bluuebunny/crossref_metadata_2025_split huggingface dataset:
Study of a noncovalent trp repressor: DNA operator complex by electrospray ionization time‐of‐flight mass spectrometry
Authors: Potter Noelle , Chernushevich Igor , Ens Werner , Standing Kenneth G. , Donald Lynda J. , Ayed Ayeda , Duckworth Harry W. , Arrowsmith Cheryl H. 
Published: February 2009

AbstractElectrospray ionization time‐of‐flight mass spectrometry (ESI‐TOF MS) has been used to study noncovalent interactions between the trp apo‐repressor (TrpR), its co‐repressor tryptophan and its specific operator DNA. In 5 mM ammonium acetate, TrpR was detected as a partially unfolded monomer. In the presence of a 21‐base‐pair DNA possessing the two symmetrically arranged CTAG consensus sequences required for specific TrpR binding, a homodimer‐dsDNA complex with a 1:1 stoichiometry was observed. Co‐repressor was not needed for the complex to form under our experimental conditions. Collision induced dissociation (CID‐MS) revealed that this complex was very stable in the gas phase since dissociation was achieved only at energies that also broke covalent bonds. We saw no evidence for the presence of the six water molecules that mediate the interaction between the protein and the DNA in the crystal structure. To check the binding specificity of the TrpR for its target DNA, a competitive experiment was undertaken: the protein was mixed with an equimolar amount of three different DNAs in which the two CTAG sequences were separated by 2, 4, and 6 bp, respectively. Only the DNA with the correct consensus spacing of 4 bp was able to form stable interactions with TrpR. This experiment demonstrates the potential of ESI‐MS to test the sequence‐specificity of protein‐DNA complexes. The interactions between the TrpR‐DNA complex and 5‐methyl‐, L‐ and D‐tryptophan were also investigated. Two molecules of 5‐methyl‐ or L‐tryptophan were bound with high affinity to the TrpR‐DNA complex. On the other hand, D‐tryptophan appeared to bind to the complex with poor specificity and poor affinity.
Formation and properties of mixed disulfides between thioredoxin reductase from Escherichia coli and thioredoxin: Evidence that cysteine‐138 functions to initiate dithiol‐disulfide interchange and to accept the reducing equivalent from reduced flavin
Authors: Veine Donna M. , Mulrooney Scott B. , Wang Pan‐Fen , Williams Charles H. 
Published: February 2009

AbstractMutation of one of the cysteine residues in the redox active disulfide of thioredoxin reductase from Escherichia coli results in C135S with Cys138 remaining or C138S with Cys135 remaining. The expression system for the genes encoding thioredoxin reductase, wild‐type enzyme, C135S, and C138S has been re‐engineered to allow for greater yields of protein. Wild‐type enzyme and C135S were found to be as previously reported, whereas discrepancies were detected in the characteristics of C138S. It was shown that the original C138S was a heterogeneous mixture containing C138S and wild‐type enzyme and that enzyme obtained from the new expression system is the correct species. C138S obtained from the new expression system having 0.1% activity and 7% flavin fluorescence of wild‐type enzyme was used in this study. Reductive titrations show that, as expected, only 1 mol of sodium dithionite/mol of FAD is required to reduce C138S. The remaining thiol in C135S and C138S has been reacted with 5,5′‐dithiobis‐(2‐nitrobenzoic acid) to form mixed disulfides. The half time of the reaction was <5 s for Cys138 in C135S and approximately 300 s for Cys135 in C138S showing that Cys138 is much more reactive. The resulting mixed disulfides have been reacted with Cys32 in C35S mutant thioredoxin to form stable, covalent adducts C138S‐C35S and C135S‐C35S. The half times show that Cys138 is approximately fourfold more susceptible to attack by the nucleophile. These results suggest that Cysl38 may be the thiol initiating dithiol‐disulfide interchange between thioredoxin reductase and thioredoxin.
Crystal structures of the psychrophilic α‐amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor
Authors: Aghajari Nushin , Haser Richard , Feller Georges , Gerday Charles 
Published: February 2009

AbstractOn page 565 in Volume 7, Number 3, 1998, under Molecular fold and calcium binding site, the domain numbering was wrongly reported. The correct numbering is domain A: residues 1 to 86 and 147 to 356, and domain B: residues 87 to 146. Domain C was correctly cited and remains as reported.
Determination of the amino acid requirements for a protein hinge in triosephosphate isomerase
Authors: Sun Jeonghoon , Sampson Nicole S. 
Published: February 2009

AbstractWe have determined the sequence requirements for a protein hinge in triosephosphate isomerase. The codons encoding the hinge at the C‐terminus of the active‐site lid of triosephosphate isomerase were replaced with a genetic library of all possible 8, 000 amino acid combinations. The most active of these 8, 000 mutants were selected using in vivo complementation of a triosephosphate isomerase deficient strain ofE. coli, DF502. Approximately 3% of the mutants complement DF502 with an activity that is above 70% of wild‐type activity. The sequences of these hinge mutants reveal that the solutions to the hinge flexibility problem are varied. Moreover, these preferences are sequence dependent; that is, certain pairs occur frequently. They fall into six families of similar sequences. In addition to the hinge sequences expected on the basis of phylogenetic analysis, we selected three new families of 3‐amino‐acid hinges: X(A/S)(L/K/ M), X(aromatic/β‐branched)(L/K), and XP(S/N). The absence of these hinge families in the more than 60 known species of triosephosphate isomerase suggests that during evolution, not all of sequence space is sampled, perhaps because there is no neutral mutation pathway to access the other families.
A conformational equilibrium in a protein fragment caused by two consecutive capping boxes: 1H‐, 13C‐NMR, and mutational analysis
Authors: Guerois Raphaël , Cordier‐Ochsenbein Françoise , Neumann Jean‐Michel , Sanson Alain , Baleux Françoise , Huynh‐Dinh Tam 
Published: February 2009

AbstractThe conformational properties of an 18 residues peptide spanning the entire sequence, L1KTPA5QFDAD10ELRAA15MKG, of the first helix (A‐helix) of domain 2 of annexin I, were thoroughly investigated. This fragment exhibits several singular features, and in particular, two successive potential capping boxes, T3XXQ6 and D8XXE11. The former corresponds to the native hydrogen bond network stabilizing the α helix N‐terminus in the protein; the latter is a non‐native capping box able to break the helix at residue D8, and is observed in the domain 2 partially folded state. Using 2D‐NMR techniques, we showed that two main populations of conformers coexist in aqueous solution. The first corresponds to a single helix extending from T3 to K17. The second corresponds to a broken helix at residue D8. Four mutants, T3A, F7A, D8A, and E11A, were designed to further analyze the role of key amino acids in the equilibrium between the two ensembles of conformers. The sensitivity of NMR parameters to account for the variations in the populations of conformers was evaluated for each peptide. Our data show the δ13Cα chemical shift to be the most relevant parameter. We used it to estimate the population ratio in the various peptides between the two main ensembles of conformers, the full helix and the broken helix. For the WT, E11A, and F7A peptides, these ratios are respectively 35/65, 60/40, 60/40. Our results were compared to the data obtained from helix/coil transition algorithms.
About the pKa of the active‐site histidine in flavocytochrome b2 (yeast L‐lactate dehydrogenase)
Authors: Rao K. Sudhindra , Lederer Florence 
Published: February 2009

AbstractFlavocytochrome b2 or L‐lactate dehydrogenase from yeasts catalyzes the oxidation of L‐lactate at the expense of monoelectronic acceptors such as cytochrome c, its physiological partner. When incubated in the presence of both L‐lactate and a keto acid, the enzyme catalyzes a transhydrogenation reaction wherein only the flavin is involved. During this reaction, the substrate α‐hydrogen is transferred not only to the solvent but also in part to the keto acid, which acts as reverse substrate. Thus, when bound to the reduced enzyme, this hydrogen is sticky. In the context of a carbanion mechanism, it resides on Nϵ of His373, the active site base. We have shown before that a correlation between the amount of intermolecular hydrogen transfer from [2‐3H] lactate and the keto acid reverse substrate concentration enables the determination of the first‐order rate constant, kHe for exchange of the substrate‐derived protein‐bound hydrogen with bulk solvent (Urban P, Lederer F, 1985, J Biol Chem 260:11115‐11122). In this work, we show that the exchange with the solvent appears to be independent of the phosphate buffer concentration in the range from 40 to 500 mM. It is thus probable that exchange occurs directly with water molecules. The second‐order rate constant for exchange is then 0.16 (±0.03) M−1 s−1. Using the Eigen equation, this figure yields a pKa of 9.1 ± 0.1 for His373 in the reduced enzyme, compared to a probable value of 6.0 or less in the oxidized enzyme (Suzuki H, Ogura YC, 1970, J Biochem 67:291‐295). The mechanistic significance of these results is discussed.
A biomimetic strategy in the synthesis and fragmentation of cyclic protein
Authors: Tam James P. , Lu Yi‐An 
Published: February 2009

AbstractThis paper describes a simple biomimetic strategy to prepare small cyclic proteins containing multiple disulfide bonds. Our strategy involves intramolecular acyl transfer reactions to assist both the synthesis and fragmentation of these highly constrained cyclic structures in aqueous solution. To illustrate our strategy, we synthesized the naturally occurring circulin B and cyclopsychotride (CPT), both consisting of 31 amino acid residues tightly packed in a cystine‐knot motif with three disulfide bonds and an end‐to‐end cyclic form. The synthesis of these small cyclic proteins can be achieved by orthogonal ligation of free peptide thioester via the thia zip reaction, which involves a series of reversible thiol‐thiolactone exchanges to arrive at an α‐amino thiolactone, which then undergoes an irreversible, spontaneous ring contraction through an S, N‐acyl migration to form the cyclic protein. A two‐step disulfide formation strategy is employed for obtaining the desired disulfide‐paired products. Partial acid hydrolysis through intramolecular acyl transfer of X‐Ser, X‐Thr, Asp‐X, and Glu‐X sequences is used to obtain the assignment of the circulins disulfide bond connectives. Both synthetic circulin B and CPT are identical to the natural products and, thus, the total synthesis confirms the disulfide connectivity of circulin B and CPT contain a cystine‐knot motif of 1‐4, 2‐5, and 3‐6. In general, our strategy, based on the convergence of chemical proteolysis and aminolysis of peptide bonds through acyl transfer, is biomimetic and provides a useful approach for the synthesis and characterization of large end‐to‐end cyclic peptides and small proteins.
Porin mutants with new channel properties
Authors: Schmid Benedikt , Maveyraud Laurent , Krömer Markus , Schulz Georg E. 
Published: February 2009

AbstractThe general diffusion porin from Rhodopseudomonas blastica was produced in large amounts in Escherichia coli inclusion bodies and (re)natured to the exact native structure. Here, we report on 13 mutants at the pore eyelet giving rise to new diffusion properties as measured in planar lipid bilayer experiments. The crystal structures of seven of these mutants were established. The effects of charge‐modifying mutations at the pore eyelet are consistent with the known selectivity for cations. Deletions of 16 and 27 residues of the constriction loop L3 resulted in labile trimers and pores. The reduction of the eyelet cross section by introducing tryptophans gave rise to a closely correlated decrease of the conductivities. A mutant with six newly introduced tryptophans in the eyelet closed its pore in a defined manner within seconds under a voltage of 20 mV, suggesting the existence of two states. The results indicate that the pore can be engineered in a rational manner.
Structure and stability of the P93G variant of ribonuclease A
Authors: Schultz L. Wayne , Hargraves Steven R. , Klink Tony A. , Raines Ronald T. 
Published: February 2009

AbstractThe peptide bonds preceding Pro 93 and Pro 114 of bovine pancreatic ribonuclease A (RNase A) are in the cis conformation. The trans‐to‐cis isomerization of these bonds had been indicted as the slow step during protein folding. Here, site‐directed mutagenesis was used to replace Pro 93 or Pro 114 with a glycine residue, and the crystalline structure of the P93G variant was determined by X‐ray diffraction analysis to a resolution of 1.7 Å. This structure is essentially identical to that of the wild‐type protein, except for the 91‐94 β‐turn containing the substitution. In the wild‐type protein, the β‐turn is of type V1a. In the P93G variant, this turn is of type II with the peptide bond preceding Gly 93 being trans. The thermal stabilities of the P93G and P1 14G variants were assessed by differential scanning calorimetry and thermal denaturation experiments monitored by ultraviolet spectroscopy. The value of ΔΔGm, which reports on the stability lost in the variants, is 1.5‐fold greater for the P114G variant than for the P93G variant. The greater stability of the P93G variant is likely due to the relatively facile accommodation of residues 91‐94 in a type II turn, which has a preference for a glycine residue in its i + 2 position.
The discoidin domain family revisited: New members from prokaryotes and a homology‐based fold prediction
Authors: Baumgartner Stefan , Hofmann Kay , Bucher Philipp , Chiquet‐Ehrismann Ruth 
Published: February 2009

AbstractMembers of the discoidin (DS) domain family, which includes the C1 and C2 repeats of blood coagulation factors V and VIII, occur in a great variety of eukaryotic proteins, most of which have been implicated in cell‐adhesion or developmental processes. So far, no three‐dimensional structure of a known example of this extracellular module has been determined, limiting the usefulness of identifying a new sequence as member of this family. Here, we present results of a recent search of the protein sequence database for new DS domains using generalized profiles, a sensitive multiple alignment‐based search technique. Several previously unrecognized DS domains could be identified by this method, including the first examples from prokaryotic species. More importantly, we present statistical, structural, and functional evidence that the Dl domain of galactose oxidase whose three‐dimensional structure has been determined at 1.7 Å resolution, is a distant member of this family. Taken together, these findings significantly expand the concept of the DS domain, by extending its taxonomic range and by implying a fold prediction for all its members. The proposed alignment with the galactose oxidase sequence makes it possible to construct homology‐based three‐dimensional models for the most interesting examples, as illustrated by an accompanying paper on the C1 and C2 domains of factor V.
Identification of a frizzled‐like cysteine rich domain in the extracellular region of developmental receptor tyrosine kinases
Authors: Saldanha Jose , Singh Juswinder , Mahadevan Daruka 
Published: February 2009

AbstractIn Drosophila, members of the Frizzled family of tissue‐polarity genes encode proteins that appear to function as cell‐surface receptors for Wnts. The Frizzled genes belong to the seven transmembrane class of receptors (7TMR) and have on their extracellular region a cysteine‐rich domain that has been implicated as the Wnt binding domain. This region has a characteristic spacing of ten cysteines, which has also been identified in FrzB (a secreted antagonist of Wnt signaling) and Smoothened (another 7TMR, which is involved in suppression of the hedgehog pathway). We have identified, using BLAST, sequence similarity between the cysteine‐rich domain of Frizzled and several receptor tyrosine kinases, which have roles in development. These include the muscle‐specific receptor tyrosine kinase (MuSK), the neuronal specific kinase (NSK2), and RORl and ROR2. At present, the ligands for these developmental tyrosine kinases are unknown. Our results suggest that Wnt‐like ligands may bind to these developmental tyrosine kinases
An internal affinity‐tag for purification and crystallization of the siderophore receptor fhua, integral outer membrane protein from escherichia coli K‐12
Authors: Ferguson Andrew D. , Coulton James W. , Breed Jason , Diederichs Kay , Welte Wolfram 
Published: February 2009

AbstractFhuA (Mr 78, 992, 714 amino acids), siderophore receptor for ferrichrome‐iron in the outer membrane of Escherichia coli, was affinity tagged, rapidly purified, and crystallized. To obtain FhuA in quanties sufficient for crystallization, a hexahisti‐dine tag was genetically inserted into the fhuA gene after amino acid 40.5, which resides in a known surface‐exposed loop. Recom‐binant FhuA40.5.H6 was overexpressed in an E. coli strain that is devoid of several major porins and using metal‐chelate chroma‐tography was purified in large amounts to homogeneity. FhuA crystals were grown using the hanging drop vapor diffusion technique and were suitable for X‐ray diffraction analysis. On a rotating anode X‐ray source, diffraction was observed to 3.0 Å resolution. The crystals belong to space group P61 or P65 with unit cell dimensions of a = b = 174 Å, c = 88 Å (α = β = 90°,γ = 120°).
The interaction of eIF4E with 4E‐BP1 is an induced fit to a completely disordered protein
Authors: Fletcher C. Mark , Wagner Gerhard 
Published: February 2009

Abstract4E binding protein 1 (4E‐BP1) inhibits translation by binding to the initiation factor eIF4E and is mostly or completely unstructured in both free and bound states. We wished to determine whether the free protein has local structure that could be involved in eIF4E binding. Assignments were obtained using double and triple resonance NMR methods. Residues 4–10, 43–46, and 56–65 could not be assigned, primarily because of a high degree of 1H and 15N chemical shift overlap. Steady‐state {1H}−15N NOEs were measured for 45 residues in the assigned regions. Except for the two C‐terminal residues, the NOEs were between −0.77 and −1.14, indicating a high level of flexibility. Furthermore, the {1H}−15N NOE spectrum recorded with presaturation contained no strong positive signals, making it likely that no other residues have positive or smaller negative NOEs. This implies that 4E‐BPI has no regions of local order in the absence of eIF4E. The interaction therefore appears to be an induced fit to a completely disordered protein molecule.
Detecting distant relatives of mammalian LPS‐binding and lipid transport proteins
Authors: Beamer Lesa J. , Fischer Daniel , Eisenberg David 
Published: February 2009

AbstractIn mammals, a family of four lipid binding proteins has been previously defined that includes two lipopolysaccharide binding proteins and two lipid transfer proteins. The first member of this family to have its three‐dimensional structure determined is bactericidal/permeability‐increasing protein (BPI). Using both the sequence and structure of BPI, along with recently developed sequence‐sequence and sequence‐structure similarity search methods, we have identified 13 distant members of the family in a diverse set of eukaryotes, including rat, chicken, Caenorhabditis elegans, and Biomphalaria galbrata. Although the sequence similarity between these 13 new members and any of the 4 original members of the BPI family is well below the “twilight zone,” their high sequence‐structure compatibility with BPI indicates they are likely to share its fold. These findings broaden the BPI family to include a member found in retina and brain, and suggest that a primitive member may have contained only one of the two similar domains of BPI.
Conserved sequence motifs among bacterial, eukaryotic, and archaeal phosphatases that define a new phosphohydrolase superfamily
Authors: Thaller Maria Cristina , Schippa Serena , Rossolini Gian Maria 
Published: February 2009

AbstractMembers of a new molecular family of bacterial nonspecific acid phosphatases (NSAPs), indicated as class C, were found to share significant sequence similarities to bacterial class B NSAPs and to some plant acid phosphatases, representing the first example of a family of bacterial NSAPs that has a relatively close eukaryotic counterpart. Despite the lack of an overall similarity, conserved sequence motifs were also identified among the above enzyme families (class B and class C bacterial NSAPs, and related plant phosphatases) and several other families of phosphohydrolases, including bacterial phosphoglycolate phosphatases, histidinol‐phosphatase domains of the bacterial bifunctional enzymes imidazole‐glycerolphosphate dehydratases, and bacterial, eukaryotic, and archaeal phosphoserine phosphatases and threalose‐6‐phosphatases. These conserved motifs are clustered within two domains, separated by a variable spacer region, according to the pattern [FILMAVT]‐D‐[ILFRMVY]‐D‐[GSNDE]‐[TV]‐[ILVAM]‐[ATS VILMC]‐X‐{YFWHKR}‐X‐{YFWHNQ}‐X( 102, 191 )‐{KRHNQ}‐G‐D‐{FYWHILVMC}‐{QNH}‐{FWYGP}‐D‐{PSNQYW}. The dephosphorylating activity common to all these proteins supports the definition of this phosphatase motif and the inclusion of these enzymes into a superfamily of phosphohydrolases that we propose to indicate as “DDDD” after the presence of the four invariant aspartate residues. Database searches retrieved various hypothetical proteins of unknown function containing this or similar motifs, for which a phosphohydrolase activity could be hypothesized.
All in the family: Structural and evolutionary relationships among three modular proteins with diverse functions and variable assembly
Authors: Bergdoll Marc , Eltis Lindsay D. , Cameron Alexander D. , Dumas Philippe , Bolin Jeffrey T. 
Published: February 2009

AbstractThe crystal structures of three proteins of diverse function and low sequence similarity were analyzed to evaluate structural and evolutionary relationships. The proteins include a bacterial bleomycin resistance protein, a bacterial extradiol dioxygenase, and human glyoxalase I. Structural comparisons, as well as phylogenetic analyses, strongly indicate that the modem family of proteins represented by these structures arose through a rich evolutionary history that includes multiple gene duplication and fusion events. These events appear to be historically shared in some cases, but parallel and historically independent in others. A significant early event is proposed to be the establishment of metal‐binding in an oligomeric ancestor prior to the first gene fusion. Variations in the spatial arrangements of homologous modules are observed that are consistent with the structural principles of three‐dimensional domain swapping, but in the unusual context of the formation of larger monomers from smaller dimers or tetramers. The comparisons support a general mechanism for metalloprotein evolution that exploits the symmetry of a homooligomeric protein to originate a metal binding site and relies upon the relaxation of symmetry, as enabled by gene duplication, to establish and refine specific functions.