Showing papers in "Biochemistry in 1994"
TL;DR: Overall, higher order parameters were not found in the peptide-bound form, indicating that on average, picosecond-time-scale disorder is not reduced upon binding peptide, and the relaxation data of the SH2-phosphopeptide complex were fit with fewer exchange terms than the uncomplexed form.
Abstract: The backbone dynamics of the C-terminal SH2 domain of phospholipase C gamma 1 have been investigated. Two forms of the domain were studied, one in complex with a high-affinity binding peptide derived from the platelet-derived growth factor receptor and the other in the absence of this peptide. 2-D 1H-15N NMR methods, employing pulsed field gradients, were used to determine steady-state 1H-15N NOE values and T1 and T2 15N relaxation times. Backbone dynamics were characterized by the overall correlation time (tau m), order parameters (S2), effective correlation times for internal motions (tau e), and, if required, terms to account for motions on a microsecond-to-millisecond-time scale. An extended two-time-scale formalism was used for residues having relaxation data and that could not be fit adequately using a single-time-scale formalism. The overall correlation times of the uncomplexed and complexed forms of SH2 were found to be 9.2 and 6.5 ns, respectively, suggesting that the uncomplexed form is in a monomer-dimer equilibrium. This was subsequently confirmed by hydrodynamic measurements. Analysis of order parameters reveals that residues in the so-called phosphotyrosine-binding loop exhibited higher than average disorder in both forms of SH2. Although localized differences in order parameters were observed between the uncomplexed and complexed forms of SH2, overall, higher order parameters were not found in the peptide-bound form, indicating that on average, picosecond-time-scale disorder is not reduced upon binding peptide. The relaxation data of the SH2-phosphopeptide complex were fit with fewer exchange terms than the uncomplexed form. This may reflect the monomer-dimer equilibrium that exists in the uncomplexed form or may indicate that the complexed form has lower conformational flexibility on a microsecond-to-millisecond-time scale.
2,064 citations
TL;DR: This Perspectives includes a brief review of BP photochemistry and a selection of specific applications of these photoprobes, which address questions in protein, nucleic acid, and lipid biochemistry.
Abstract: The photoactivatable aryl ketone derivatives have been rediscovered as biochemical probes in the last 5 years. The expanding use of benzophenone (BP) photoprobes can be attributed to three distinct chemical and biochemical advantages. First, BPs are chemically more stable than diazo esters, aryl azides, and diazirines. Second, BPs can be manipulated in ambient light and can be activated at 350-360 nm, avoiding protein-damaging wavelengths. Third, BPs react preferentially with unreactive C-H bonds, even in the presence of solvent water and bulk nucleophiles. These three properties combine to produce highly efficient covalent modifications of macromolecules, frequently with remarkable site specificity. This Perspectives includes a brief review of BP photochemistry and a selection of specific applications of these photoprobes, which address questions in protein, nucleic acid, and lipid biochemistry.
828 citations
TL;DR: Three naturally occurring polymorphisms of the human beta 2-adrenergic receptor caused by missense mutations encoding for amino acids 16 and 27 of the extracellular N-terminus of the receptor are delineated and the functional consequences of these polymorphisms by site-directed mutagenesis and the recombinant expression of these receptors in Chinese hamster fibroblasts are studied.
Abstract: We have recently delineated three naturally occurring polymorphisms of the human beta 2-adrenergic receptor caused by missense mutations encoding for amino acids 16 and 27 of the extracellular N-terminus of the receptor. We have studied the functional consequences of these polymorphisms by site-directed mutagenesis and the recombinant expression of these receptors in Chinese hamster fibroblasts. The polymorphisms consist of substitutions of Gly for Arg at amino acid 16 (Arg16-->Gly), Glu for Gln at amino acid 27 (Gln27-->Glu), and a combination of both substitutions. All three mutated receptors displayed normal agonist binding and functional coupling to Gs, resulting in the stimulation of adenylyl cyclase activity. However, these mutations markedly altered the degree of agonist-promoted downregulation of receptor expression. After 24-h exposure to 10 microM isoproterenol, wild-type beta 2AR underwent a 26 +/- 3% reduction in receptor density. In contrast, Arg16-->Gly underwent a 41 +/- 3% reduction. Gln27-->Glu, on the other hand, was found to be completely resistant to downregulation. Arg16-->Gly+Gln27-->Glu also underwent an increased downregulation compared to wild-type beta 2AR (39 +/- 4%). The rates of new receptor synthesis after irreversible alkylation were not different between these receptors, nor were the rates of agonist-promoted receptor internalization to the intracellular pool. Gln27-->Glu cellular mRNA minimally increased during agonist exposure, and wild-type beta 2AR and the other mutated receptor mRNAs did not change, which infer that the aberrant downregulation patterns of these polymorphisms may be due to the altered degradation of receptor protein.(ABSTRACT TRUNCATED AT 250 WORDS)
806 citations
TL;DR: The method employs a set of statistical tables (log likelihoods) complied from well-characterized membrane protein data, and a novel dynamic programming algorithm to recognize membrane topology models by expectation maximization.
Abstract: This paper describes a new method for the prediction of the secondary structure and topology of integral membrane proteins based on the recognition of topological models. The method employs a set of statistical tables (log likelihoods) complied from well-characterized membrane protein data, and a novel dynamic programming algorithm to recognize membrane topology models by expectation maximization. The statistical tables show definite biases toward certain amino acid species on the inside, middle, and outside of a cellular membrane. Using a set of 83 integral membrane protein sequences taken from a variety of bacterial, plant, and animal species, and a strict jackknifing procedure, where each protein (along with any detectable homologues) is removed from the training set used to calculate the tables before prediction, the method successfully predicted 64 of the 83 topologies, and of the 37 complex multispanning topologies 34 were predicted correctly.
795 citations
TL;DR: Observations suggest that the membrane fatty acid desaturases and hydrocarbon hydroxylases have a related protein fold, possibly arising from a common ancestral origin.
Abstract: The eukaryotic fatty acid desaturases are iron-containing enzymes that catalyze the NAD-(P)H- and O2-dependent introduction of double bonds into methylene-interrupted fatty acyl chains. Examination of deduced amino acid sequences for the membrane desaturases from mammals, fungi, insects, higher plants, and cyanobacteria has revealed three regions of conserved primary sequence containing HX(3 or 4)H,HX(2 or 3)HH, and HX(2 or 3)HH. This motif is also present in the bacterial membrane enzymes alkane hydroxylase (omega-hydroxylase) and xylene monooxygenase. Hydropathy analyses indicate that these enzymes contain up to three long hydrophobic domains which would be long enough to span the membrane bilayer twice. The conserved His-containing regions have a consistent positioning with respect to these potential membrane spanning domains. Taken together, these observations suggest that the membrane fatty acid desaturases and hydrocarbon hydroxylases have a related protein fold, possibly arising from a common ancestral origin. In order to examine the functional role of these conserved His residues, we have made use of the ability of the rat delta 9 desaturase gene to complement a yeast strain deficient in the delta 9 desaturase gene function (ole1). By site-directed mutagenesis, eight conserved His residues in the rat delta 9 desaturase were individually converted to Ala. Each His-->Ala mutation failed to complement the yeast ole1 mutant. In contrast, mutation of three nonconserved flanking His residues or a partially conserved Arg residue within the conserved motif to Ala allowed for complementation of the ole1 phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)
782 citations
TL;DR: This work explains domain movements in proteins in terms of the repertoire of low-energy conformation changes that are known to occur in proteins, and describes the basic elements of this repertoire, hinge and shear motions, and shows how they can be combined to produce domain movements.
Abstract: We survey all the known instances of domain movements in proteins for which there is crystallographic evidence for the movement. We explain these domain movements in terms of the repertoire of low-energy conformation changes that are known to occur in proteins. We first describe the basic elements of this repertoire, hinge and shear motions, and then show how the elements of the repertoire can be combined to produce domain movements. We emphasize that the elements used in particular proteins are determined mainly by the structure of the interfaces between the domains.
736 citations
TL;DR: DNA photolyase repairs DNA by utilizing the energy of visible light to break the cyclobutane ring of the dimer and restores the dipyrimidine and the functional form of flavin ready for a new cycle of catalysis.
Abstract: Cyclobutane pyrimidine dimers (Pyr Pyr) are the major DNA photoproducts induced by the UV component of solar radiation. Photoreactivating enzyme (DNA photolyase) repairs DNA by utilizing the energy of visible light to break the cyclobutane ring of the dimer. Photolyases are monomeric proteins of 50-60 kDa with stoichiometric amounts of two noncovalent chromophore/cofactors. One of these cofactors is FADH-, and the second chromophore is either methenyltetrahydrofolate (MTHF) or 8-hydroxy-5-deazariboflavin (8-HDF). The enzyme binds the DNA substrate in a light-independent reaction, the second chromophore of the bound enzyme absorbs a visible photon and, by dipole-dipole interaction, transfers energy to FADH- which, in turn, transfers an electron to Pyr Pyr in DNA; the Pyr Pyr- splits and back electron transfer restores the dipyrimidine and the functional form of flavin ready for a new cycle of catalysis.
696 citations
TL;DR: Using a novel procedure, the enzyme has been purified to homogeneity from bovine lung with a heme content of approximately 1 heme/heterodimer, and .NO increases the Vmax of sGC by 100-200-fold, probably by interacting with aHeme moiety on the enzyme.
Abstract: Nitric oxide (.NO) is a recently discovered signaling agent which plays a role in many biological processes such as vasodilation and neuronal synaptic transmission. The only receptor characterized thus far for .NO is the soluble form of guanylate cyclase (sGC). .NO increases the Vmax of sGC by 100-200-fold, probably by interacting with a heme moiety on the enzyme. Although several procedures exist for purifying sGC, these procedures result in preparations with low heme contents. Using a novel procedure, the enzyme has been purified to homogeneity from bovine lung with a heme content of approximately 1 heme/heterodimer. The UV-visible spectrum of the enzyme contains a Soret peak centered at 431 nm and a single broad alpha/beta peak at 555 nm indicative of a 5-coordinate ferrous heme with histidine as the axial ligand. The heme moiety does not bind oxygen but will readily bind .NO to form a 5-coordinate complex or carbon monoxide (CO) to form a 6-coordinate complex. Oxidation of the heme with ferricyanide shifts the Soret to 393 nm, due most likely to the formation of a 5-coordinate ferric heme. In the ferric state, the heme will apparently not bind water but will bind cyanide with reduced affinity compared to methemoglobin and metmyoglobin. Purified enzyme containing 1 heme/heterodimer is activated 130-fold by .NO and 4.4-fold by CO.
639 citations
TL;DR: The role of the neuronal microtubule-associated protein tau has been studied by generating a series of tau constructs differing in one or several of its subdomains: length and composition of the repeat domains, extensions of the repeats in the N- or C-terminal direction, constructs without repeats, assembly vs projection domain, and number of N- terminal inserts.
Abstract: The role of the neuronal microtubule-associated protein tau has been studied by generating a series of tau constructs differing in one or several of its subdomains: length and composition of the repeat domains, extensions of the repeats in the N- or C-terminal direction, constructs without repeats, assembly vs projection domain, and number of N-terminal inserts. The interaction of the mutant tau proteins with microtubules was judged by several independent methods. (i) Direct binding assays between tau and taxol-stabilized microtubules yield dissociation constants and stoichiometries. (ii) Light scattering and X-ray scattering of assembling microtubule solutions reflect the capacity of tau to promote microtubule nucleation, elongation, and bundling in bulk solution. (iii) Dark field microscopy of assembling microtubules allows one to assess the efficiency of nucleation and bundling separately. The repeat region alone, the N-terminal domains alone, or the C-terminal tail alone binds only weakly to microtubules. However, binding is strongly enhanced by combinations such as the repeat region plus one or both of the flanking regions which could be viewed as "jaws" for tau on the microtubule surface (the proline-rich domain P upstream of the repeats and the "fifth" repeat R' downstream). Such combinations make tau's binding productive in terms of microtubule assembly and stabilization, while the combination of the flanking regions without repeats binds only unproductively. Efficient nucleation parallels strong binding in most cases, i.e., when a construct binds tightly to microtubules, it also nucleates them efficiently and vice versa. In addition, the proline-rich domain P in combination with the repeats R or the flanking domain R' causes pronounced bundling. This effect disappears when the N-terminal domains (acidic or basic) are added on, suggesting that the tau isoforms are not "bundling proteins" in the proper sense. In spite of the wide range of binding strength and nucleation efficiency, the stoichiometries of binding are rather reproducible (around 0.5 tau/tubulin dimer); this is in remarkable contrast to the effect of certain types of phosphorylation which can strongly reduce the stoichiometry.
628 citations
TL;DR: In situ hybridization reveals intense labeling of MT-IV mRNA in the differentiating spinous layer of cornified epithelia, whereas MT-I is expressed predominantly in the basal, proliferative layer; thus, there is a switch in MT isoform synthesis during differentiation of these epithelias.
Abstract: A new member of the metallothionein (MT) gene family was discovered that lies about 20 kb 5' of the MT-III gene in both mouse and human. The MT-IV proteins are highly conserved in both species and have a glutamate insertion at position 5 relative to the classical MT-I and MT-II proteins. Murine MT-IV mRNA appears to be expressed exclusively in stratified squamous epithelia associated with oral epithelia, esophagus, upper stomach, tail, footpads, and neonatal skin. The MT derived from tongue epithelium contains both zinc and copper. Many of these epithelia develop parakeratosis during zinc deficiency in the rat. In situ hybridization reveals intense labeling of MT-IV mRNA in the differentiating spinous layer of cornified epithelia, whereas MT-I is expressed predominantly in the basal, proliferative layer; thus, there is a switch in MT isoform synthesis during differentiation of these epithelia. We suggest that MT-IV plays a special role in regulating zinc metabolism during the differentiation of stratified epithelia.
533 citations
TL;DR: The probe was used to measure the rate of Pi release during a single turnover of ATP hydrolysis with actomyosin subfragment 1 from rabbit skeletal muscle, to determine to what extent Pi release contributes to the rate limitation of this ATPase.
Abstract: A probe has been developed that can rapidly measure micromolar concentrations of inorganic phosphate (Pi), in particular to follow the release of Pi in real time from enzymes such as phosphatases. Its application is described to investigate the mechanism of actomyosin subfragment 1 ATPase. The probe uses the A197C mutant of Escherichia coli phosphate binding protein (PBP), generated by oligonucleotide-directed mutagenesis. A new fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC), was attached to the single cysteine to produce the reporter molecule that was purified free of unlabeled protein and unattached MDCC. The labeled protein has an excitation maximum at 425 nm and emission maximum at 474 nm in the absence of Pi, shifting to 464 nm with a 5.2-fold increase in fluorescence (lambda max/lambda max) when complexed with Pi at pH 7.0, low ionic strength, 22 degrees C. The fluorescence increase is not much altered by change to pH 8 or by increase in ionic strength to 1 M. Pi binds tightly (Kd approximately 0.1 microM) and rapidly (1.36 x 10(8) M-1 s-1) and the dissociation rate constant is 21 s-1, at pH 7.0, low ionic strength, 22 degrees C. A variety of phosphate esters were tested to investigate the specificity of the MDCC-PBP and none gave a significant fluorescence increase at 100 microM or higher concentration. ATP weakly inhibited the Pi-induced fluorescence change, indicating that it binds at least 3000-fold weaker than Pi. Because Pi is a widespread contaminant, the probe is used in conjunction with a "Pi mop", consisting of 7-methylguanosine and purine nucleoside phosphorylase, to remove free Pi from solutions by its conversion to ribose 1-phosphate. Because the equilibrium constant of this reaction is > 100, free Pi can be reduced below 0.1 microM. The probe was used to measure the rate of Pi release during a single turnover of ATP hydrolysis with actomyosin subfragment 1 from rabbit skeletal muscle, to determine to what extent Pi release contributes to the rate limitation of this ATPase. Using a stopped-flow apparatus, a small lag prior to rapid Pi release was detected at pH 7.0, low ionic strength, between 5 and 22 degrees C at both high and low [ATP]. For measurements of a single turnover at low [ATP], the observed rate increased with [actin], showing saturation with a Km with respect to actin of 26 microM.(ABSTRACT TRUNCATED AT 400 WORDS)
TL;DR: Using a simulated annealing procedure in sequence space, sequences are designed to have sufficiently low energy in a given target conformation, which plays the role of the native structure in this study.
Abstract: We have studied the folding mechanism of lattice model 36-mer proteins. Using a simulated annealing procedure in sequence space, we have designed sequences to have sufficiently low energy in a given target conformation, which plays the role of the native structure in our study. The sequence design algorithm generated sequences for which the native structures is a pronounced global energy minimum. Then, designed sequences were subjected to lattice Monte Carlo simulations of folding. In each run, starting from a random coil conformation, the chain reached its native structure, which is indicative that the model proteins solve the Levinthal paradox. The folding mechanism involved nucleation growth. Formation of a specific nucleus, which is a particular pattern of contacts, is shown to be a necessary and sufficient condition for subsequent rapid folding to the native state. The nucleus represents a transition state of folding to the molten globule conformation. The search for the nucleus is a rate-limiting step of folding and corresponds to overcoming the major free energy barrier. We also observed a folding pathway that is the approach to the native state after nucleus formation; this stage takes about 1% of the simulation time. The nucleus is a spatially localized substructure of the native state having 8 out of 40 native contacts. However, monomers belonging to the nucleus are scattered along the sequence, so that several nucleus contacts are long-range while other are short-range. A folding nucleus was also found in a longer chain 80-mer, where it also constituted 20% of the native structure. The possible mechanism of folding of designed proteins, as well as the experimental implications of this study is discussed.
TL;DR: These studies establish the biochemical identity of native and recombinant MIF and provide a first insight into the three-dimensional structural properties of this critical inflammatory mediator.
Abstract: The cytokine macrophage migration inhibitory factor (MIF) has been identified to be secreted by the pituitary gland and the monocyte/macrophage and to play an important role in endotoxic shock. Despite the recent molecular cloning of a human T-cell MIF, characterization of the biochemical and biological properties of this protein has remained incomplete because substantial quantities of purified, recombinant, or native MIF have not been available. We describe the cloning of mouse MIF from anterior pituitary cells (AtT-20) and the purification of native MIF from mouse liver by sequential ion exchange and reverse-phase chromatography. For comparison purposes, human MIF was cloned from the Jurkat T-cell line and also characterized. Mouse and human MIF were highly homologous (90% identity over 115 amino acids). Recombinant mouse and human MIF were expressed in Escherichia coli and purified in milligram quantities by a simple two-step procedure. The molecular weight of native mouse MIF (12.5 kDa monomer) was identical with that of recombinant mouse MIF as assessed by gel electrophoresis and mass spectroscopy. No significant post-translational modifications were detected despite the presence of two potential N-linked glycosylation sites. Recombinant MIF inhibited monocyte migration in a dose-dependent fashion, and both recombinant and native MIF-exhibited comparable biological activities. MIF induced the secretion of tumor necrosis factor-alpha and stimulated nitric oxide production by macrophages primed with interferon-gamma. Circular dichroism spectroscopy revealed that bioactive mouse and human MIF exhibit a highly ordered, three-dimensional structure with a significant percentage of beta-sheet and alpha-helix conformation. Guanidine hydrochloride-induced unfolding experiments demonstrated that MIF is of low to moderate thermodynamic stability. These studies establish the biochemical identity of native and recombinant MIF and provide a first insight into the three-dimensional structural properties of this critical inflammatory mediator.
TL;DR: The structure of the 56-residue B1 immunoglobulin-binding domain from streptococcal protein G has been determined in two different crystal forms, and crystallographic models differ from NMR-derived models in several local regions, primarily in the loop involving residues 46-51.
Abstract: The structure of the 56-residue B1 immunoglobulin-binding domain from streptococcal protein G has been determined in two different crystal forms. The crystal structures were deduced by molecular replacement, based on the structure of the B2 domain (Brookhaven accession code 1PGX). Final R values are 0.174 and 0.198 for orthorhombic and trigonal forms, for diffraction data from 6.0 to 2.07 A and from 6 to 1.92 A, respectively. The orthorhombic crystals have an unusually high packing density for protein crystals, with Vm = 1.66 and a solvent content of 26%. The protein structure is found to be very similar (rms deviation 0.25 A for 56 C alpha's) in the two crystal forms, with an efficiently packed hydrophobic core between a four-stranded beta-sheet and a four-turn alpha-helix. The B1 domain has the same fold and general structure as the B2 domain (rms deviations 0.36 and 0.39 A), despite the six residue differences between them. The crystallographic models differ from NMR-derived models in several local regions, primarily in the loop involving residues 46-51; other significant variations are observed in the helix and in the structure of bound water. The primary crystal contact is the same in both crystal forms, involving both sheet edges to form extended beta-sheets throughout the crystals.
TL;DR: The crystal structure of avian recombinant FPS, the first three-dimensional structure for any prenyltransferase, was determined to 2.6-A resolution, and suggests that the conserved aspartate residues participate in substrate binding of catalysis.
Abstract: The synthesis of farnesyl diphosphate (FPP), a key intermediate in the isoprenoid biosynthetic pathway required for the synthesis of cholesterol and in the formation of prenylated proteins, is catalyzed by the enzyme farnesyl diphosphate synthase (FPS). The crystal structure of avian recombinant FPS, the first three-dimensional structure for any prenyltransferase, was determined to 2.6-A resolution. The enzyme exhibits a novel fold composed entirely of alpha-helices joined by connecting loops. The enzyme's most prominent structural feature is the arrangement of 10 core helices around a large central cavity. Two aspartate-rich sequences that are highly conserved among the isoprenyl diphosphate synthase family of prenyltransferases, and are essential for enzymatic activity, were found on opposite walls of this cavity, with the aspartate side chains approximately 12 A apart and facing each other. The location and metal ion binding properties of these sequences suggest that the conserved aspartate residues participate in substrate binding of catalysis.
TL;DR: The results of these studies provide a thermodynamic scale for the relative beta-sheet forming propensities of the amino acids that shows a clear correlation with the beta- sheet preferences derived from statistical surveys of proteins of known structure.
Abstract: The results of a study to measure the beta-sheet forming propensities of the 20 naturally occurring amino acids are presented. The protein host for these studies is the 56 amino acid B1 domain of staphylococcal IgG binding protein G [Fahnestock, S.R., Alexander, P., Nagle, J., & Filpula, D. (1986) J. Bacteriol. 167, 870-880]. This protein was selected because it exhibits a reversible two-state thermal denaturation transition and its structure is known at high resolution. A suitable guest position in the protein was identified, and its neighboring environment was modified to minimize the potential for artifactual interactions. All 20 amino acids were individually substituted at the guest site, and their effect on the protein's thermal stability was determined. NMR was used to verify the structural integrity of several of the proteins with different amino acid substitutions at the guest site. The results of these studies provide a thermodynamic scale for the relative beta-sheet forming propensities of the amino acids that shows a clear correlation with the beta-sheet preferences derived from statistical surveys of proteins of known structure.
TL;DR: Molecular modeling of the structure of Temozolomide indicates that the prodrug can make a favorable noncovalent encounter with DNA and the known structure-activity relationships as well as the biological and clinical properties of temozolmide can be interpreted in terms of this model.
Abstract: The hypothesis that the antitumor prodrug temozolomide is ring-opened to MTIC which then further breaks down to a reactive diazonium ion at guanine-rich sequences in DNA has been probed by NMR spectroscopy and computational techniques. Temozolomide is stable at acid pH but decomposes to MTIC at pH > 7; in contrast, MTIC is stable at alkaline pH values but rapidly fragments in a methylating mode at pH < 7. The proximate methylating agent is the reactive methyldiazonium species. Runs of guanine residues represent an accessible nucleophilic microenvironment in DNA site-specific conversion of the prodrug temozolomide to MTIC possibly via an activated water molecule in the major groove. Molecular modeling of the structure of temozolomide indicates that the prodrug can make a favorable noncovalent encounter with DNA. The known structure-activity relationships as well as the biological and clinical properties of temozolomide can be interpreted in terms of this model.
TL;DR: General features of the nitrogenase system, including conformational coupling of nucleotide hydrolysis, aspects of the cluster structures, and the general spatial organization of redox centers within the protein subunits, are relevant to a wide range of biochemical systems.
Abstract: Biological nitrogen fixation is catalyzed by the nitrogenase enzyme system which consists of
two metalloproteins, the iron (Fe-) protein and the molybdenum-iron (MoFe-) protein. Together, these
proteins mediate the ATP-dependent reduction of dinitrogen to ammonia. Recent crystallographic analyses
of Fe-protein and MoFe-protein have revealed the polypeptide fold and the structure and organization of
the unusual metal centers in nitrogenase. These structure provide a molecular framework for addressing
the mechanism of the nitrogenase-catalyzed reaction. General features of the nitrogenase system, including
conformational coupling of nucleotide hydrolysis, aspects of the cluster structures, and the general spatial
organization of redox centers within the protein subunits, are relevant to a wide range of biochemical
systems.
TL;DR: The exact behavior of a given protein at low pH is a complex interplay between a variety of stabilizing and destabilizing forces, some of which are very sensitive to the environment.
Abstract: A systematic investigation of the effect of acid on the denaturation of some 20 monomeric proteins indicates that several different types of conformational behavior occur, depending on the protein, the acid, the presence of salts or denaturant, and the temperature. Three major types of effects were observed. Type I proteins, when titrated with HCl in the absence of salts, show two transitions, initially unfolding in the vicinity of pH 3-4 and then refolding to a molten globule-like conformation, the A state, at lower pH. Two variations in this behavior were noted: some type I proteins, when titrated with HCl in the absence of salts, show only partial unfolding at pH 2 before the transition to the molten globule state; others of this class form an A state that is a less compact from of the molten globule state. In the presence of salts, these proteins transform directly from the native state to the molten globule conformation. Type II proteins, upon acid titration, do not fully unfold but directly transform to the molten globule state, typically in the vicinity of pH 3. Type III proteins show no significant unfolding to pH as low as 1, but may be caused to behave similarly to type I in the presence of urea. Thus, the exact behavior of a given protein at low pH is a complex interplay between a variety of stabilizing and destabilizing forces, some of which are very sensitive to the environment. In particular, the protein conformation is quite sensitive to salts (anions) that affect the electrostatic interactions, denaturants, and temperature, which cause additional global destabilization.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: Using the FLP/FRT system for site-specific recombination and the wild-type recognition site (FRT) in conjunction with certain mutant FRT sites, it becomes possible to provoke, with high yield, a double-reciprocal crossover event in cultured mammalian cells.
Abstract: Using the FLP/FRT system for site-specific recombination and the wild-type recognition site (FRT) in conjunction with certain mutant FRT sites, it becomes possible to provoke, with high yield, a double-reciprocal crossover event in cultured mammalian cells. It is demonstrated that this technology enables a targeting of expression cassettes to appropriate chromosomal reference sites in the recipient cell to improve the concepts of reverse genetics. The design of mutant FRT sites promoting such a process will be delineated. Our results show that the five spacer mutations tested are functional as the wild type but differ in the extent of their cross-recombination, which has to be minimized for their simultaneous usage.
TL;DR: The nifS gene product is a pyridoxal phosphate binding enzyme that catalyzes the desulfurization of L-cysteine to yield L-alanine and sulfur in Azotobacter vinelandii and it is suggested that NIFS participates in the biosynthesis of the nitrogenase metalloclusters by providing the inorganic sulfur required for Fe-S core formation.
Abstract: The nifS gene product (NIFS) is a pyridoxal phosphate binding enzyme that catalyzes the desulfurization of L-cysteine to yield L-alanine and sulfur In Azotobacter vinelandii this activity is required for the full activation of the nitrogenase component proteins Because the nitrogenase component proteins, Fe protein and MoFe protein, both contain metalloclusters which are required for their respective activities, it is suggested that NIFS participates in the biosynthesis of the nitrogenase metalloclusters by providing the inorganic sulfur required for Fe-S core formation [Zheng, L, White, R H, Cash, V L Jack, R F, & Dean, D R (1993) Proc Natl Acad Sci USA 90, 2754-2758] In the present study the mechanism for the desulfurization of L-cysteine catalyzed by NIFS was determined in the following ways First, the substrate analogs, L-allylglycine and vinylglycine, were shown to irreversibly inactivate NIFS by formation of a gamma-methylcystathionyl or cystathionyl residue, respectively, through nucleophilic attack by an active site cysteinyl residue on the corresponding analog-pyridoxal phosphate adduct Second, this reactive cysteinyl residue, which is required for L-cysteine desulfurization activity, was identified as Cys325 by the specific alkylation of that residue and by site-directed mutagenesis experiments Third, the formation of an enzyme-bound cysteinyl persulfide was identified as an intermediate in the NIFS-catalyzed reaction Fourth, evidence was obtained for an enamine intermediate in the formation of L-alanine(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: Indole diterpenes are the most potent nonpeptidyl inhibitors of maxi-K channels identified to date, although tremorgenicity may be unrelated to channel block.
Abstract: Tremorgenic indole alkaloids produce neurological disorders (e.g., staggers syndromes) in ruminants. The mode of action of these fungal mycotoxins is not understood but may be related to their known effects on neurotransmitter release. To determine whether these effects could be due to inhibition of K+ channels, the interaction of various indole diterpenes with high-conductance Ca(2+)-activated K+ (maxi-K) channels was examined. Paspalitrem A, paspalitrem C, aflatrem, penitrem A, and paspalinine inhibit binding of [125I]charybdotoxin (ChTX) to maxi-K channels in bovine aortic smooth muscle sarcolemmal membranes. In contrast, three structurally related compounds, paxilline, verruculogen, and paspalicine, enhanced toxin binding. As predicted from the binding studies, covalent incorporation of [125I]ChTX into the 31-kDa subunit of the maxi-K channel was blocked by compounds that inhibit [125I]ChTX binding and enhanced by compounds that stimulate [125I]ChTX binding. Modulation of [125I]ChTX binding was due to allosteric mechanisms. Despite their different effects on binding of [125I]ChTX to maxi-K channels, all compounds potently inhibited maxi-K channels in electrophysiological experiments. Other types of voltage-dependent or Ca(2+)-activated K+ channels examined were not affected. Chemical modifications of paxilline indicate a defined structure-activity relationship for channel inhibition. Paspalicine, a deshydroxy analog of paspalinine lacking tremorgenic activity, also potently blocked maxi-K channels. Taken together, these data suggest that indole diterpenes are the most potent nonpeptidyl inhibitors of maxi-K channels identified to date. Some of their pharmacological properties could be explained by inhibition of maxi-K channels, although tremorgenicity may be unrelated to channel block.
TL;DR: Data indicate that 2C19 is the principal determinant of (S)-mephenytoin 4'-hydroxylase activity in human liver.
Abstract: The present study assesses the role of members of the human CYP2C subfamily in the 4'-hydroxylation of (S)-mephenytoin. When recombinant CYP2C proteins were expressed using a yeast cDNA expression system, 2C19 stereospecifically 4'-hydroxylated (S)-mephenytoin with a turnover number at least 10 times higher than that of human liver microsomes. 2C9 (both Ile359 and Leu359 alleles) and 2C18 (Thr385 and Met385 alleles) metabolized this substrate at a rate 100-fold lower than 2C19, and metabolism by these 2C proteins was not stereospecific for the S-enantiomer. 2C8 exhibited very little mephenytoin 4'-hydroxylase activity. In contrast, the Ile359 allele of 2C9 had a high turnover number for the hydroxylation of tolbutamide, while the Leu359 allele was less active toward this substrate. Immunoblot analysis of 16 human liver donor samples indicated that (S)-mephenytoin 4'-hydroxylase activity correlated with the hepatic CYP2C19 content, but it did not correlate with the hepatic content of CYP2C9. Moreover, direct sequencing of the polymerase chain reaction (PCR) products of 2C9 mRNA from six of these human livers through areas of known allelic variations indicated that the identity of the allele of 2C9 (Cys144 vs Arg, Tyr358 vs Cys, Ile359 vs Leu, or Gly417 vs Asp) did not appear to influence (S)-mephenytoin 4'-hydroxylase activity in these samples. These data indicate that 2C19 is the principal determinant of (S)-mephenytoin 4'-hydroxylase activity in human liver.
TL;DR: The 2.0-å resolution crystal structure of a 1:1 complex between the bacterial ribonuclease, barnase, and a Cys-->Ala(40,82) double mutant of its intracellular polypeptide inhibitor, barstar is solved and shows that the overall structural response to barnase-binding is significant.
Abstract: We have solved, refined, and analyzed the 2.0-a resolution crystal structure of a 1:1 complex between the bacterial ribonuclease, barnase, and a Cys-->Ala(40,82) double mutant of its intracellular polypeptide inhibitor, barstar. Barstar inhibits barnase by sterically blocking the active site with a helix and adjacent loop segment. Almost half of the 14 hydrogen bonds between barnase and barstar involve two charged residues, and a third involve one charged partner. The electrostatic contribution to the overall binding energy is considerably greater than for other protein-protein interactions. Consequently, the very high rate constant for the barnase-barstar association (10(8) s-1 M-1) is most likely due to electrostatic steering effects. The barnase active-site residue His102 is located in a pocket on the surface of barstar, and its hydrogen bonds with Asp39 and Gly31 residues of barstar are directly responsible for the pH dependence of barnase-barstar binding. There is a high degree of complementarity both of the shape and of the charge of the interacting surfaces, but neither is perfect. The surface complementarity is slightly poorer than in protease-inhibitor complexes but a little better than in antibody-antigen interactions. However, since the burial of solvent in the barnase-barstar interface improves the fit significantly by filling in the majority of gaps, as well as stabilizing unfavorable electrostatic interactions, its role seems to be more important than in other protein-protein complexes. The electrostatic interactions between barnase and barstar are very similar to those between barnase and the tetranucleotide d(CGAC). In the barnase-barstar complex, the two phosphate-binding sites in the barnase active site are occupied by Asp39 and Gly43 of barstar. However, barstar has no equivalent for a guanine base of an RNA substrate, resulting in the occupation of the guanine recognition site in the barnase-barstar complex by nine ordered water molecules. Upon barnase-barstar binding, entropy losses resulting from the immobilization of segments of the protein chain and the energetic costs of conformational changes are minimized due to the essentially preformed active site of barnase. However, a certain degree of flexibility within the barnase active site is required to allow for the structural differences between barnase-barstar binding and barnase-RNA binding. A comparison between the bound and the free barstar structure shows that the overall structural response to barnase-binding is significant. This response can be best described as outwardly oriented, rigid-body movements of the four alpha-helices of barstar, resulting in the structure of bound barstar being somewhat expanded.
TL;DR: X-ray diffraction studies yielded 3 A resolution crystal structures of hemagglutinin in complex with four of the synthetic analogs and with the naturally occurring cell-surface saccharide (alpha 2-3)sialyllactose, which could lead to the design of tight binding inhibitors of possible therapeutic value.
Abstract: The interaction between influenza virus hemagglutinin and its cell-surface receptor, 5-N-acetylneuraminic acid (sialic acid), was probed by the synthesis of 12 sialic acid analogs, including derivatives at the 2-carboxylate, 5-acetamido, 4-, 7-, and 9-hydroxyl, and glycosidic positions. The equilibrium dissociation constants of these analogs were determined by nuclear magnetic resonance spectroscopy. Ligand modifications that reduced or abolished binding included the replacement of the 2-carboxylate with a carboxamide, the substitution of azido or N-benzyloxycarbonyl groups for the 5-acetamido group, and the replacement of the 9-hydroxyl with amino or O-acetyl moieties. Modifications having little effect on binding included the introduction of longer chains at the 4-hydroxyl position, the replacement of the acetamido methyl group with an ethyl group, and the removal of the 7-hydroxyl group. X-ray diffraction studies yielded 3 A resolution crystal structures of hemagglutinin in complex with four of the synthetic analogs [alpha-2-O-methyl-, 4-O-acetyl-alpha-2-O-methyl-, 9-amino-9-deoxy-alpha-2-O-methyl-, and alpha-2-O-(4'-benzylamidocarboxybutyl)-N-acetylneuraminic acid] and with the naturally occurring cell-surface saccharide (alpha 2-3)sialyllactose. The X-ray studies unambiguously establish the position and orientation of bound sialic acid, indicate the position of the lactose group of (alpha 2-3)sialyllactose, and suggest the location of an alpha-glycosidic chain (4'-benzylamidocarboxybutyl) that increases the binding affinity of sialic acid by a factor of about 3. Although the protein complexed with alpha-2-O-methylsialic acid contains the mutation Gly-135-->Arg near the ligand binding site, the mutation apparently does not affect the ligand's position. The X-ray studies allow us to interpret the binding affinities in terms of the crystallographic structure. The results suggest further experiments which could lead to the design of tight binding inhibitors of possible therapeutic value.
TL;DR: Measurements have shown that the presence of TMAO has no effect on the interaction of urea with the protein in either the native or the unfolded (reduced carboxymethylated RNase T1) state, and the effects of the two cosolvents on the transition temperature were found to be essentially the algebraic sum of their effects when used individually.
Abstract: Many organisms accumulate low molecular weight substances known as osmolytes when they experience environmental water stress. The main classes of osmolytes are sugars, polyhydric alcohols, amino acids and their derivatives, and methylamines, and all are known to be protein stabilizers. However, marine cartilaginous fishes and the coelacanth use, as osmolytes, a combination of urea and methylamines, i.e., a denaturant and a stabilizer, in a 2:1 molar ratio. Preferential binding and thermal denaturation measurements in the presence of each cosolvent separately and in their mixtures have been carried out using ribonuclease T1 (RNase T1) as the protein. At a 2:1 molar ratio of urea and trimethylamine N-oxide (TMAO), the effects of the two cosolvents on the transition temperature (Tm) were found to be essentially the algebraic sum of their effects when used individually. Preferential interaction measurements of urea, TMAO and urea in its 2:1 molar ratio mixture with TMAO, have shown that the presence of TMAO has no effect on the interaction of urea with the protein in either the native or the unfolded (reduced carboxymethylated RNase T1) state. The preferential interaction of TMAO in the presence of urea could not be measured for technical reasons. Calculations of transfer free energy in the two end states of the denaturation reaction have shown that 2 M urea destabilizes RNase T1 by 3.8 +/- 0.3 kcal/mol whether 1 M TMAO is present or not. The contribution of 1 M TMAO to stabilization is calculated to be 3.1 kcal/mol in the presence of 2 M urea and is measured to be 2.7 kcal/mol in its absence.
TL;DR: Kinetic analyses of these two substrates show that 7,8-benzoflavone increases the Vmax of phenanthrene metabolism without changing the Km and that Phenanthrene decreases the V max of 7, 8-benZ oflavone metabolism without increasing the KM, providing the first evidence that two different molecules can be simultaneously bound to the same P450 active site.
Abstract: A unique characteristic of the CYP3A subfamily of cytochrome P450 enzymes is their ability to be activated by certain compounds. It is reported that CYP3A4-catalyzed phenanthrene metabolism is activated by 7,8-benzoflavone and that 7,8-benzoflavone serves as a substrate for CYP3A4. Kinetic analyses of these two substrates show that 7,8-benzoflavone increases the Vmax of phenanthrene metabolism without changing the Km and that phenanthrene decreases the Vmax of 7,8-benzoflavone metabolism without increasing the Km. These results suggest that both substrates (or substrate and activator) are simultaneously present in the active site. Both compounds must have access to the active oxygen, since neither phenanthrene nor 7,8-benzoflavone can competitively inhibit the other substrate. These data provide the first evidence that two different molecules can be simultaneously bound to the same P450 active site. Additionally, structure-activity relationship studies were performed with derivatives of 7,8-benzoflavone structure. The effects of 13 different compounds on the regioselectivity of phenanthrene, chrysene, and benzo[a]pyrene metabolism were determined. Of the 13 compounds studied, 6 were activators, 2 were partial activators, and 5 were inhibitors. Analyses of the data suggest that (1) naphthalene substituted with a ketone in the 2-position can activate 3A4 and (2) the presence of an activator results in a narrower effective substrate binding site. Since the CYP3A enzymes are very important in drug metabolism, the possibility of activation, and autoactivation, must be considered when in vitro-in vivo correlations are made and when possible drug interactions are considered.
TL;DR: The results support the viability of TFE titration as a tool for extrapolation of quantitative helix-coil equilibrium constants for peptides with little or no apparent helical content in aqueous solution.
Abstract: The formation of local secondary structure is an essential step in the folding of a polypeptide from a random coil to a well-defined native conformation. Detection of hidden structural propensities in amino acid sequences may provide important insight into how this is accomplished. 2,2,2-Trifluoroethanol (TFE) has been shown to induce helical structure in polypeptides, and TFE titration has been used as a qualitative probe for helical tendency. We have investigated the propensity of five synthetic peptides to adopt helical structure in TFE. The free energy of helix formation exhibits linear dependence on the mole ratio of TFE to water, and the constant of proportionality (m-value) can be perturbed systematically by altering the peptide length and unsystematically by altering the temperature. Three peptides with closely related sequences but different N-cap residues show different titration behavior from 5 to 75 degrees C, suggesting that TFE acts only within the context of a preexisting helix-coil equilibrium. These observations can be reconciled with a model for TFE/H2O exchange at peptide binding sites. Our results support the viability of TFE titration as a tool for extrapolation of quantitative helix-coil equilibrium constants for peptides with little or no apparent helical content in aqueous solution.
TL;DR: Oligonucleotide ligands described here represent an initial set of lead compounds in the ongoing effort toward the development of potent and specific VEGF antagonists.
Abstract: The proliferation of new blood vessels (angiogenesis) is a process that accompanies many pathological conditions including rheumatoid arthritis and solid tumor growth. Among angiogenic cytokines that have been identified to date, vascular endothelial growth factor (VEGF) is one of the most potent. We used SELEX [systematic evolution of ligands by exponential enrichment; Tuerk, C., & Gold, L. (1990) Science 249, 505-510] to identify RNA ligands that bind to VEGF in a specific manner with affinities in the low nanomolar range. Ligands were selected from a starting pool of about 10(14) RNA molecules containing 30 randomized positions. Isolates from the affinity-enriched pool were grouped into six distinct families on the basis of primary and secondary structure similarities. Minimal sequence information required for high-affinity binding to VEGF is contained in 29-36-nucleotide motifs. Binding of truncated (minimal) high-affinity ligands to VEGF is competitive with that of other truncated ligands and heparin. Furthermore, truncated ligands from the six ligand families inhibit binding of [125I]VEGF to its cell-surface receptors. Oligonucleotide ligands described here represent an initial set of lead compounds in our ongoing effort toward the development of potent and specific VEGF antagonists.
TL;DR: It is concluded that the majority of the ADRP mutants have folding defects, and nearly all of the mutants studied show abnormal sensitivity to light compared to the wild type, and they activate transducin less efficiently.
Abstract: Autosomal dominant retinitis pigmentosa (ADRP) is a hereditary form of retinitis pigmentosa which accounts for about 15% of all types of the latter disease. Recently, close to 50 mutations, mostly point mutations, have been identified in the rhodopsin gene in ADRP patients. We have introduced these mutations in the synthetic bovine rhodopsin gene and herein report on the expression of the mutant genes in COS-1 cells and studies in vitro of the properties of the expressed opsins. The mutant phenotypes fall into three classes: Class I mutants are expressed in COS-1 cells at wild-type levels, form the normal rhodopsin chromophore with 11-cis-retinal, and are transported to the cell surface. However, on illumination, they activate transducin inefficiently. Class II mutants remain in the endoplasmic reticulum and do not bind 11-cis-retinal to form the chromophore. Class III mutants are expressed at low levels and form rhodopsin chromophore only poorly. They also remain in the endoplasmic reticulum and, as expected, show high mannose glycosylation. Nearly all of the mutants studied show abnormal sensitivity to light compared to the wild type, and they activate transducin less efficiently. We conclude that the majority of the ADRP mutants have folding defects.