scispace - formally typeset
Search or ask a question

Showing papers in "Biochemistry in 1998"


Journal ArticleDOI
TL;DR: Improved thermodynamic parameters for prediction of RNA duplex formation are derived from optical melting studies of 90 oligoribonucleotide duplexes containing only Watson-Crick base pairs, and on average, the new parameters predict DeltaG degrees37, DeltaH degrees, DeltaS degrees, and TM within the limit of the model.
Abstract: Improved thermodynamic parameters for prediction of RNA duplex formation are derived from optical melting studies of 90 oligoribonucleotide duplexes containing only Watson−Crick base pairs. To test end or base composition effects, new sets of duplexes are included that have identical nearest neighbors, but different base compositions and therefore different ends. Duplexes with terminal GC pairs are more stable than duplexes with the same nearest neighbors but terminal AU pairs. Penalizing terminal AU base pairs by 0.45 kcal/mol relative to terminal GC base pairs significantly improves predictions of ΔG°37 from a nearest-neighbor model. A physical model is suggested in which the differential treatment of AU and GC ends accounts for the dependence of the total number of Watson−Crick hydrogen bonds on the base composition of a duplex. On average, the new parameters predict ΔG°37, ΔH°, ΔS°, and T M within 3.2%, 6.0%, 6.8%, and 1.3 °C, respectively. These predictions are within the limit of the model, based on...

1,142 citations


Journal ArticleDOI
TL;DR: This study explores the proposal that PTPs may be regulated by reversible reduction/oxidation involving cellular oxidants such as hydrogen peroxide (H2O2) and proposes a chemical mechanism for reversible inactivation involving a cysteine sulfenic acid intermediate.
Abstract: Protein tyrosine phosphatases (PTPs) catalyze the hydrolysis of phosphotyrosine from specific signal-transducing proteins. Although regulatory mechanisms for protein kinases have been described, no general mechanism for controlling PTPs has been demonstrated. Numerous reports have shown that cellular redox status plays an important role in tyrosine phosphorylation-dependent signal transduction pathways. This study explores the proposal that PTPs may be regulated by reversible reduction/oxidation involving cellular oxidants such as hydrogen peroxide (H2O2). Recent reports indicated that H2O2 is transiently generated during growth factor stimulation and that H2O2 production is concomitant with relevant tyrosine phosphorylation. By use of recombinant enzymes, the effects of H2O2 on three PTPs [PTP1, LAR (leukocyte antigen-related), and VHR (vaccinia H1-related)] and three distinct serine/threonine protein phosphatases (PPs: PP2Cα, calcineurin, and λ phosphatase) were determined. Hydrogen peroxide had no app...

937 citations


Journal ArticleDOI
TL;DR: It is found that the interactions of four tryptophan analogues with phosphatidylcholine membranes find that the analogues reside in the vicinity of the glycerol group where they all cause similar modest changes in acyl chain organization and that hydrocarbon penetration was not increased by reduction of hydrogen bonding or electric dipole interaction ability.
Abstract: One of the ubiquitous features of membrane proteins is the preference of tryptophan and tyrosine residues for membrane surfaces that presumably arises from enhanced stability due to distinct interfacial interactions. The physical basis for this preference is widely believed to arise from amphipathic interactions related to imino group hydrogen bonding and/or dipole interactions. We have examined these and other possibilities for tryptophan's interfacial preference by using 1H magic angle spinning (MAS) chemical shift measurements, two-dimensional (2D) nuclear Overhauser effect spectroscopy (2D-NOESY) 1H MAS NMR, and solid state 2H NMR to study the interactions of four tryptophan analogues with phosphatidylcholine membranes. We find that the analogues reside in the vicinity of the glycerol group where they all cause similar modest changes in acyl chain organization and that hydrocarbon penetration was not increased by reduction of hydrogen bonding or electric dipole interaction ability. These observations ...

905 citations


Journal ArticleDOI
TL;DR: These studies provide a framework for deriving detailed structural and dynamical information from tryptophan fluorescence intensity and lifetime data in peptides and proteins.
Abstract: A general approach to dissecting the complex photophysics of tryptophan is presented and used to elucidate the effects of amino acid functional groups on tryptophan fluorescence. We have definitively identified the amino acid side chains that quench tryptophan fluorescence and delineated the respective quenching mechanisms in a simple model system. Steady-state and time-resolved fluorescence techniques, photochemical H-D exchange experiments, and transient absorption techniques were used to measure individual contributions to the total nonradiative rate for deactivation of the excited state, including intersystem crossing, solvent quenching, and excited-state proton and electron transfer rates. Eight amino acid side chains representing six functional groups quench 3-methylindole fluorescence with a 100-fold range in quenching rate constant. Lysine and tyrosine side chains quench by excited-state proton transfer; glutamine, asparagine, glutamic and aspartic acid, cysteine, and histidine side chains quench by excited-state electron transfer. These studies provide a framework for deriving detailed structural and dynamical information from tryptophan fluorescence intensity and lifetime data in peptides and proteins.

802 citations


Journal ArticleDOI
TL;DR: In contrast to other phosphopantetheinyl transferases (PPTases) previously examined, Sfp will modify the apo forms of heterologous recombinant proteins, including the PCP domain of Saccharomyces cerevisiae Lys2, the aryl carrier protein (ArCP) domain of Escherichia coli EntB, and the E. coli acyl carrierprotein (ACP) subunit, suggesting Sfp as a good candidate forheterologous coexpression
Abstract: The Bacillus subtilis enzyme Sfp, required for production of the lipoheptapeptide antibiotic surfactin, posttranslationally phosphopantetheinylates a serine residue in each of the seven peptidyl carrier protein domains of the first three subunits (SrfABC) of surfactin synthetase to yield docking sites for amino acid loading and peptide bond formation. With recombinant Sfp and 16−17-kDa peptidyl carrier protein (PCP) domains excised from the SrfB1 and SrfB2 modules as apo substrates, kcat values of 56−104 min-1 and Km values of 1.3−1.8 μM were determined, indicating equivalent recognition of the adjacent PCP domains by Sfp. In contrast to other phosphopantetheinyl transferases (PPTases) previously examined, Sfp will modify the apo forms of heterologous recombinant proteins, including the PCP domain of Saccharomyces cerevisiae Lys2 (involved in lysine biosynthesis), the aryl carrier protein (ArCP) domain of Escherichia coli EntB (involved in enterobactin biosynthesis), and the E. coli acyl carrier protein (...

622 citations


Journal ArticleDOI
TL;DR: It is demonstrated by CD and tryptophan fluorescence spectroscopy that copper induces changes to the tertiary structure of SHaPrP(29-231) and proposed that the binding of two copper ions to four octarepeats induces a more defined structure to this region.
Abstract: The infectious isoform of the prion protein (PrPSc) is derived from cellular PrP (PrPC) in a conversion reaction involving a dramatic reorganization of secondary and tertiary structure. While our understanding of the pathogenic role of PrPSc has grown, the normal physiologic function of PrPC still remains unclear. Using recombinant Syrian hamster prion protein [SHaPrP(29-231)], we investigated metal ions as possible ligands of PrP. Near-UV circular dichroism spectroscopy (CD) indicates that the conformation of SHaPrP(29-231) resembles PrPC purified from hamster brain. Here we demonstrate by CD and tryptophan (Trp) fluorescence spectroscopy that copper induces changes to the tertiary structure of SHaPrP(29-231). Binding of copper quenches the Trp fluorescence emission significantly, shifts the emission spectrum to shorter wavelengths, and also induces changes in the near-UV CD spectrum of SHaPrP(29-231). The binding sites are highly specific for Cu2+, as indicated by the lack of a change in Trp fluorescence emission with Ca2+, Co2+, Mg2+, Mn2+, Ni2+, and Zn2+. Binding of Cu2+ also promotes the conformational shift from a predominantly alpha-helical to a beta-sheet structure. Equilibrium dialysis experiments indicate a binding stoichiometry of approximately 2 copper molecules per PrP molecule at physiologically relevant concentrations, and pH titration of Cu2+ binding suggests a role for histidine as a chelating ligand. NMR spectroscopy has recently demonstrated that the octarepeats (PHGGGWGQ) in SHaPrP(29-231) lack secondary or tertiary structure in the absence of Cu2+. Our results suggest that each Cu2+ binds to a structure defined by two octarepeats (PHGGGWGQ) with one histidine and perhaps one glycine carbonyl chelating the ion. We propose that the binding of two copper ions to four octarepeats induces a more defined structure to this region.

525 citations


Journal ArticleDOI
TL;DR: The structural biology described herein for Abeta(1-40) suggests that the C-terminal two-thirds of the peptide is an alpha-helix in membrane-like environments, deprotonation of two acidic amino acids in the helix promotes a helix-coil conformational transition that precedes aggregation.
Abstract: The three-dimensional solution structure of the 40 residue amyloid beta-peptide, Abeta(1-40), has been determined using NMR spectroscopy at pH 5.1, in aqueous sodium dodecyl sulfate (SDS) micelles. In this environment, which simulates to some extent a water-membrane medium, the peptide is unstructured between residues 1 and 14 which are mainly polar and likely solvated by water. However, the rest of the protein adopts an alpha-helical conformation between residues 15 and 36 with a kink or hinge at 25-27. This largely hydrophobic region is likely solvated by SDS. Based on the derived structures, evidence is provided in support of a possible new location for the transmembrane domain of Abeta within the amyloid precursor protein (APP). Studies between pH 4.2 and 7.9 reveal a pH-dependent helix-coil conformational switch. At the lower pH values, where the carboxylate residues are protonated, the helix is uncharged, intact, and lipid-soluble. As the pH increases above 6. 0, part of the helical region (15-24) becomes less structured, particularly near residues E22 and D23 where deprotonation appears to facilitate unwinding of the helix. This pH-dependent unfolding to a random coil conformation precedes any tendency of this peptide to aggregate to a beta-sheet as the pH increases. The structural biology described herein for Abeta(1-40) suggests that (i) the C-terminal two-thirds of the peptide is an alpha-helix in membrane-like environments, (ii) deprotonation of two acidic amino acids in the helix promotes a helix-coil conformational transition that precedes aggregation, (iii) a mobile hinge exists in the helical region of Abeta(1-40) and this may be relevant to its membrane-inserting properties and conformational rearrangements, and (iv) the location of the transmembrane domain of amyloid precursor proteins may be different from that accepted in the literature. These results may provide new insight to the structural properties of amyloid beta-peptides of relevance to Alzheimer's disease.

509 citations


Journal ArticleDOI
TL;DR: The results demonstrate that various P450 isoforms may exhibit atypical enzyme kinetics depending on the substrate(s) employed and that these results may be explained by a model which includes simultaneous binding of two substrate molecules in the active site.
Abstract: Some cytochrome P450 catalyzed reactions show atypical kinetics, and these kinetic processes can be grouped into five categories: activation, autoactivation, partial inhibition, substrate inhibition, and biphasic saturation curves. A two-site model in which the enzyme can bind two substrate molecules simultaneously is presented which can be used to describe all of these observed kinetic properties. Sigmoidal kinetic characteristics were observed for carbamazepine metabolism by CYP3A4 and naphthalene metabolism by CYPs 2B6, 2C8, 2C9, and 3A5 as well as dapsone metabolism by CYP2C9. Naphthalene metabolism by CYP3A4 and naproxen metabolism by CYP2C9 demonstrated nonhyperbolic enzyme kinetics suggestive of a low Km, low Vmax component for the first substrate molecule and a high Km, high Vmax component for the second substrate molecule. 7,8-Benzoflavone activation of phenanthrene metabolism by CYP3A4 and dapsone activation of flurbiprofen and naproxen metabolism by CYP2C9 were also observed. Furthermore, part...

505 citations


Journal ArticleDOI
TL;DR: In this paper, the effect of liposome surfacency on cellular uptake is examined, which is generally believed to be mediated by adsorption of the liposomes onto the cell surface and subsequent endocytosis.
Abstract: The cellular uptake of liposomes is generally believed to be mediated by adsorption of liposomes onto the cell surface and subsequent endocytosis. This report examines the effect of liposome surfac...

500 citations


Journal ArticleDOI
TL;DR: Observations are consistent with previous results using chronically rejecting human renal allografts and provide a compelling argument supporting the involvement of peroxynitrite during this pathophysiologic condition.
Abstract: Previous studies from our laboratory have demonstrated that the mitochondrial protein manganese superoxide dismutase is inactivated, tyrosine nitrated, and present as higher molecular mass species during human renal allograft rejection. To elucidate mechanisms whereby tyrosine modifications might result in loss of enzymatic activity and altered structure, the effects of specific biological oxidants on recombinant human manganese superoxide dismutase in vitro have been evaluated. Hydrogen peroxide or nitric oxide had no effect on enzymatic activity, tyrosine modification, or electrophoretic mobility. Exposure to either hypochlorous acid or tetranitromethane (pH 6) inhibited (approximately 50%) enzymatic activity and induced the formation of dityrosine and higher mass species. Treatment with tetranitromethane (pH 8) inhibited enzymatic activity 67% and induced the formation of nitrotyrosine. In contrast, peroxynitrite completely inhibited enzymatic activity and induced formation of both nitrotyrosine and dityrosine along with higher molecular mass species. Combination of real-time spectral analysis and electrospray mass spectroscopy revealed that only three (Y34, Y45, and Y193) of the nine total tyrosine residues in manganese superoxide dismutase were nitrated by peroxynitrite. Inspection of X-ray crystallographic data suggested that neighboring glutamate residues associated with two of these tyrosines may promote targeted nitration by peroxynitrite. Tyr34, which is present in the active site, appeared to be the most susceptible residue to peroxynitrite-mediated nitration. Collectively, these observations are consistent with previous results using chronically rejecting human renal allografts and provide a compelling argument supporting the involvement of peroxynitrite during this pathophysiologic condition.

493 citations


Journal ArticleDOI
TL;DR: It is indicated that cells in patients that express the MDR1 transporter will be relatively resistant to the anti-viral effects of the HIV-1 protease inhibitors, and that absorption, excretion, and distribution of these inhibitors in the body may be affected by the multidrug transporter.
Abstract: The FDA approved HIV-1 protease inhibitors, ritonavir, saquinavir, and indinavir, are very effective in inhibiting HIV-1 replication, but their long-term efficacy is unknown. Since in vivo efficacy depends on access of these drugs to intracellular sites where HIV-1 replicates, we determined whether these protease inhibitors are recognized by the MDR1 multidrug transporter (P-glycoprotein, or P-gp), thereby reducing their intracellular accumulation. In vitro studies in isolated membrane preparations from insect cells infected with MDR1-expressing recombinant baculovirus showed that these inhibitors significantly stimulated P-gp-specific ATPase activity and that this stimulation was inhibited by SDZ PSC 833, a potent inhibitor of P-gp. Furthermore, photoaffinity labeling of P-gp with the substrate analogue [125I]iodoarylazidoprazosin (IAAP) was inhibited by all three inhibitors. Cell-based approaches to evaluate the ability of these protease inhibitors to compete for transport of known P-gp substrates showe...

Journal ArticleDOI
TL;DR: The structures of the trigonal crystal form of bovine beta-lactoglobulin variant A at pH 6.2, 7.1, and 8.2 have been determined by X-ray diffraction methods and provide a structural basis for a variety of pH-dependent chemical, physical, and spectroscopic phenomena, collectively known as the Tanford transition.
Abstract: The structures of the trigonal crystal form of bovine β-lactoglobulin variant A at pH 6.2, 7.1, and 8.2 have been determined by X-ray diffraction methods at a resolution of 2.56, 2.24, and 2.49 A, respectively. The corresponding values for R (Rfree) are 0.192 (0.240), 0.234 (0.279), and 0.232 (0.277). The C and N termini as well as two disulfide bonds are clearly defined in these models. The glutamate side chain of residue 89 is buried at pH 6.2 and becomes exposed at pH 7.1 and 8.2. This conformational change, involving the loop 85−90, provides a structural basis for a variety of pH-dependent chemical, physical, and spectroscopic phenomena, collectively known as the Tanford transition.

Journal ArticleDOI
TL;DR: It is suggested that the peptide imposes positive curvature strain, facilitating the formation of a torus-type pore, and that the presence of negative curvature-inducing lipids inhibits pore formation.
Abstract: Magainin 2, an antimicrobial peptide from the Xenopus skin, kills bacteria by permeabilizing the cell membranes. We have proposed that the peptide preferentially interacts with acidic phospholipids to form a peptide-lipid supramolecular complex pore, which allows mutually coupled transbilayer traffic of ions, lipids, and peptides, thus simultaneously dissipating transmembrane potential and lipid asymmetry [Matsuzaki, K., Murase, O., Fujii, N., and Miyajima, K. (1996) Biochemistry 35, 11361-11368]. In this paper, we examined the effect of membrane curvature strain on pore formation. Magainin effectively forms the pore only in phosphatidylglycerol bilayers at low peptide-to-lipid ratios, well below 1/100. In contrast, the permeabilization of phosphatidylserine, phosphatidic acid, or cardiolipin bilayers occurred at much higher peptide-to-lipid ratios (1/50 to 1/10) with some morphological change of the vesicles. The latter three classes of phospholipids are known to form hexagonal II structures under conditions of reduced interlipid electrostatic repulsions. Incorporation of phosphatidylethanolamine also inhibited the magainin-induced pore formation in the inhibitory order of dioleoylphosphatidylethanolamine > dielaidoylphosphatidylethanolamine. Addition of a small amount of palmitoyllysophosphatidylcholine enhanced the peptide-induced permeabilization of phosphatidylglycerol bilayers. Magainin greatly raised the bilayer to hexagonal II phase transition temperature of dipalmitoleoylphosphatidylethanolamine. These results suggest that the peptide imposes positive curvature strain, facilitating the formation of a torus-type pore, and that the presence of negative curvature-inducing lipids inhibits pore formation.

Journal Article
TL;DR: In this article, the advantages and limitations of the use of fluorescence methods for the quantitative determination of the thermodynamics of protein unfolding transitions (i.e., induced by temperature or chemical denaturant) are discussed.
Abstract: The advantages and some limitations of the use of fluorescence methods for the quantitative determination of the thermodynamics of protein unfolding transitions (i.e., induced by temperature or chemical denaturant) are discussed. Advantages include the sensitivity, multi-dimensional nature of the data, wide amenable concentration range, high signal-to-noise, rapidity of measurement, and adaptability to a variety of sample compartments. Aside from the need for a probe, some problems associated with the method involve the handling of baselines for the pre- and post-transition regions and the difficulty (shared by most other methods) of discerning whether the transition is two-state or multi-state.

Journal ArticleDOI
TL;DR: Kinetic measurements reveal that the enzymes strongly prefers cleavage of the substrate over ligation of the two cleavage products and that the enzyme's catalytic efficiency is limited by the rate of substrate binding.
Abstract: We previously reported the in vitro selection of a general-purpose RNA-cleaving DNA enzyme that exhibits a catalytic efficiency (kcat/KM) exceeding that of any other known nucleic acid enzyme [Santoro, S. W. and Joyce, G. F. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4262-4266]. This enzyme contains approximately 30 deoxynucleotides and can cleave almost any RNA substrate under simulated physiological conditions, recognizing the substrate through two Watson-Crick binding domains. The kinetics of cleavage under conditions of varying pH, choice of divalent metal cofactor, and divalent metal concentration are consistent with a chemical mechanism involving metal-assisted deprotonation of a 2'-hydroxyl of the substrate, leading to substrate cleavage. Kinetic measurements reveal that the enzyme strongly prefers cleavage of the substrate over ligation of the two cleavage products and that the enzyme's catalytic efficiency is limited by the rate of substrate binding. The enzyme displays a high level of substrate specificity, discriminating against RNAs that contain a single base mismatch within either of the two substrate-recognition domains. With appropriate design of the substrate-recognition domains, the enzyme exhibits a potent combination of high substrate sequence specificity and selectivity, high catalytic efficiency, and rapid catalytic turnover.

Journal Article
TL;DR: In this paper, the biological significance of free radical generation induced by viral and bacterial infections was discussed, where the level of xanthine oxidase (XO) at the infected sites was elevated to a great extent.
Abstract: In recent years, accumulated evidence indicates that free radical species and nitric oxide (NO) or its derivatives are the key denominators in carcinogenesis. Our present topics discussed in this article will focus on the biological significance of free radical generation induced by viral and bacterial infections. In influenza virus infection in mice, the level of xanthine oxidase (XO) at the infected sites was elevated to a great extent. The timing of paralleled induction of XO with that of inducible NO synthase (iNOS) indicates efficient simultaneous reaction: NO + O2*- --> ONOO- (peroxynitrite). Peroxynitrite formation was identified by immunostaining of nitrotyrosine at the local site of infected organs. Peroxynitrite exhibits unique chemical reactivities such as protein nitration, DNA-strand breakage, guanine nitration, etc., which may then bring about not only cytotoxic effect but also mutagenesis. Numbers of evidence in vitro and in vivo show that treatment with chemical carcinogens such as carbon tetrachloride and heterocyclic amines also generated superoxide. The chronic inflammatory reactions, e.g., zymosan- and silica-induced granuloma, revealed very similar free radical generation in vivo. In addition, most experimental solid tumors have elevated levels of iNOS in the tumor tissue, and NO thus generated facilitates vascular permeability, which accelerates nutritional supply to the tumor tissue and hence sustains the rapid tumor growth. These circumstantial evidences suggest that inflammatory responses induced by various pathogens would accelerate mutagenesis as well as tissue damage, whereas NO also sustains more effectively solid tumor growth when normal cells are transformed to tumor or carcinoma cells by the host-derived free radical species.

Journal ArticleDOI
TL;DR: Modulation of PLD2 activity by synucleins may play a role in some aspects of the pathophysiologies that characterize these neurodegenerative diseases.
Abstract: Two widely expressed mammalian phosphatidylcholine (PC)-specific phospholipases D (PLD), PLD1 and PLD2, have been identified. Recombinantly expressed PLD2 has high basal activity and is insensitive to GTP-binding protein activators of PLD1 [Colley, W. C., et al. (1997) Curr. Biol. 7, 191-201]. To investigate the regulation of PLD2 we isolated PLD2, from mouse brain by immunoaffinity chromatography. The native and recombinant proteins have indistinguishable properties: PLD2 is potently activated by phosphoinositides with a vicinal 4,5-phosphate pair but is not stimulated by guanosine 5'-O-(3-thio triphosphate)-activated ADP-ribosylation factor-1, Rho family GTP-binding proteins, or protein kinases C-alpha, or -beta1. We used recombinant PLD2 in a reconstitution assay to search for regulators in cell and tissue extracts. Bovine brain contains a heat-stable protein factor that inhibits PLD2 activity in vitro. This factor was purified to homogeneity and identified as a mixture of alpha- and beta-synucleins by microsequencing and Western blotting. Recombinantly expressed alpha- and beta-synucleins inhibit PLD2 activity in vitro (K0.5 10 nM). Inhibition is not overcome by the protein or lipid activators of PLD1. Synucleins have been implicated in Parkinson's and Alzheimer's diseases. Our findings suggest that inhibition of PLD2 may be a function of synucleins. Modulation of PLD2 activity by synucleins may play a role in some aspects of the pathophysiologies that characterize these neurodegenerative diseases.

Journal ArticleDOI
TL;DR: The structural model is consistent with data from site-directed mutagenesis and binding of carbohydrate analogues, and allows the rational design of therapeutic Gb3 analogues that block the attachment of toxin to cells.
Abstract: Shiga-like toxin I (SLT-I) is a virulence factor of Escherichia coli strains that cause disease in humans. Like other members of the Shiga toxin family, it consists of an enzymatic (A) subunit and five copies of a binding subunit (the B-pentamer). The B-pentamer binds to a specific glycolipid, globotriaosylceramide (Gb3), on the surface of target cells and thereby plays a crucial role in the entry of the toxin. Here we present the crystal structure at 2.8 A resolution of the SLT-I B-pentamer complexed with an analogue of the Gb3 trisaccharide. The structure reveals a surprising density of binding sites, with three trisaccharide molecules bound to each B-subunit monomer of 69 residues. All 15 trisaccharides bind to one side of the B-pentamer, providing further evidence that this side faces the cell membrane. The structural model is consistent with data from site-directed mutagenesis and binding of carbohydrate analogues, and allows the rational design of therapeutic Gb3 analogues that block the attachment ...

Journal ArticleDOI
TL;DR: The unique selectivity of SNX-482 suggests its usefulness in studying the diversity, function, and pharmacology of class E and/or R-type Ca2+ channels.
Abstract: We describe the first potent and selective blocker of the class E Ca2+channel. SNX-482, a novel 41 amino acid peptide present in the venom of the African tarantula, Hysterocrates gigas, was identif...

Journal ArticleDOI
TL;DR: The processing of a number of ubiquitin derivatives by two human UCH isozymes is reported and it is found that these enzymes show little discrimination based on the P1' amino acid, except that proline is cleaved slowly.
Abstract: Ubiquitin C-terminal hydrolases (UCH) are deubiquitinating enzymes which hydrolyze C-terminal esters and amides of ubiquitin. Here we report the processing of a number of ubiquitin derivatives by two human UCH isozymes (isozymes L1 and L3) and find that these enzymes show little discrimination based on the P1‘ amino acid, except that proline is cleaved slowly. Ubiquitinyllysine derivatives linked by the α- or e-amino group are hydrolyzed at identical rates. Isozyme-specific hydrolytic preferences are only evident when the leaving group is large. The ubiquitin gene products can be cotranslationally processed by one or both of these UCH isozymes, and purified UbCEP52 can be hydrolyzed by UCH isozyme L3. Binding of nucleic acid by UbCEP52 converts it to a form resistant to processing by these enzymes, apparently because of the formation of a larger, more tightly folded substrate. Consistent with this postulate is the observation that these enzymes do not hydrolyze large ubiquitin derivatives such as Ne-ubiqu...

Journal ArticleDOI
TL;DR: A quantitative assay system that permits monitoring of the assembly of PHFs in real time by the fluorescence of dyes such as thioflavine S or T is reported, and it is shown that the assembly is optimal at pH approximately 6 and low ionic strengths and increases steeply with temperatures above 30 degreesC, indicating that it is an entropy-driven process.
Abstract: Alzheimer's disease is characterized by the progressive deposition of two types of fibers in the affected brains, the amyloid fibers (consisting of the Aβ peptide, generating the amyloid plaques) a...

Journal ArticleDOI
TL;DR: In this article, a very accurate addition (called structure X here) to the B-DNA dodecamer family of X-ray structures was described, which confirmed the observation of Drew and Dickerson [(1981) J. Mol. Biol. 151, 535−556] that the spine of hydration in AT tract DNA is two layers deep.
Abstract: We describe a very accurate addition (called structure X here) to the B-DNA dodecamer family of X-ray structures. Our results confirm the observation of Drew and Dickerson [(1981) J. Mol. Biol. 151, 535−556] that the spine of hydration in AT tract DNA is two layers deep. However, our results suggest that the primary spine is partially occupied by sodium ions. We suggest that many sequence-dependent features of DNA conformation are mediated by site specific binding of cations. For example, preferential localization of cations, as described here within the minor groove of structure X, is probably the structural origin of AT tract bending and groove narrowing. The secondary spine, which does not interact directly with the DNA, is as geometrically regular as the primary spine, providing a model for transmission of sequence information into solvent regions. A fully hydrated magnesium ion located in the major groove of structure X appears to pull cytosine bases partially out from the helical stack, exposing π-s...

Journal ArticleDOI
TL;DR: An improved basis for predicting the rates of thiol-disulfide exchange reactions is provided and ways in which differences in the rate of competing reactions can be either minimized, to simplify the analysis of disulfide-coupled folding reactions, or enhanced, to favor formation of particular disulfides are suggested.
Abstract: Thiol-disulfide exchange reactions are required for many aspects of cellular metabolism including the folding of disulfide-bonded proteins, electron transfer, and numerous regulatory mechanisms. To identify factors influencing the rates of these reactions in polypeptides, the reactivities of Cys thiols in 16 model peptides were measured. For each of the peptides, which contained single Cys residues with thiol pKas ranging from 7.4 to 9.1, the rates of exchange with four disulfide-bonded compounds were measured. In reactions with two of the disulfide reagents, cystine and 2-hydroxyethyl disulfide, the peptide thiols displayed Bronsted correlations between reaction rate and pKa similar to those observed previously with model compounds (betanuc = 0.5 and 0.3, respectively). For two reagents with net charges, oxidized glutathione and cystamine, however, the apparent Bronsted coefficients were 0 and 0.8, respectively. These observations are in striking contrast with those obtained with model compounds, for which the Bronsted coefficients for the nucleophilic thiolates are largely independent of the disulfide-containing compound. The differences in the apparent Bronsted coefficients can be largely accounted for by electrostatic interactions between charged groups on the peptides and disulfide reagents and demonstrate that such interactions can play a dominant role in determining the rates of thiol-disulfide exchange in biological molecules. The results presented here provide an improved basis for predicting the rates of these reactions and suggest ways in which differences in the rates of competing reactions can be either minimized, to simplify the analysis of disulfide-coupled folding reactions, or enhanced, to favor formation of particular disulfides.

Journal ArticleDOI
TL;DR: The data indicate that SeMet quenching of Trp fluorescence could become a simple and useful tool for studies of protein folding, and protein-protein andprotein-peptide interactions.
Abstract: The two interaction surfaces of the dumbbell-shaped calcium-regulatory protein calmodulin (CaM) are rich in the amino acid Met. In this work we have used fluorescence spectroscopy to study the role of these Met residues in binding the single Trp residue that is found in many CaM-binding domain peptides. This approach is facilitated by the absence of Trp residues in CaM. In addition to the wild-type protein, we studied CaM containing the unnatural amino acid selenomethionine (SeMet), which was biosynthetically substituted for its nine Met residues. Furthermore, a CaM mutant protein in which all four Met residues in the C-terminal domain were mutated to Leu, and the N-terminal domain contained either Met or the unnatural SeMet, was studied. The Trp fluorescence quantum yield of many Trp-containing CaM-binding peptides increases upon binding to calcium-CaM. Moreover, the emission wavelength of the Trp fluorescence is blue-shifted from 353 to 325−333 nm. These parameters indicate movement of Trp from a solven...

Journal ArticleDOI
TL;DR: 3,4,9, 10-perylenetetracarboxylic diimide-based ligands are identified as potent inhibitors of human telomerase by using a primer extension assay that does not use PCR-based amplification of the telomersase primer extension products.
Abstract: The single-stranded (TTAGGG)n tail of human telomeric DNA is known to form stable G-quadruplex structures. Optimal telomerase activity requires the nonfolded single-stranded form of the primer, and stabilization of the G-quadruplex form is known to interfere with telomerase binding. We have identified 3,4,9, 10-perylenetetracarboxylic diimide-based ligands as potent inhibitors of human telomerase by using a primer extension assay that does not use PCR-based amplification of the telomerase primer extension products. A set of NMR titrations of the ligand into solutions of G-quadruplexes using various oligonucleotides related to human telomeric DNA showed strong and specific binding of the ligand to the G-quadruplex. The exchange rate between bound and free DNA forms is slow on the NMR time scale and allows the unequivocal determination of the binding site and mode of binding. In the case of the 5'-TTAGGG sequence, the ligand-DNA complex consists of two quadruplexes oriented in a tail-to-tail manner with the ligand sandwiched between terminal G4 planes. Longer telomeric sequences, such as TTAGGGTT, TTAGGGTTA, and TAGGGTTA, form 1:1 ligand-quadruplex complexes with the ligand bound at the GT step by a threading intercalation mode. On the basis of 2D NOESY data, a model of the latter complex has been derived that is consistent with the available experimental data. The determination of the solution structure of this telomerase inhibitor bound to telomeric quadruplex DNA should help in the design of new anticancer agents with a unique and novel mechanism of action.

Journal ArticleDOI
TL;DR: Treatment of rat heart mitochondria with HNE resulted in the selective inhibition of KGDH and pyruvate dehydrogenase (PDH), while other NADH-linked dehydrogenases and electron chain complexes were unaffected.
Abstract: Previous research has established that 4-hydroxy-2-nonenal (HNE), a highly toxic product of lipid peroxidation, is a potent inhibitor of mitochondrial respiration. HNE exerts its effects on respiration by inhibiting alpha-ketoglutarate dehydrogenase (KGDH). Because of the central role of KGDH in metabolism and emerging evidence that free radicals contribute to mitochondrial dysfunction associated with numerous diseases, it is of great interest to further characterize the mechanism of inhibition. In the present study, treatment of rat heart mitochondria with HNE resulted in the selective inhibition of KGDH and pyruvate dehydrogenase (PDH), while other NADH-linked dehydrogenases and electron chain complexes were unaffected. KGDH and PDH are structurally and catalytically similar multienzyme complexes, suggesting a common mode of inhibition. To determine the mechanism of inhibition, the effects of HNE on purified KGDH and PDH were examined. These studies revealed that inactivation by HNE was greatly enhanced in the presence of substrates that reduce the sulfur atoms of lipoic acid covalently bound to the E2 subunits of KGDH and PDH. In addition, loss of enzyme activity induced by HNE correlated closely with a decrease in the availability of lipoic acid sulfhydryl groups. Use of anti-lipoic acid antibodies indicated that HNE modified lipoic acid in both purified enzyme preparations and mitochondria and that this modification was dependent upon the presence of substrates. These results therefore identify a potential mechanism whereby free radical production and subsequent lipid peroxidation lead to specific modification of KGDH and PDH and inhibition of NADH-linked mitochondrial respiration.

Journal ArticleDOI
TL;DR: It is found that S-nitrosothiols can react with thiols to generate nitroxyl (HNO) and the corresponding disulfide, which results in the generation of other species including NO, sulfinamide, and hydroxylamine.
Abstract: S-Nitrosothiols have been implicated to play key roles in a variety of physiological processes. The potential physiological importance of S-nitrosothiols prompted us to examine their reaction with thiols. We find that S-nitrosothiols can react with thiols to generate nitroxyl (HNO) and the corresponding disulfide. Further reaction of HNO with the remaining S-nitrosothiol and thiol results in the generation of other species including NO, sulfinamide, and hydroxylamine. Mechanisms are proposed that rationalize the observed products.

Journal ArticleDOI
TL;DR: The average membrane location of a series of diphenylhexatriene (DPH)-derived membrane probes was analyzed by measuring the quenching of DPH fluorescence with aseries of nitroxide-labeled lipids.
Abstract: The average membrane location of a series of diphenylhexatriene (DPH)-derived membrane probes was analyzed by measuring the quenching of DPH fluorescence with a series of nitroxide-labeled lipids i...

Journal ArticleDOI
TL;DR: It is proposed that HIV-1 resistance to AZT results from the selectively decreased binding of AZTTP and the increased pyroph phosphorolytic cleavage of chain-terminated viral DNA by the mutant RT at physiological pyrophosphate levels, resulting in a net decrease in chain termination.
Abstract: The multiple mutations associated with high-level AZT resistance (D67N, K70R, T215F, K219Q) arise in two separate subdomains of the viral reverse transcriptase (RT), suggesting that these mutations may contribute differently to overall resistance. We compared wild-type RT with the D67N/K70R/T215F/K219Q, D67N/K70R, and T215F/K219Q mutant enzymes. The D67N/K70R/T215F/K219Q mutant showed increased DNA polymerase processivity; this resulted from decreased template/primer dissociation from RT, and was due to the T215F/K219Q mutations. The D67N/K70R/T215F/K219Q mutant was less sensitive to AZTTP (IC50 approximately 300 nM) than wt RT (IC50 approximately 100 nM) in the presence of 0.5 mM pyrophosphate. This change in pyrophosphate-mediated sensitivity of the mutant enzyme was selective for AZTTP, since similar Km values for TTP and inhibition by ddCTP and ddGTP were noted with wt and mutant RT in the absence or in the presence of pyrophosphate. The D67N/K70R/T215F/K219Q mutant showed an increased rate of pyrophosphorolysis (the reverse reaction of DNA synthesis) of chain-terminated DNA; this enhanced pyrophosphorolysis was due to the D67N/K70R mutations. However, the processivity of pyrophosphorolysis was similar for the wild-type and mutant enzymes. We propose that HIV-1 resistance to AZT results from the selectively decreased binding of AZTTP and the increased pyrophosphorolytic cleavage of chain-terminated viral DNA by the mutant RT at physiological pyrophosphate levels, resulting in a net decrease in chain termination. The increased processivity of viral DNA synthesis may be important to enable facile HIV replication in the presence of AZT, by compensating for the increased reverse reaction rate.

Journal ArticleDOI
TL;DR: The structural basis for understanding a novel mechanism of ester hydrolysis is provided and the functional importance of the unusually modified amino acid is explained.
Abstract: Human lysosomal arylsulfatase A (ASA) is a prototype member of the sulfatase family. These enzymes require the posttranslational oxidation of the −CH2SH group of a conserved cysteine to an aldehyde...