yeast poly(A) polymerases to 8‐azido‐ATP. Mn2+ strongly stimulated this reaction due to a 50‐fold lower Ki for 8‐azido‐ATP in the presence of Mn2+. Mutations of the highly conserved Asp residues 113, 115, and 167, critical for metal binding in the catalytic domain of bovine poly(A) polymerase, led to a strong reduction of cross‐linking efficiency, and Mn2+ no longer stimulated the reaction. Sites of 8‐azido‐ATP crosslinking were mapped in different poly(A) polymerases by CNBr‐cleavage and analysis of tryptic peptides by mass spectroscopy. The main cross‐link in Schizosaccharomyces pombe poly (A) polymerase could be assigned to the peptide DLELSDNNLLK (amino acids 167‐177). Database searches with sequences surrounding the cross‐link site detected significant homologies to other nucleotidyltransferase families, suggesting a conservation of the nucleotide‐binding fold among these families of enzymes. Mutations in the region of the “helical turn motif” (a domain binding the triphosphate moiety of the nucleotide) and in the suspected nucleotide‐binding helix of bovine poly (A) polymerase impaired ATP binding and catalysis. The results indicate that ATP is bound in part by the helical turn motif and in part by a region that may be a structural analog to the fingers domain found in many polymerases.
The novel fluorescent CDP‐analogue (Pβ)MABA‐CDP is a specific probe for the NMP binding site of UMP/CMP kinase
Authors: Rudolph Markus G. , Veit THOMAS J.H. , Reinstein Jochen 
Published: February 2009

AbstractDirect thermodynamic and kinetic investigations of the binding of nucleotides to the nucleoside monophosphate (NMP) site of NMP kinases have not been possible so far because a spectroscopic probe was not available. By coupling a fluorescent N‐methylanthraniloyl‐ (mant) group to the β‐phosphate of CDP via a butyl linker, a CDP analogue [(Pβ)MABA‐CDP] was obtained that still binds specifically to the NMP site of UmpKdicty, because the base and the ribose moieties, which are involved in specific interactions, are not modified. This allows the direct determination of binding constants for its substrates in competition experiments.
Spectral contribution of the individual tryptophan of αB‐crystallin: A study by site‐directed mutagenesis
Authors: Liang Jack J‐N. , Sun Tian‐Xiao , Akhtar Nila J. 
Published: February 2009

AbstractThere are two tryptophan residues in the lens αB‐crystallin, Trp9 and Trp60. We prepared two Trp → Phe substituted mutants, W9F and W60F, for use in a spectroscopic study. The two tryptophan residues contribute to Trp fluorescence and near‐ultraviolet circular dichroism (UV CD) differently. The major difference in the near‐UV CD is the contribution of 1La of Trp: it is positive in W60F but becomes negative in W9F. Further analysis of the near‐UV CD shows an increased intensity in the region of 270–280 nm for W60F, suggesting that the Tyr48 is affected by the W60F mutation. It appears that Trp60 is located in a more rigid environment than Trp9, which agrees with a recent structural model in which Trp60 is in a β‐strand.
Synthesis, folding, and structure of the β‐turn mimic modified B1 domain of streptococcal protein G
Authors: Odaert Benoit , Jean Fabienne , Melnyk Oleg , Tartar Andre , Lippens Guy , Boutillon Christophe , Buisine Eric 
Published: February 2009

AbstractThe mechanism of β‐sheet formation remains a fundamental issue in our understanding of the protein folding process, but is hampered by the often encountered kinetic competition between folding and aggregation. The role of local versus nonlocal interactions has been probed traditionally by mutagenesis of both turn and strand residues. Recently, rigid organic molecules that impose a correct chain reversal have been introduced in several small peptides to isolate the importance of the long‐range interactions. Here, we present the incorporation of a well‐studied β‐turn mimic, designated as the dibenzofuran‐based (DBF) amino acid, in the B1 domain of streptococcal protein G (B1G), and compare our results with those obtained upon insertion of the same mimic into the N‐terminal β‐hairpin of B1G (O Melnyk et al., 1998, Lett Pept Sci 5:147–150). The DBF‐B1G domain conserves the structure and the functional and thermodynamical properties of the native protein, whereas the modified peptide does not adopt a native‐like conformation. The nature of the DBF flanking residues in the modified B1G domain prevents the β‐turn mimic from acting as a strong β‐sheet nucleator, which reinforces the idea that the native β‐hairpin formation is not driven by the β‐turn formation, but by tertiary interactions.
Mass spectrometric characterization of oat phytochrome A: Isoforms and posttranslational modifications
Authors: Lapko Veniamin N. , Jiang Xiang‐Yu , Smith David L. , Song Pill‐Soon 
Published: February 2009

AbstractAt least four mRNAs for oat phytochrome A (phyA) are present in etiolated oat tissue. The complete amino acid sequences of two phyA isoforms (A3 and A4) and the N‐terminal amino acid sequence of a third isoform (A5) were deduced from cDNA sequencing (Hershey et al., 1985). In the present study, heterogeneity of phyA on a protein level was studied by tryptic mapping using electrospray ionization mass‐spectrometry (ESIMS). The total tryptic digest of iodoacetamide‐modified phyA was fractionated by gel filtration chromatography followed by reversed‐phase high‐performance liquid chromatography. ESIMS was used to identify peptides. Amino acid sequences of the peptides were confirmed or determined by collision‐induced dissociation mass spectrometry (CID MS), MS/MS, or by subdigestion of the tryptic peptides followed by ESIMS analysis. More than 97% of the phyA3 sequence (1, 128 amino acid residues) was determined in the present study. Mass‐spectrometric analysis of peptides unique to each form showed that phyA purified from etiolated oat seedling is represented by three isoforms A5, A3, and A4, with ratio 3.4:2.3:1.0. Possible light‐induced changes in phytochrome in vivo phosphorylation site at Ser7 (Lapko VN et al., 1997, Biochemistry 36:10595–10599) as well at Ser17 and Ser598 (known as in vitro phosphorylation sites) were also analyzed. The extent of phosphorylation at Ser7 appears to be the same for phyA isolated from dark‐grown and red‐light illuminated seedlings. In addition to Ser7, Ser598 was identified as an in vivo phosphorylation site in oat phyA. Ser598 phosphorylation was found only in phyA from the red light‐treated seedlings, suggesting that the protein phosphorylation plays a functional role in the phytochrome A‐mediated light‐signal transduction.
Auracyanin a from the thermophilic green gliding photosynthetic bacterium chloroflexus aurantiacus represents an unusual class of small blue copper proteins
Authors: Driessche Gonzalez Van , Hu Wei , Werken Gerrit Van De , Selvaraj Fabiyola , Mcmanus James D. , Blankenship Robert E. , Van Beeumen Jozef J. 
Published: February 2009

AbstractThe amino acid sequence of the small copper protein auracyanin A isolated from the thermophilic photosynthetic green bacterium Chloroflexus aurantiacus has been determined to be a polypeptide of 139 residues. His58, Cys123, His128, and Met132 are spaced in a way to be expected if they are the evolutionary conserved metal ligands as in the known small copper proteins plastocyanin and azurin. Secondary structure prediction also indicates that auracyanin has a general β‐barrel structure similar to that of azurin from Pseudomonas aeruginosa and plastocyanin from poplar leaves. However, auracyanin appears to have sequence characteristics of both small copper protein sequence classes. The overall similarity with a consensus sequence of azurin is roughly the same as that with a consensus sequence of plastocyanin, namely 30.5%. We suggest that auracyanin A, together with the B forms, is the first example of a new class of small copper proteins that may be descendants of an ancestral sequence to both the azurin proteins occurring in prokaryotic nonphotosynthetic bacteria and the plastocyanin proteins occurring in both prokaryotic cyanobacteria and eukaryotic algae and plants. The N‐terminal sequence region 1–18 of auracyanin is remarkably rich in glycine and hydroxy amino acids, and required mass spectrometric analysis to be determined. The nature of the blocking group X is not yet known, although its mass has been determined to be 220 Da. The auracyanins are the first small blue copper proteins found and studied in anoxygenic photosynthetic bacteria and are likely to mediate electron transfer between the cytochrome bc1 complex and the photosynthetic reaction center.
Evidence for a copper‐coordinated histidine–tyrosine cross‐link in the active site of cytochrome oxidase
Authors: Buse Gerhard , Soulimane Tewfik , Dewor Manfred , Meyer Helmut E. , Blüggel Martin 
Published: February 2009

AbstractFollowing hints from X‐ray data (Ostermeier C et al., 1997, Proc Natl Acad Sci USA 94:10547–10553; Yoshikawa S et al., 1998, Science 280:1723–1729), chemical evidence is presented from four distantly related cytochrome‐c oxidases for the existence of a copperB‐coordinated His240–Tyr244) cross‐link at the O2‐activating Heme Fea3–CuB center in the catalytic subunit I of the enzyme. The early evolutionary invention of this unusual structure may have prevented demaging OH‐radical release at e–‐transfer to dioxygen and thus have enabled O2 respiration.
Study of the stability and unfolding mechanism of BBA1 by molecular dynamics simulations at different temperatures
Authors: Wang Lu , Duan Yong , Shortle Rebecca , Imperiali Barbara , Kollman Peter A. 
Published: February 2009

AbstractBBA1 is a designed protein that has only 23 residues. It is the smallest protein without disulfide bridges that has a well‐defined tertiary structure in solution. We have performed unfolding molecular dynamics simulations on BBA1 and some of its mutants at 300, 330, 360, and 400 K to study their kinetic stability as well as the unfolding mechanism of BBA1. It was shown that the unfolding simulations can provide insights into the forces that stabilize the protein. Packing, hydrophobic interactions, and a salt bridge between Asp12 and Lys16 were found to be important to the protein' stability. The unfolding of BBA1 goes through two major steps: (1) disruption of the hydrophobic core and (2) unfolding of the helix. The β‐hairpin remains stable in the unfolding because of the high stability of the type II' turn connecting the two β‐strands.
A novel recombinant single‐chain hepatitis C virus ns3‐ns4a protein with improved helicase activity
Authors: Howe ANITA Y.M. , Chase Robert , Taremi S. Shane , Risano Christine , Beyer Brian , Malcolm Bruce , Lau JOHNSON Y.N. 
Published: February 2009

AbstractHepatitis C virus (HCV) nonstructural protein 3 (NS3) has been shown to possess protease and helicase activities and has also been demonstrated to spontaneously associate with nonstructural protein NS4A (NS4A) to form a stable complex. Previous attempts to produce the NS3/NS4A complex in recombinant baculovirus resulted in a protein complex that aggregated and precipitated in the absence of nonionic detergent and high salt. A single‐chain form of the NS3/NS4A complex (His‐NS4A21–32‐GSGS‐NS33–631) was constructed in which the NS4A core peptide is fused to the N‐terminus of the NS3 protease domain as previously described (Taremi et al., 1998). This protein contains a histidine tagged NS4A peptide (a.a. 21–32) fused to the full‐length NS3 (a.a. 3–631) through a flexible tetra amino acid linker. The recombinant protein was expressed to high levels inEscherichia coli, purified to homogeneity, and examined for NTPase, nucleic acid unwinding, and proteolytic activities. The single‐chain recombinant NS3‐NS4A protein possesses physiological properties equivalent to those of the NS3/NS4A complex except that this novel construct is stable, soluble and sixfold to sevenfold more active in unwinding duplex RNA. Comparison of the helicase activity of the single‐chain recombinant NS3‐NS4A with that of the full‐length NS3 (without NS4A) and that of the helicase domain alone suggested that the presence of the protease domain and at least the NS4A core peptide are required for optimal unwinding activity.
Packed protein bilayers in the 0.90 å resolution structure of a designed alpha helical bundle
Authors: Privé Gilbert G. , Anderson Daniel H. , Wesson Laura , Cascio Duilio , Eisenberg David 
Published: February 2009

AbstractA 12‐residue peptide designed to form an α‐helix and self‐associate into an antiparallel 4‐α‐helical bundle yields a 0.9 Å crystal structure revealing unanticipated features. The structure was determined by direct phasing with the “Shake‐and‐Bake” program, and contains four crystallographically distinct 12‐mer peptide molecules plus solvent for a total of 479 atoms. The crystal is formed from nearly ideal α‐helices hydrogen bonded head‐to‐tail into columns, which in turn pack side‐by‐side into sheets spanning the width of the crystal. Within each sheet, the α‐helices run antiparallel and are closely spaced (9–10 Å center‐to‐center). The sheets are more loosely packed against each other (13–14 Å between helix centers). Each sheet is amphiphilic: apolar leucine side chains project from one face, charged lysine and glutamate side chains from the other face. The sheets are stacked with two polar faces opposing and two apolar faces opposing. The result is a periodic biomaterial composed of packed protein bilayers, with alternating polar and apolar interfaces. All of the 30 water molecules in the unit cell lie in the polar interface or between the stacked termini of helices. A section through the sheet reveals that the helices packed at the apolar interface resemble the four‐α‐helical bundle of the design, but the helices overhang parts of the adjacent bundles, and the helix crossing angles are less steep than intended (7–11° rather than 18°).
Analysis of the extent of unfolding of denatured insulin‐like growth factor
Authors: Chang Jui‐Yoa , WALTER MÄRKI  , Lai Por‐Hsiung 
Published: February 2009

AbstractInsulin‐like growth factor (IGF‐1) contains three disulfide bonds. In the presence of denaturant and thiol catalyst, IGF‐1 shuffles its native disulfide bonds and denatures to form a mixture of scrambled isomers. The composition of scrambled IGF varies under different denaturing conditions. Among the 14 possible scrambled IGF isomers, the yield of the beads‐form isomer is shown to be directly proportional to the strength of the denaturing condition. This paper demonstrates a new approach to quantify the extent of unfolding of the denatured protein.
Folding of barstar C40A/C82A/P27A and catalysis of the peptidyl‐prolylcis/transisomerization by human cytosolic cyclophilin (Cyp18)
Authors: Golbik Ralph , Fischer Gunter , Fersht Alan R. 
Published: February 2009

AbstractRefolding of b*C40A/C82A/P27A is comprised of several kinetically detectable folding phases. The slowest phase in refolding originates fromtrans → cisisomerization of the Tyr47–Pro48 peptide bond being incisconformation in the native state. This refolding phase can be accelerated by the peptidyl‐prolylcis/transisomerase human cytosolic cyclophilin (Cyp18) with akcat/KMof 254, 000 M–1s–1. The fast refolding phase is not influenced by the enzyme.
The disulfide‐coupled folding pathway of apamin as derived from diselenide‐quenched analogs and intermediates
Authors: Pegoraro Stefano , Fiori Stella , Cramer Jörg , Rudolph‐Böhner Sabine , Moroder Luis 
Published: February 2009

AbstractThe sequence of apamin, an 18 residue bee venom toxin, encloses all the information required for the correct disulfide‐coupled folding into the cystine‐stabilized α‐helical motif. Three apamin analogs, each containing a pair of selenocysteine residues replacing the related cysteines, were synthesized to mimic the three possible apamin isomers with two crossed, parallel, or consecutive disulfides, respectively. Refolding experiments clearly revealed that the redox potential of selenocysteine prevails over the sequence encoded structural information for proper folding of apamin. Thus, selenocysteine can be used as a new device to generate productive and nonproductive folding intermediates of peptides and proteins. In fact, disulfides are selectively reduced in presence of the diselenide and the conformational features derived from these intermediates as well as from the three‐dimensional (3D) structures of the selenocysteine‐containing analogs with their nonnatural networks of diselenide/disulfide bridges allowed to gain further insight into the subtle driving forces for the correct folding of apamin that mainly derive from local conformational preferences.
NMR study of Ni2+ binding to the H‐N‐H endonuclease domain of colicin E9
Authors: Hannan Jonathan P. , Whittaker Sara B.‐M. , Davy Sharon L. , Kühlmann Ulrike C. , Pommer Ansgar J. , Hemmings Andrew M. , James Richard , Kleanthous Colin , Moore Geoffrey R. 
Published: February 2009

AbstractNi2+ affinity columns are widely used for protein purification, but they carry the risk that Ni2+ ions may bind to the protein, either adventitiously or at a physiologically important site. Dialysis against ethylenediaminetetraacetic acid (EDTA) is normally used to remove metal ions bound adventitiously to proteins; however, this approach does not always work. Here we report that a bacterial endonuclease, the DNase domain of colicin E9, binds Ni2+ acquired from Ni2+ affinity columns, and appears to bind [Ni(EDTA)(H2O)n]2− at low ionic strength. NMR was used to detect the presence of both Ni2+ coordinated to amino acid side chains and [Ni(EDTA)(H2O)n]2−. Dialysis against ≥0.2 MNaCl was required to remove the [Ni(EDTA)(H2O)n]2−. The NMR procedure we have used to characterize the presence of Ni2+ and [Ni (EDTA)(H2O)n]2− should be applicable to other proteins where there is the possibility of binding paramagnetic metal ions that are present to expedite protein purification. In the present case, the binding of Ni2+ seems likely to be physiologically relevant, and the NMR data complement recent X‐ray crystallographic evidence concerning the number of histidine ligands to bound Ni2+.
Stable proline box motif at the N‐terminal end of α‐helices
Authors: Viguera A.R. , Serrano L. 
Published: February 2009

AbstractWe describe a novel N‐terminal α‐helix local motif that involves three hydrophobic residues and a Pro residue (Pro‐box motif). Database analysis shows that when Pro is the N‐cap of an α‐helix the distribution of amino acids in adjacent positions changes dramatically with respect to the average distribution in an α‐helix, but not when Pro is at position N1. N‐cap Pro residues are usually associated to Ile and Leu, at position N', Val at position N3 and a hydrophobic residue (h) at position N4. The side chain of the N‐cap Pro packs against Val, while the hydrophobic residues at positions N' and N4 make favorable interactions. To analyze the role of this putative motif (sequence fingerprint hPXXhh), we have synthesized a series of peptides and analyzed them by circular dichroism (CD) and NMR. We find that this motif is formed in peptides, and that the accompanying hydrophobic interactions contribute up to 1.2 kcal/mol to helix stability. The fact that some of the residues in this fingerprint are not good N‐cap and helix formers results in a small overall stabilization of the α‐helix with respect to other peptides having Gly as the N‐cap and Ala at N3 and N4. This suggests that the Pro‐box motif will not specially contribute to protein stability but to the specificity of its fold. In fact, 80% of the sequences that contain the fingerprint sequence in the protein database are adopting the described structural motif, and in none of them is the helix extended to place Pro at the more favorable N1 position.