bluuebunny/crossref_metadata_2025_split huggingface dataset:
Crystal structures of the active site mutant (Arg‐243 → Ala) in the T and R allosteric states of pig kidney fructose‐1,6‐bisphosphatase expressed in escherichia coli
Authors: Stec Boguslaw , Abraham Reimar , Giroux Eugene , Kantrowitz Evan R. 
Published: February 2009

AbstractThe active site of pig kidney fructose‐l,6‐bisphosphatase (EC 3.1.3.11) is shared between subunits, Arg‐243 of one chain interacting with fructose‐1,6‐bisphosphate or fructose‐2,6‐bisphosphate in the active site of an adjacent chain. In this study, we present the X‐ray structures of the mutant version of the enzyme with Arg‐243 replaced by alanine, crystallized in both T and R allosteric states. Kinetic characteristics of the altered enzyme showed the magnesium binding and inhibition by AMP differed slightly; affinity for the substrate fructose‐1,6‐bisphosphate was reduced 10‐fold and affinity for the inhibitor fructose‐2,6‐bisphosphate was reduced 1,000‐fold (Giroux E, Williams MK, Kantrowitz ER, 1994, J Biol Chem 269:31404–31409). The X‐ray structures show no major changes in the organization of the active site compared with wild‐type enzyme, and the structures confirm predictions of molecular dynamics simulations involving Lys‐269 and Lys‐274. Comparison of two independent models of the T form structures have revealed small but significant changes in the conformation of the bound AMP molecules and small reorganization of the active site correlated with the presence of the inhibitor. The differences in kinetic properties of the mutant enzyme indicate the key importance of Arg‐243 in the function of fructose‐1,6‐bisphosphatase. Calculations using the X‐ray structures of the Arg‐243 Ala enzyme suggest that the role of Arg‐243 in the wild‐type enzyme is predominantly electrostatic in nature.
Contribution of the tyrosines to the structure and function of the human U1A N‐terminal RNA binding domain
Authors: Kranz James K. , Lu Jirong , Hall Kathleen B. 
Published: February 2009

AbstractRNA binding domains (RBDs) are members of a large family of proteins that share minimal sequence conservation but adopt an αβ sandwich global fold. Defining the contributions of specific amino acids to RBD structure and RNA binding is critical to understanding the functions of these proteins. In these experiments with the human U1A N‐terminal RNA binding domain (RBD1), the contributions from each of its four tyrosines to protein structure, stability, and RNA binding were measured. Each tyrosine was substituted with phenylalanine and one other selected residue, and the resulting proteins were characterized by chemical denaturation to measure their unfolding free energy, by binding free energies to the wild‐type RNA hairpin, and by 19F NMR to probe for structural changes. Features of the protein identified in these experiments include a possible tyrosine/lysine contact in an α‐helix, which may be an example of an energetically favorable aromatic/amino side chain interaction. One long loop of the protein, which shows unusual 15N backbone and tyrosine side‐chain dynamics, is implicated in protein:protein association. The diverse interactions of the four tyrosine residues in the organization of RBD1 illustrate how each member of this family of proteins will have unique molecular details that contribute to function.
Preparation and properties of pure, full‐length Ic1R protein of escherichia coli. Use of time‐of‐flight mass spectrometry to investigate the problems encountered
Authors: Donald Lynda J. , Chernushevich Igor V. , Zhou Jie , Verentchikov Anatoli , Poppe‐Schriemer Nancy , Hosfield David J. , Westmore John B. , Ens Werner , Duckworth Harry W. , Standing Kenneth G. 
Published: February 2009

AbstractIclR protein, the repressor of the aceBAK operon of Escherichia coli, has been examined by time‐of‐flight mass spectrometry, with ionization by matrix assisted laser desorption or by electrospray. The purified protein was found to have a smaller mass than that predicted from the base sequence of the cloned icl R gene. Additional measurements were made on mixtures of peptides derived from IclR by treatment with trypsin and cyanogen bromide. They showed that the amino acid sequence is that predicted from the gene sequence, except that the protein has suffered truncation by removal of the N‐terminal eight or, in some cases, nine amino acid residues. The peptide bond whose hydrolysis would remove eight residues is a typical target for the E. coli protease OmpT. We find that, by taking precautions to minimize OmpT proteolysis, or by eliminating it through mutation of the host strain, we can isolate full‐length IclR protein (lacking only the N‐terminal methionine residue). Full‐length IclR is a much better DNA‐binding protein than the truncated versions: it binds the aceBAK operator sequence 44‐fold more tightly, presumably because of additional contacts that the N‐terminal residues make with the DNA. Our experience thus demonstrates the advantages of using mass spectrometry to characterize newly purified proteins produced from cloned genes, especially where proteolysis or other covalent modification is a concern. This technique gives mass spectra from complex peptide mixtures that can be analyzed completely, without any fractionation of the mixtures, by reference to the amino acid sequence inferred from the base sequence of the cloned gene.
Crystal structure of a biologically inactive mutant of toxic shock syndrome toxin‐1 at 2.5 A resolution
Authors: Papageorgiou Anastassios C. , Quinn Conrad P. , Beer David , Brehm Rossalyn D. , Tranter Howard S. , Bonventre Peter F. , Acharya K.Ravi 
Published: February 2009

AbstractToxic shock syndrome toxin‐1 (TSST‐1) is one of a family of staphylococcal exotoxins recognized as microbial superantigens. The toxin plays a dominant role in the genesis of toxic shock in humans through a massive activation of the immune system. This potentially lethal illness occurs as a result of the interaction of TSST‐1 with a significant proportion of the T‐cell repertoire. TSST‐1, like other superantigens, can bind directly to class II major histocompatibility (MHC class II) molecules prior to its interaction with entire families of Vβ chains of the T‐cell receptor (TCR). The three‐dimensional structure of a mutant (His‐135‐Ala) TSST‐1 was compared with the structure of the native (wild‐type) TSST‐1 at 2.5 Å resolution. The replacement of His 135 of TSST‐1 with an Ala residue results in the loss of T‐cell mitogenicity and toxicity in experimental animals. This residue, postulated to be directly involved in the toxin‐TCR interactions, is located on the major helix α2, which forms the backbone of the molecule and is exposed to the solvent. In the molecular structure of the mutant toxin, the helix α2 remains unaltered, but the His to Ala modification causes perturbations on the neighboring helix α1 by disrupting helix‐helix interactions. Thus, the effects on TCR binding of the His 135 residue could actually be mediated, wholly or in part, by the α1 helix.
Structural characterization of the tumor suppressor p16, an ankyrin‐like repeat protein
Authors: Boice Judith A. , Fairman Robert 
Published: February 2009

AbstractThe p16 protein has been identified as a tumor suppressor that functions by inhibiting the cyclin‐dependent kinases CDK4 and CDK6. Deletions or point mutations in the p16 gene have been found in a number of human cancers, emphasizing its importance in regulating cell cycle progression. Inhibition by p16 occurs through protein‐protein interactions with its targets. This is not surprising, since p16 is thought to contain ankyrin‐like repeats, motifs implicated in protein‐protein interactions. Our goal was to identify structural characteristics of p16 not only as an important step towards understanding CDK4 inhibition but also to explore the role of ankyrin repeats in the p16 structure, as no detailed structure of any protein containing these motifs has been reported. We have expressed, refolded, and purified p16 from E. coli and have shown it to be functionally active by specific binding to CDK4. Analytical ultracentrifugation has shown that p16 weakly self‐associates to form dimers with a Kd = 270 μM. The CD spectrum indicates that the protein is composed of 33% α‐helix, 22% β‐sheet, 19% β‐turn, and 27% other (which includes aromatic and random coil contributions). Further CD experiments suggest that p16 exhibits low structural stability with a ΔG of ‐2.3 kcal/mol. This weak stability is a consequence of a highly dynamic structure as measured by ANS‐binding, NMR hydrogen‐deuterium exchange, and fluorescence. It is possible that a well‐defined tertiary structure is imparted upon the binding of p16 to CDK4.
An optimized g‐tensor for rhodobacter capsulatus cytochrome c2 in solution: A structural comparison of the reduced and oxidized states
Authors: Zhao Dezheng , Mackenzie Neil E. , Hutton Harold M. , Cusanovich Michael A. 
Published: February 2009

AbstractThe optimized g‐tensor parameters for the oxidized form of Rhodobacter capsulatus cytochrome c2 in solution were obtained using a set (50) of backbone amide protons. Dipolar shifts for more than 500 individual protons of R. capsulatus cytochrome c2 have been calculated by using the optimized g‐tensor and the X‐ray crystallographic coordinates of the reduced form of R. capsulatus cytochrome c2. The calculated results for dipolar shifts are compared with the observed paramagnetic shifts. The calculated and the observed data are in good agreement throughout the entire protein, but there are significant differences between calculated and experimental results localized to the regions in the immediate vicinity of the heme ligand and the region of the front crevice of the protein (residues 44–50, 53–57, and 61–68). The results not only indicate that the overall solution structures are very similar in both the reduced and oxidized states, but that these structures in solution are similar to the crystal structure. However, there are small structural changes near the heme and the rearrangement of certain residues that result in changes in their hydrogen bonding concomitant with the change in the oxidation states; this was also evident in the data for the NH exchange rate measurements for R. capsulatus cytochrome c2.
Denaturants can accelerate folding rates in a class of globular proteins
Authors: Camacho Carlos J. , Thirumalai D. 
Published: February 2009

AbstractWe present a lattice Monte Carlo study to examine the effect of denaturants on the folding rates of simplified models of proteins. The two‐dimensional model is made from a three‐letter code mimicking the presence of hydrophobic, hydrophilic, and cysteine residues. We show that the rate of folding is maximum when the effective hydrophobic interaction ϵH is approximately equal to the free energy gain ϵS upon forming disulfide bonds. In the range 1 ≤ ϵH/ϵS ≤ 3, multiple paths that connect several intermediates to the native state lead to fast folding. It is shown that at a fixed temperature and ϵS the folding rate increases as ϵH decreases. An approximate model is used to show that ϵH should decrease as a function of the concentration of denaturants such as urea or guanidine hydrochloride. Our simulation results, in conjunction with this model, are used to show that increasing the concentration of denaturants can lead to an increase in folding rates. This occurs because denaturants can destabilize the intermediates without significantly altering the energy of the native conformation. Our findings are compared with experiments on the effects of denaturants on the refolding of bovine pancreatic trypsin inhibitor and ribonuclease T1. We also argue that the phenomenon of denaturant‐enhanced folding of proteins should be general.
Identification of protein kinase A phosphorylation sites on NBD1 and R domains of CFTR using electrospray mass spectrometry with selective phosphate ion monitoring
Authors: Townsend R. Reid , Lipniunas Peter H. , Tulk Barry M. , Verkman A.S. 
Published: February 2009

AbstractHPLC‐electrospray mass spectrometry was used to identify the phosphorylated sites on a bacterially expressed cystic fibrosis transmembrane conductance regulator (CFTR) fragment containing the first nucleotide binding domain (NBD1) and the regulatory domain (R). Tryptic digests of NBD1‐R (CFTR residues 404–830) were analyzed after protein kinase A (PKA) treatment for all possible peptides and phosphopeptides (a total of 118 species) containing Ser residues within “high‐probability” PKA consensus sequences: R‐R/K‐X‐S/T, R‐X‐X‐S/T, and R‐X‐S/T. Three criteria were used to assign phosphorylated sites: (1) an 80‐Da increase in the predicted average molecular weight of the tryptic peptides; (2) co‐elution with the PO3− ion induced by stepped energy collision; and (3) the relative elution positions of the phosphorylated and unmodified peptides. Ser residues within the eight dibasic sites in the NBD1 and R domains (positions 422, 660, 700, 712, 737, 768, 795, and 813) were phosphorylated, a pattern similar to that observed for full‐length CFTR. The serine at position 753, which in CFTR is phosphorylated in vivo, was not phosphorylated. The remaining potential PKA sites, Ser489, Ser519, Ser557, Ser670, and Thr788, were not phosphorylated. The “low‐probability” PKA sites (those not containing an Arg residue) were not phosphorylated. The results suggest that isolated domains of CFTR developed useful models for investigating the biochemical and structural effects of phosphorylation within CFTR. The mass spectrometry approach in this study should prove useful for defining phosphorylation sites of CFTR in vitro and in vivo.
High‐ and low‐temperature unfolding of human high‐density apolipoprotein A‐2
Authors: Gursky Olga , Atkinson David 
Published: February 2009

AbstractHuman plasma apolipoprotein A‐2 (apoA‐2) is the second major protein of the high‐density lipoproteins that mediate the transport and metabolism of cholesterol. Using CD spectroscopy and differential scanning calorimetry, we demonstrate that the structure of lipid‐free apoA‐2 in neutral low‐salt solutions is most stable at ∼25 °C and unfolds reversibly both upon heating and cooling from 25 °C. High‐temperature unfolding of apoA‐2, monitored by far‐UV CD, extends from 25–85 °C with midpoint Th = 56 ± 2 °C and vant Hoff's enthalpy ΔH(Th) = 17 ± 2 kcal/mol that is substantially lower than the expected enthalpy of melting of the α‐helical structure. This suggests low‐cooperativity apoA‐2 unfolding. The apparent free energy of apoA‐2 stabilization inferred from the CD analysis of the thermal unfolding, ΔGapp(25°) = 0.82 ± 0.15 kcal/mol, agrees with the value determined from chemical denaturation. Enhanced low‐temperature stability of apoA‐2 observed upon increase in Na2HPO4 concentration from 0.3 mM to 50 mM or addition of 10% glycerol may be linked to reduced water activity. The close proximity of the heat and cold unfolding transitions, that is consistent with low ΔGAPP (25°), indicates that lipid‐free apoA‐2 has a substantial hydrophobic core but is only marginally stable under near‐physiological solvent conditions. This suggests that in vivo apoA‐2 transfer is unlikely to proceed via the lipid‐free state. Low ΔH(Th) and low apparent ΔCp ∼ 0.52 kcal/mol‐K inferred from the far‐UV CD analysis of apoA‐2 unfolding, and absence of tertiary packing interactions involving Tyr groups suggested by near‐UV CD, are consistent with a molten globular‐like state of lipid‐free apoA‐2.
Binding sites for blood coagulation factor Xa and protein S involving residues 493–506 in factor Va
Authors: Heeb Mary J. , Kojima Yumi , Hackeng Tilman M. , Griffin John H. 
Published: February 2009

AbstractInactivation due to cleavage of Factor Va (FVa) at Arg 506 by activated protein C (APC) helps to downregulate blood coagulation. To identify potential functional roles of amino acids near Arg 506, synthetic overlapping pentadecapeptides comprising FVa heavy chain residues 481–525 were tested for their ability to inhibit prothrombin activation by prothrombinase complexes [Factor Xa (FXa):FVa:phospholipids:Ca2+]. The most potent inhibition was observed for peptide VP493 (residues 493–506), with 50% inhibition at 2.5 μM. VP493 also inhibited FXa in plasma in FXa‐1‐stage clotting assays by 50% at 3 μM. When the C‐terminal carboxamide group of VP493 was replaced by a carboxyl group, most prothrombinase inhibitory activity was lost. VP493 preincubated with FXa inhibited prothrombinase with a pattern of mixed inhibition. Homologous peptides from Factor VIII sequences did not inhibit prothrombinase. Affinity‐purified antibodies to VP493 inhibited prothrombinase activity and prolonged FXa‐1‐stage clotting times. VP493 also blocked the ability of protein S to inhibit prothrombinase independently of APC. Immobilized VP493 bound specifically with similar affinity to both FXa and protein S (Kd ∼ 40 nM), but did not measurably bind prothrombin or APC. These studies suggest that FVa residues 493–506 contribute to binding sites for both FXa and protein S, providing a rationale for the ability of protein S to negate the protective effect of FXa toward APC cleavage of FVa. Possible loss of this FVa binding site for FXa due to cleavage at Arg 506 by APC may help explain why this cleavage causes 40% decrease in FVa activity and facilitates inactivation of FVa.
Cation selective promotion of tubulin polymerization by alkali metal chlorides
Authors: Wolff J. , Sackett Dan L. , Knipling Leslie 
Published: February 2009

AbstractA role for charge‐based interactions in protein stability at the monomer or dimer level is well known. We show here that such interactions can also be important for the higher‐order structures of microtubule assembly. Alkali metal chlorides increase the rate of polymerization of pure tubulin driven by either taxol or dimethyl sulfoxide. The effect is cation selective, exhibiting a sequence Na+> K+> Li+> Cs+, with optimal concentrations for Na+at ∽ 160 mM. Hofmeister anion effects are additive with these rate stimulations. Sodium is less potent than guanidinium ion stimulation reported previously, but produces a larger fraction of normal microtubules. Alkali metal cations lower the critical concentration by a factor of ∽2, produce cold reversible polymers whose formation is sensitive to podophyllotoxin inhibition, increase the fraction of polymers present as microtubules from ∽0.9 to 0.99, and reverse or prevent urea‐induced depolymerization of microtubules. In the presence of microtubule‐associated proteins, the promotion of polymerization is no longer cation selective. In the polymerization of tubulin S, in which the acidic C termini of both monomers have been cleaved, the cation enhancement is markedly decreased, although selectivity persists.Because the selectivity sequence is similar to that of the coil/helix transition of polyglutamic acid, we suggest that a major part, although not all, of the cation selective enhancement of polymerization results from shielding of the glutamate‐rich C termini of the tubulin monomers.
Fluorescence energy transfer measurement of distances between ligand binding sites of tubulin and its implication for protein‐protein interaction
Authors: Bhattacharya Anusree , Roy Siddhartha , Bhattacharyya Bhabatarak 
Published: February 2009

Abstract9‐(Dicyanovinyl) julolidine (DCVJ) is a fluorescent probe, which binds to a unique site on the tubulin dimer and exhibits different properties that are dependent upon its oligomeric state (Kung & Reed, 1989). DCVJ binds to tubulin, the tubulin‐colchicine complex, and the tubulin‐ruthenium red complex equally well, but binds tighter to the ANS‐tubulin complex than to tubulin alone. The energy transfer studies indicate a small amount of energy transfer with colchicine, but a significant energy transfer with ANS. It was shown previously that ruthenium red binds near the C‐terminal tail region of the α‐subunit. Ruthenium red causes major quenching of fluorescence of the tubulin‐DCVJ complex, suggesting proximity of binding sites.The derived distances are consistent with DCVJ binding near the αβ interface, but on the opposite face of the colchicine binding site. Location of the binding site correlates with the observed effect of a different polymerized state of tubulin on the DCVJ spectroscopic properties. The effect of dimer‐dimer association on DCVJ binding, at high protein concentrations (Kung & Reed, 1989), suggests that such an association may occur through lateral contacts of the elongated tubulin dimer, at least in a significant fraction of the cases. Transmission of ANS‐induced conformational change to the DCVJ binding site, which is near important dimer‐dimer contact sites, makes it possible that such conformational changes may be responsible for polymerization inhibition by anilino‐naphthalene sulfonates.
Extended repertoire of permissible peptide ligands for HLA‐B*2702
Authors: Raghavan Malini , Lebrón José A. , Johnson Jennifer L. , Bjorkman Pamela J. 
Published: February 2009

AbstractRecognition of self peptides bound to the class I major histocompatibility complex molecule HLA‐B27 is thought to trigger proliferation of autoreactive T cells and result in autoimmune arthritic diseases. Previous work from other laboratories established that a predominant feature of endogenous peptides eluted from purified B27 is an arginine at position 2. We studied the binding of peptides containing both natural and unnatural amino acids by the subtype HLA‐B*2702, with the goal of gaining insight into peptide binding by this B27 subtype that is associated with susceptibility to arthritic disease. A soluble form of B*2702 was depleted of endogeneous peptides. We tested the binding of peptides substituted with cysteine, homocysteine, or an α‐amino‐ϵ‐mercapto hexanoic acid side chain (Amh) instead of the naturally occurring arginine at position 2, to determine whether the peptide sulfhydryl residue could be covalently linked to cysteine 67 in the B*2702 binding cleft. Although none of the altered peptide sequences bound covalently to B*2702, the affinities of the homocysteine‐ and Amh‐substituted peptides were close to that of the native peptide sequence. Substitutions at position 2 with other side chains, such as glutamine and methionine, also resulted in peptides that bound with only slightly reduced affinity. These results demonstrate that peptide side chains other than arginine at position 2 can be accomodated within the B*2702 peptide binding site with only minor reductions in affinity. This extended repertoire of permissible B27‐binding peptides should be taken into account for a consideration of disease‐associated peptide sequences.
Water mediated protein‐DNA interactions: The relationship of thermodynamics to structural detail
Authors: Morton Craig J. , Ladbury John E. 
Published: February 2009

AbstractThe elucidation of a relationship between the thermodynamic parameters and the structural changes accompanying bio‐molecular interactions could lead to predictive algorithms. For example, based on some knowledge of the structure of a target molecule the affinities of ligands could be determined with obvious implications for the pharmaceutical industry. In attempting to relate the thermodynamic and structural changes on formation of a protein‐DNA complex, the correlation between change in heat capacity and burial of surface area has proved successful. However, this correlation appears to break down when water molecules are included in the binding interface. Here we present data that support the hypothesis that bound water molecules have to be considered as contributing to the change in heat capacity and could, thus, be used in ligand design.