Showing papers in "Biochemistry in 1995"

Short-chain dehydrogenases/reductases (SDR).

Abstract: Short-chain dehydrogenases/reductases (SDR) constitute a large protein family. Presently, at least 57 characterized, highly different enzymes belong to this family and typically exhibit residue identities only at the 15-30% level, indicating early duplicatory origins and extensive divergence. In addition, another family of 22 enzymes with extended protein chains exhibits part-chain SDR relationships and represents enzymes of no less than three EC classes. Furthermore, subforms and species variants are known of both families. In the combined SDR superfamily, only one residue is strictly conserved and ascribed a crucial enzymatic function (Tyr 151 in the numbering system of human NAD(+)-linked prostaglandin dehydrogenase). Such a function for this Tyr residue in SDR enzymes in general is supported also by chemical modifications, site-directed mutagenesis, and an active site position in those tertiary structures that have been characterized. A lysine residue four residues downstream is also largely conserved. A model for catalysis is available on the basis of these two residues. Binding of the coenzyme, NAD(H) or NADP(H), is in the N-terminal part of the molecules, where a common GlyXXXGlyXGly pattern occurs. Two SDR enzymes established by X-ray crystallography show a one-domain subunit with seven to eight beta-strands. Conformational patterns are highly similar, except for variations in the C-terminal parts. Additional structures occur in the family with extended chains. Some of the SDR molecules are known under more than one name, and one of the enzymes has been shown to be susceptible to native, chemical modification, producing reduced Schiff base adducts with pyruvate and other metabolic keto derivatives. Most SDR enzymes are dimers and tetramers. In those analyzed, the area of major subunit contacts involves two long alpha-helices (alpha E, alpha F) in similar and apparently strong subunit interactions. Future possibilities include verification of the proposed reaction mechanism and tracing of additional relationships, perhaps also with other protein families. Short-chain dehydrogenases illustrate the value of comparisons and diversified research in generating unexpected discoveries.

Kinetics of human soluble and membrane-bound catechol O-methyltransferase: a revised mechanism and description of the thermolabile variant of the enzyme.

Abstract: Human soluble (S) and membrane-bound (MB) catechol O-methyltransferase (COMT, EC 2.1.1.6) enzymes have been expressed at sufficiently high levels in Escherichia coli and in baculovirus-infected insect cells to allow kinetic characterization of the enzyme forms. The use of tight-binding inhibitors such as entacapone enabled the estimation of actual enzyme concentrations and, thereby, comparison of velocity parameters, substrate selectivity, and regioselectivity of the methylation of both enzyme forms. Kinetics of the methylation reaction of dopamine, (-)-noradrenaline, L-dopa, and 3,4-dihydroxybenzoic acid was studied in detail. Here, the catalytic number (Vmax) of S-COMT was somewhat higher than that of MB-COMT for all four substrates. The Km values varied considerably, depending on both substrate and enzyme form. S-COMT showed about 15 times higher Km values for catecholamines than MB-COMT. The distinctive difference between the enzyme forms was also the higher affinity of MB-COMT for the coenzyme S-adenosyl-L-methionine (AdoMet). The average dissociation constants Ks were 3.4 and 20.2 microM for MB-COMT and S-COMT, respectively. Comparison between the kinetic results and the atomic structure of S-COMT is presented, and a revised mechanism for the reaction cycle is discussed. Two recently published human COMT cDNA sequences differed in the position of S-COMT amino acid 108, the residue being either Val-108 [Lundstrom et al. (1991) DNA Cell. Biol. 10, 181-189] or Met-108 [Bertocci et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1416-1420].(ABSTRACT TRUNCATED AT 250 WORDS)

A DNA aptamer that binds adenosine and ATP.

Abstract: We have used in vitro selection to isolate adenosine/ATP-binding DNA sequences from a pool of approximately 2 x 10(14) different random-sequence single-stranded DNA molecules. One of these aptamers has been characterized and binds adenosine in solution with a dissociation constant of 6 +/- 3 microM. Experiments with ATP analogs indicate that functional groups on both the base and the sugar of ATP are involved in the ligand/aptamer interaction. The binding domain of this aptamer was localized to a 42 base sequence by deletion analysis. A pool of mutagenized versions of this sequence was then synthesized and screened for functional adenosine binding sequences; comparison of the selected variants revealed two highly conserved guanosine-rich regions, two invariant adenosine residues, and two regions of predominantly Watson--Crick covariation. This data led us to propose a model of the ATP-binding DNA structure which is based on a stable framework composed of two stacked G-quartets. The two highly conserved adenosine residues may stack between the top G-quartet and the two short stems, forming a pocket in which the adenosine or ATP ligand binds. Site-directed mutagenesis, base analog substitution studies, and the design of highly divergent but functional sequences provide support for this model.

Thermodynamic Parameters To Predict Stability of RNA/DNA Hybrid Duplexes

Abstract: The thermodynamic parameters (delta H degree, delta S degree, and delta G degree 37) for 16 nearest-neighbor sets and one initiation factor are presented here in order to predict stability of RNA/DNA hybrid duplexes. To determine the nearest-neighbor parameters, thermodynamics for 68 different hybrid sequences (136 single-stranded oligonucleotides) with 5-13 nucleotide length including several duplexes with identical nearest-neighbors were measured by UV melting procedure. These sequences were selected to have many different combinations of nearest-neighbor pairs, and so that the number of the 16 nearest-neighbor sequences in the oligomers were as equal as possible. The structures of the hybrids were also investigated by measuring circular dichroism spectra. Comparing delta G degree 37 values of the hybrids with DNA/DNA and RNA/RNA parameters reported previously (Breslauer, K.J., Frank, R., Blocker, H., & Marky, L.A. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3746-3750; Freier, S.M., Kierzek, R., Jaeger, J.A., Sugimoto, N., Caruthers, M.H., Neilson, T., & Turner, D.H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9373-9377), RNA/RNA double helix is the most stable of the three kinds of helixes with the same nearest-neighbor sequences. Which is more stable between DNA/DNA and RNA/DNA hybrid duplexes depends on its sequence. Calculated thermodynamic values of hybrid formation with the present parameters reproduce the experimental values within reasonable errors.

X-ray structures of the myosin motor domain of Dictyostelium discoideum complexed with MgADP.BeFx and MgADP.AlF4-.

Abstract: The three-dimensional structures of the truncated myosin head from Dictyostelium discoideum myosin II complexed with beryllium and aluminum fluoride and magnesium ADP are reported at 2.0 and 2.6 A resolution, respectively. Crystals of the beryllium fluoride-MgADP complex belong to space group P2(1)2(1)2 with unit cell parameters of a = 105.3 A, b = 182.6 A, and c = 54.7 A, whereas the crystals of the aluminum fluoride complex belong to the orthorhombic space group C222(1) with unit cell dimensions of a = 87.9 A, b = 149.0 A, and c = 153.8 A. Chemical modification was not necessary to obtain these crystals. These structures reveal the location of the nucleotide complexes and define the amino acid residues that form the active site. The tertiary structure of the protein complexed with MgADP.BeFx is essentially identical to that observed previously in the three-dimensional model of chicken skeletal muscle myosin subfragment-1 in which no nucleotide was present. By contrast, the complex with MgADP.AlF4- exhibits significant domain movements. The structures suggest that the MgADP.BeFx complex mimics the ATP bound state and the MgADP.AlF4- complex is an analog of the transition state for hydrolysis. The domain movements observed in the MgADP.AlF4- complex indicate that myosin undergoes a conformational change during hydrolysis that is not associated with the nucleotide binding pocket but rather occurs in the COOH-terminal segment of the myosin motor domain.

Mechanism of autoxidative glycosylation: identification of glyoxal and arabinose as intermediates in the autoxidative modification of proteins by glucose

Abstract: Glycation and oxidation reactions contribute to protein modification in aging and diabetes. Formation of dicarbonyl sugars during autoxidation of glucose is the hypothetical first step in the autoxidative glycosylation and subsequent browning of proteins by glucose [Wolff, S. P., & Dean, R. T. (1987) Biochem. J. 245, 243-250]. In order to identify the dicarbonyl sugar(s) formed during autoxidation of glucose under physiological conditions, glucose was incubated in phosphate buffer (pH 7.4) at 37 degrees C under air (oxidative conditions) or nitrogen with transition metal chelators (antioxidative conditions). Dicarbonyl compounds were analyzed spectrophotometrically and by HPLC after reaction with Girard-T reagent. Carbohydrates were analyzed by gas chromatography-mass spectrometry. Both dicarbonyl sugar and arabinose concentrations increased with time and glucose concentration in incubations conducted under oxidative conditions; only trace amounts of these products were detected in glucose incubated under antioxidative conditions. HPLC analysis of adducts formed with Girard-T reagent indicated that glyoxal was the only alpha-dicarbonyl sugar formed on autoxidation of glucose. Glyoxal and arabinose accounted for > or = 50% of the glucose lost during a 21 day incubation. Neither glucosone nor its degradation product, ribulose, was detectable. Reaction of glyoxal with RNase yielded the glycoxidation product, N epsilon-(carboxymethyl)lysine, while arabinose is a source of pentosidine. Our results implicate glyoxal and arabinose as intermediates in the browning and crosslinking of proteins by glucose under oxidative conditions. They also provide a mechanism by which antioxidants and dicarbonyl trapping reagents, such as aminoguanidine, limit glycoxidation reactions and support further evaluation of these types of compounds for inhibition of chemical modification and crosslinking of proteins during aging and diabetes.

Alteration of the Myometrial Plasma Membrane Cholesterol Content with .beta.-Cyclodextrin Modulates the Binding Affinity of the Oxytocin Receptor

Abstract: To investigate the effect of cholesterol on the oxytocin receptor function in myometrial membranes, we developed a new method to alter the membrane cholesterol content. Using a methyl-substituted beta-cyclodextrin, we were able to selectively deplete the myometrial plasma membrane of cholesterol. Vice versa, incubating cholesterol-depleted membranes with a preformed soluble cholesterol-methyl-beta-cyclodextrin complex restored the cholesterol content of the plasma membrane. Binding experiments showed that, with the removal of cholesterol from the membrane, the dissociation constant for [3H]oxytocin is enhanced 87-fold (from Kd = 1.5 nM to Kd = 131 nM), therefore shifting the oxytocin receptor from high to low affinity. Increasing the cholesterol content of the cholesterol-depleted membrane again restored the high-affinity binding (Kd = 1.2 nM). The presence of 0.1 mM GTP gamma S did not significantly change the number of high-affinity binding sites for [3H]oxytocin in native plasma membranes, in membranes depleted of cholesterol, and in plasma membranes with restored cholesterol content. The number of high-affinity binding sites for the oxytocin antagonist [3H]PrOTA was dependent in the same way on the cholesterol content as for [3H]oxytocin. Substitution of the membrane cholesterol with other steroids showed a strong dependence of the oxytocin receptor function on the structure of the cholesterol molecule. The detergent-solubilized oxytocin receptor was not saturable with [3H]oxytocin even at concentrations up to 10(-6) M of radioligand. Addition of the cholesterol-methyl-beta-cyclodextrin complex to the detergent-solubilized oxytocin receptor induced a saturation of the solubilized binding sites (Bmax = 0.98 pmol/mg) for oxytocin (Kd = 16 nM).(ABSTRACT TRUNCATED AT 250 WORDS)

N epsilon-(carboxymethyl)lysine is a dominant advanced glycation end product (AGE) antigen in tissue proteins.

Abstract: Advanced glycation end products (AGEs) and glycoxidation products are formed during Maillard or browning reactions between sugars and proteins and are implicated in the pathophysiology of aging and the complications of diabetes. To determine the structure of AGEs, antibodies were prepared to protein browned by incubation with glucose and used in ELISA assays to measure AGEs formed in model reactions between bovine serum albumin (BSA) or N alpha-acetyllysine and glucose, fructose, or glyoxal. AGEs were formed from glucose and fructose only under oxidative conditions, but from glyoxal under both oxidative and antioxidative conditions. Gel permeation chromatographic analysis indicated that a similar AGE was formed in reactions of N alpha-acetyllysine with glucose, fructose, and glyoxal and that this AGE co-eluted with authentic N alpha-acetyl-N epsilon-(carboxymethyl)lysine. Amino acid analysis of AGE proteins revealed a significant content of N epsilon-(carboxymethyl)lysine (CML). In ELISA assays using polyclonal antibodies against AGE proteins, CML-BSA (approximately 25 mol of CML/mol of BSA), prepared by chemical modification of BSA, was a potent inhibitor of the recognition of AGE proteins and of AGEs in human lens proteins. We conclude that AGEs are largely glycoxidation products and that CML is a major AGE recognized in tissue proteins by polyclonal antibodies to AGE proteins.

Crystallographic and molecular-modeling studies of lipase B from Candida antarctica reveal a stereospecificity pocket for secondary alcohols.

Abstract: Many lipases are potent catalysts of stereoselective reactions and are therefore of interest for use in chemical synthesis. The crystal structures of lipases show a large variation in the shapes of their active site environments that may explain the large variation in substrate specificity of these enzymes. We have determined the three-dimensional structure of Candida antarctica lipase B (CALB) cocrystallized with the detergent Tween 80. In another crystal form, the structure of the enzyme in complex with a covalently bound phosphonate inhibitor has been determined. In both structures, the active site is exposed to the external solvent. The potential lid-forming helix alpha 5 in CALB is well-ordered in the Tween 80 structure and disordered in the inhibitor complex. The tetrahedral intermediates of two chiral substrates have been modeled on the basis of available structural and biochemical information. The results of this study provide a structural explanation for the high stereoselectivity of CALB toward many secondary alcohols.

The peptide backbone plays a dominant role in protein stabilization by naturally occurring osmolytes

Abstract: Transfer free energy measurements of amino acids from water to the osmolytes, sucrose and sarcosine, were made as a function of osmolyte concentration. From these data, transfer free energies of the amino acid side chains were obtained, and the transfer free energy of the peptide backbone was determined from solubility measurements of diketopiperazine (DKP). Using static accessible surface evaluations of the native and unfolded states of ribonuclease A, solvent exposed side chain and peptide backbone areas were multiplied by their transfer free energies and summed in order to evaluate the transfer free energy of the native and unfolded states of the protein from water to the osmolyte solutions. The results reproduced the main features of the free energy profile determined for denaturation of proteins in the presence of osmolytes. The side chains were found collectively to favor exposure to the osmolyte in comparison to exposure in water, and in this sense the side chains favor protein unfolding. The major factor which opposes and overrides the side chain preference for denaturation and results in the stabilization of proteins observed in osmolytes is the highly unfavorable exposure of polypeptide backbone on unfolding. Except for urea and guanidine hydrochloride solutions, it is shown that all organic solvents (e.g., dioxane, ethanol, ethylene glycol) and solutes (osmolytes) for which transfer free energy measurements have been determined exhibit unfavorable transfer free energy of the peptide backbone.(ABSTRACT TRUNCATED AT 250 WORDS)

1.4 Å structure of photoactive yellow protein, a cytosolic photoreceptor: Unusual fold, active site, and chromophore

Abstract: A photosensing protein directs light energy captured by its chromophore into a photocycle. The protein's structure must accommodate the photocycle and promote the resulting chemical or conformational changes that lead to signal transduction. The 1.4 A crystallographic structure of photoactive yellow protein, determined by multiple isomorphous replacement methods, provides the first view at atomic resolution of a protein with a photocycle. The alpha/beta fold, which differs from the original chain tracing, shows striking similarity to distinct parts of the signal transduction proteins profilin and the SH2 domain. In the dark state structure of photoactive yellow protein, the novel 4-hydroxycinnamyl chromophore, covalently attached to Cys69, is buried within the major hydrophobic core of the protein and is tethered at both ends by hydrogen bonds. In the active site, the yellow anionic form of the chromophore is stabilized by hydrogen bonds from the side chains of Tyr42 and buried Glu46 to the phenolic oxygen atom and by electrostatic complementarity with the positively charged guanidinium group of Arg52. Thr50 further interlocks Tyr42, Glu46, and Arg52 through a network of active site hydrogen bonds. Arg52, located in a concavity of the protein surface adjacent to the dominant patch of negative electrostatic potential, shields the chromophore from solvent and is positioned to form a gateway for the phototactic signal. Overall, the high-resolution structure of photoactive yellow protein supports a mechanism whereby electrostatic interactions create an active site poised for photon-induced rearrangements and efficient protein-mediated signal transduction.

Molecular Basis for Membrane Selectivity of an Antimicrobial Peptide, Magainin 2

Abstract: Magainin peptides, isolated from Xenopus skin, kill bacteria by permeabilizing their cell membranes whereas they do not lyse erythrocytes. To elucidate the rationale for this membrane selectivity, we compared the effects of the membrane lipid composition and the transmembrane potential on the membrane-lytic power of magainin 2 with that of hemolytic melittin. The activity of magainin to zwitterionic phospholipids constituting the erythrocyte surface was extremely weak compared with that of melittin, and acidic phospholipids are necessary for effective action. The presence of sterols reduced the susceptibility of the membrane to magainin. The generation of an inside-negative transmembrane potential enhanced magainin-induced hemolysis. We can conclude that the absence of any acidic phospholipids on the outer monolayer and the abundant presence of cholesterol, combined with the lack of the transmembrane potential, contribute to the protection of erythrocytes from magainin's attack.

Elucidation of the primary and three-dimensional structure of the uterotonic polypeptide kalata b1

Abstract: The amino acid sequence and structure of a uterotonic polypeptide extracted from the African plant Oldenlandia affinis DC have been determined. The peptide, kalata B1, consists of 29 amino acid residues and is rich in cysteine (6), threonine (5), and glycine (5), Enzyme cleavage studies show that the polypeptide backbone is cyclic. The three-dimensional solution structure has been determined using two-dimensional nuclear magnetic resonance (NMR) spectroscopy and distance-restrained simulated annealing, Kalata B1 is composed mainly of beta-strands connected by tight turns, forming regions of beta-sheet, except in the case of one section which forms a longer, less structured loop. The tertiary fold, together with the disulfides that form a sulfur core, produces a striking and unusual surface in which the majority of the hydrophobic residues form. a solvent-exposed patch. The hydrophobic side of kalata B1 is flanked by two diametrically opposed and opposite-charged residues. The structure calculations have been used to predict the previously unknown disulfide bond connectivities using two approaches. In the first, a family of structures was calculated on the basis of NOE constraints without the assumption of a specific disulfide connectivity. The resultant structures were examined to determine whether the calculated position of the sulfur atoms suggested that one set of disulfide connectivities was more likely than the other, theoretically possible, sets. In the second approach, a separate family of structures (50 per set) was calculated for each of the 15 possible disulfide-bonded molecules. The resultant families of structures were compared to see whether one was favored over the others, Both approaches led to the same global fold, and the most likely disulfide connectivity is predicted to be 5-22, 13-27, and 17-29. In the calculated structure the cyclic peptide backbone is folded back onto itself and braced with disulfide pairs across diagonally opposed beta-strands, This structure involves one of the disulfide bonds (5-22) threading through the eight amino acid loop formed by the other two disulfide bonds and the peptide fragments connecting them.

Enzymatic characterization of the target for isoniazid in Mycobacterium tuberculosis.

Abstract: The inhA gene has been recently shown to encode a common protein target for isoniazid and ethionamide action in Mycobacterium tuberculosis. In this paper, we demonstrate that the M. tuberculosis InhA protein catalyzes the NADH-specific reduction of 2-trans-enoyl-ACP, essential for fatty acid elongation. This enzyme preferentially reduces long-chain substrates (12-24 carbons), consistent with its involvement in mycolic acid biosynthesis. Steady-state kinetic studies showed that the two substrates bind to InhA via a sequential kinetic mechanism, with the preferred ordered addition of NADH and the enoyl substrate. The chemical mechanism involves stereospecific hydride transfer of the 4S hydrogen of NADH to the C3 position of the 2-trans-enoyl substrate, followed by protonation at C2 of an enzyme-stabilized enolate intermediate. Kinetic and microcalorimetric analysis demonstrates that the binding of NADH to the S94A mutant InhA, known to confer resistance to both isoniazid and ethionamide, is altered. This difference can account for the isoniazid-resistance phenotype, with the formation of a binary InhA-NADH complex required for drug binding. Isoniazid binding to either the wild-type or S94A mutant InhA could not be detected by titration microcalorimetry, suggesting that this compound is a prodrug, which must be converted to its active form.

Prolines and amyloidogenicity in fragments of the Alzheimer's peptide beta/A4.

Abstract: Although it is well accepted that the structure of amyloid fibrils is dominated by some form of antiparallel beta-sheet, there are few details on the secondary structural arrangements of the constituent peptides and how these peptides pack together in the fibril. We describe here the use of scanning proline mutagenesis to map the secondary structural roles of each residue in amyloidogenic peptide fragments of the Alzheimer's amyloid peptide beta/A4. In two series of fragments related to residues 15-23 and 12-26 of beta/A4, we show that Pro replacement of any residue in the amyloidogenic sequence LVFFAED, corresponding to residues 17-23, leads to essentially complete loss of fibril formation and to excellent peptide solubility. Since peptidyl-prolyl bonds are incapable of forming standard extended chain conformations, the results suggest that residues 17-23 make up the beta-sheet core of the fibrils formed by these fragments. In contrast to the proline replacements, alanine substitutions at residues 17, 18, and 20 have no effect on fibril formation, while replacement of Phe19 reduces fibril formation to 15% of the level found for the wild type sequence. Scanning proline mutagenesis should play a useful role in mapping the secondary structural features of larger amyloidogenic peptide sequences, including longer, physiologically relevant forms of beta/A4. In addition, these results suggest explanations for some amyloidogenic effects observed in disease-related peptides and also suggest a possible role for aggregation-inhibiting insertion of prolines in protein evolution and protein design.

Design of lipoxin A4 stable analogs that block transmigration and adhesion of human neutrophils.

Abstract: Lipoxins (LX) are bioactive eicosanoids that carry a tetraene structure and serve as regulators of inflammation, in part by inhibiting neutrophil migration and adhesion. Lipoxin A4 is rapidly regulated by conversion to inactive LX metabolites via local metabolism that involves dehydrogenation as the predominant route. Here, several LXA4 analogs were designed that resisted rapid conversion by both differentiated HL-60 cells and recombinant 15-hydroxyprostaglandin dehydrogenase, systems where native LXA4 is degraded within minutes. The rank order of conversion by recombinant dehydrogenase was LXA4 methyl ester > PGE2 approximately PGE2 methyl ester > LXA4 >>> the novel LXA4 analogs. In addition, 15(R/S)-methyl-LXA4, 15-cyclohexyl-LXA4, and 16-phenoxy-LXA4 proved to retain LXA4 bioactivity and inhibited neutrophil transmigration across polarized epithelial cell monolayers as well as adhesion to vascular endothelial cells. These results indicate that LXA4 analogs can be designed using these criteria to resist rapid transformation and to retain biological actions of native LXA4. Moreover, the results suggest that LXA4 stable analogs can be useful tools both in vitro and in vivo to evaluate LXA4 actions and therapeutic potential.

Substoichiometric binding of taxol suppresses microtubule dynamics

Abstract: We have measured the effects of taxol (10 nM to 1 microM) on the growing and shortening dynamics at the ends of individual bovine brain microtubules in vitro and have correlated the effects both with the stoichiometry of taxol binding to tubulin in microtubules and with the changes in the microtubule polymer mass. The results indicate that taxol suppresses microtubule dynamic instability differently depending upon the stoichiometry of taxol binding to the microtubules. At the lowest effective concentrations ( or = 1 microM taxol and > or = 0.1 mol of taxol bound/mol of tubulin in microtubules), microtubule mass increases sharply and dynamics is almost completely suppressed. The data support the hypothesis that binding of a molecule of taxol to a tubulin subunit in microtubules induces a conformational change in that subunit that strongly reduces its ability to dissociate when the subunit becomes exposed at the microtubule end.

Analysis of the pH Dependence of the Neonatal Fc Receptor/Immunoglobulin G Interaction Using Antibody and Receptor Variants

Abstract: The neonatal Fe receptor (FcRn) binds maternal immunoglobulin G (IgG) from ingested milk in the gut (pH 6.0-6.5) and delivers it to the bloodstream of the newborn (pH 7.0-7.5). A soluble version of FcRn reproduces the physiological pH-dependent interaction with IgG, showing high-affinity binding at pH 6.0-6.5 but weak or no binding at pH 7.0-7.5. We have studied the pH dependence of the FcRn/IgG interaction using a surface plasmon resonance assay to measure kinetic and equilibrium constants. We show that the affinity of FcRn for IgG is reduced about 2 orders of magnitude as the pH is raised from 6.0 to 7.0. A Hill plot analysis suggests that several titrating residues participate in the pH-dependent affinity transition. Histidine side chains are likely candidates for residues that titrate between pH 6.0 and 7.0, and previous biochemical and structural work identified several histidines on the Fe portion of IgG that are located at the FcRn binding site. Using mutant IgG molecules and IgG subtype variants that differ in the number of histidines at the IgG/FcRn interface, we demonstrate that IgG histidines located at the junction between the CH2 and CH3 domains (residues 310 and 433) contribute to the pH-dependent affinity transition. Experiments with a mutant FcRn molecule show that two histidines on the FcRn heavy chain (residues 250 and 251) also contribute to the pH dependence of the FcRn/IgG interaction. These results are interpreted using the crystal structures of FcRn and an FcRn/Fc complex.

Stereochemistry of Chitin Hydrolysis by a Plant Chitinase/Lysozyme and X-ray Structure of a Complex with Allosamidin : Evidence for Substrate Assisted Catalysis

Abstract: The plant enzyme hevamine has both chitinase and lysozyme activity. HPLC analysis of the products of the hydrolysis of chitopentaose shows that hevamine acts with retention of the configuration, despite the absence of a nucleophilic or stabilizing carboxylate. To analyze the stabilization of a putative oxocarbonium ion intermediate, the X-ray structure of hevamine complexed with the inhibitor allosamidin was determined at 1.85 A resolution. This structure supports the role of Glu127 as a proton donor. The allosamizoline group binds in the center of the active site, mimicking a reaction intermediate in which a positive charge at C1 is stabilized intramolecularly by the carbonyl oxygen of the N-acetyl group at C2.

Interaction of the nuclear GTP-binding protein Ran with its regulatory proteins RCC1 and RanGAP1.

Abstract: The guanine nucleotide dissociation and GTPase reactions of Ran, a Ras-related nuclear protein, have been investigated using different fluorescence techniques to determine how these reactions are stimulated by the guanine nucleotide exchange factor RCC1 and the other regulatory protein, RanGAP1 (GTPase-activating protein) The intrinsic GTPase of Ran is one-tenth of the rate of p21ras and is even lower in the Ran(Q69L) mutant Under saturating conditions the rate constant for the RanGAP1 stimulated GTPase reaction is 21 s-1 at 25 degrees C, which is a 10(5)-fold stimulation, whereas RanGAP1 has no effect on Ran(Q69L) The intrinsic guanine nucleotide dissociation rates of Ran are also very low and are likewise increased 10(5)-fold by the exchange factor RCC1 Methods to describe the reaction kinetically are presented The Ran(T24N) mutant, which is analogous to the S17N mutant of p21ras, has decreased relative affinities for both GDP/GTP and favors GDP binding However, it was found to interact almost normally with RCC1 The combination of these properties leads to stabilization of the Ran(T24N)-RCC1 complex and may result in vivo in depletion of RCC1 available for stimulating guanine nucleotide exchange

Relative Thermodynamic Stability of DNA, RNA, and DNA:RNA Hybrid Duplexes: Relationship with Base Composition and Structure

Abstract: Fourteen oligonucleotides 8-21 nucleotides in length and their complements were synthesized as DNA and RNA For each sequence, four kinds of duplexes, DNA:DNA, RNA:RNA, DNA:RNA, and RNA:DNA, were prepared Twelve sequences had AT/U content varying from 25 to 80% and dPy content in the DNA strands varying from 0 to 100% Thermodynamic stabilities of four duplexes for each sequence were determined in solution containing 100 mM Na+, 10 mM phosphate, and 01 mM EDTA, pH 71 CD spectra and electrophoretic mobility on native polyacrylamide gel were measured for most duplexes Quantitative correlations of hybrid stability both with deoxypyrimidine content and, at fixed dPy content, with the fraction of AT/U in duplexes were found We also demonstrated that hybrids with 70-80% deoxypyrimidine DNA strand and a high or moderate AT/U fraction displayed the highest relative stability compared to their RNA counterparts Relationships of relative intensities of CD bands at 210 nm and relative electrophoretic mobilities of hybrids with relative hybrid stability suggested that hybrid conformation varies continuously between A- and B-form and is the decisive factor in relative hybrid stability

Reconstitution of Membrane Proteins into Lipid-Rich Bilayered Mixed Micelles for NMR Studies

Abstract: This paper describes a study undertaken to assess the possibility and practical consequences of reconstituting integral and peripheral membrane proteins into bilayered discoidal mixed micelles ("bicelles") composed of dimyristoylphosphatidylcholine and smaller amounts of either CHAPSO or short-chain phosphatidylcholine. The amphiphilic assemblies in these mixtures are uniquely suited for use in NMR structural studies because they can be magnetically oriented with experimentally-tunable system order. The first step of this study was to test about 15 membrane-associating polypeptides and proteins for their ability to interfere with magnetic orientation of the bicellar assemblies. A variety of results were obtained ranging from no perturbation to a complete disruption of orientation. Second, the suitability of bicelles as mimics of natural bilayers was tested by reconstituting diacylglycerol kinase, an integral membrane enzyme. The kinase was observed to be functional and completely stable for at least 24 h when incubated at 38 degrees C in bicelles. Third, the NMR spectra from a number of bicelle-reconstituted proteins were examined. In some cases, 13C NMR resonances from reconstituted proteins were extremely broad and asymmetric. In other cases, resonances from reconstituted proteins were moderately broad, but much less so than resonances from proteins reconstituted into multilayers oriented by mechanical methods. In the cases of two surface-associating proteins (cytochrome c and leucine enkephalin), oriented sample 13C NMR spectra of extremely high resolution were obtained. For these proteins it was also demonstrated that the experimentally variable order of the bicellar assemblies could be exploited to provide a means of screening for detergent-specific structural perturbations, for making spectral assignments, and for measuring chemical shift anisotropies and dipolar couplings. Taken as a whole, these results indicate that bicelles may be uniquely and effectively employed as model membranes to facilitate NMR structural studies of many, but not all, membrane proteins.

Membrane thinning caused by magainin 2.

Abstract: Magainin 2 is a 23-residue antibiotic peptide found in the skin of Xeonpus laevis (African clawed frog). It belongs to a broad class of alpha-helical peptides which interact directly with the lipid bilayer. Very little is presently known about the nature of this peptide/lipid interaction on the molecular level. We have performed a sequence of lamellar X-ray diffraction experiments to provide some insight into the nature of this interaction. We have found that, at concentrations below the critical concentration for lysis, the peptide causes the membrane thickness to decrease roughly in proportion to the peptide concentration. We further show that this thinning is consistent with a model where the peptide adsorbs within the headgroup region of the lipid bilayer at these concentrations. The energy cost of this thinning may also explain why the peptide inserts at high concentrations. We have already shown that a similar interaction exists for alamethicin interacting with diphytanoylphosphatidylcholine, and it should hold for a wide variety of peptide/lipid systems.

Nested cooperativity in the ATPase activity of the oligomeric chaperonin GroEL.

Abstract: Initial rates of ATP hydrolysis by wild-type GroEL were measured as a function of ATP concentration from 0 to 0.8 mM. Two allosteric transitions are observed: one at relatively low ATP concentrations ( Ala GroEL mutant [Yifrach, O., & Horovitz, A. (1994) J. Mol. Biol. 243, 397-401]. On the basis of these observations a mathematical model for nested cooperativity in ATP hydrolysis by GroEL is developed in which there are two levels of allostery: one within each ring and the second between rings. In the first level, each hepatameric ring is in equilibrium between the T and R states, in accordance with the Monod-Wyman-Changeux (MWC) model of cooperativity [Monod et al. (1965) J. Mol. Biol. 12, 88-118]. A second level of allostery is between the rings of the GroEL particle which undergoes sequential Koshland-Nemethy-Filmer (KNF)-type transitions from the TT state via the TR state to the RR state [Koshland et al. (1966) Biochemistry 5, 365-385]. Using our model, we estimate the values of the Hill coefficient for the negative cooperativity between rings in wild-type GroEL and the Arg-196-->Ala mutant to be 0.003 (+/- 0.001) and 0.07 (+/- 0.02), respectively. The inter-ring coupling free energies in wild-type GroEL and the Arg-196-->Ala mutant are -7.5 (+/- 0.4) and -3.9 (+/- 0.3) kcal mol-1, respectively.

Solution structure of the DNA binding domain of HIV-1 integrase.

Abstract: The solution structure of the DNA binding domain of HIV-1 integrase (residues 220-270) has been determined by multidimensional NMR spectroscopy. The protein is a dimer in solution, and each subunit is composed of a five-stranded P-barrel with a topology very similar to that of the SH3 domain. The dimer is formed by a stacked @-interface comprising strands 2, 3, and 4, with the two triple-stranded antiparallel P-sheets, one from each subunit, oriented antiparallel to each other. One surface of the dimer, bounded by the loop between strands Pl and P2, forms a saddle-shaped groove with dimensions of approximately 24 x 23 x 12 8, in cross section. Lys264, which has been shown from mutational data to be involved in DNA binding, protrudes from this surface, implicating the saddle- shaped groove as the potential DNA binding site. The integrase protein of the human immunodeficiency virus (HIV) mediates a key step in the life cycle of the virus, namely the integration of a DNA copy of the viral genome into a host chromosome (see Goff, 1992; Vink & Plasterk, 1993; for reviews). HIV-1’ integrase is composed of three functional domains (Bushman et al., 1993; Engelman et al., 1993; van Gent et al., 1993; Vink et al., 1993): a small N-terminal domain that contains a His2Cys2 zinc binding motif, a central catalytic domain whose crystal structure has recently been solved (Dyda et al., 1994), and a C-terminal DNA binding domain. While the catalytic core domain can carry out a simple polynucleotidyl transfer termed disintegra- tion (Bushman et al., 1993), all three domains are required for the 3’ processing and DNA strand transfer activities that accomplish integration of the viral genome (Drelich et al., 1992; Schauer & Billich, 1992; Vink et al., 1993). Conse- quently, the N- and C-terminal domains provide additional potentially useful targets for rational drug design aimed at inhibiting HIV integration into the host genome. The function of the N-terminal domain is at present unknown, and in the case of the related Rous Sarcoma virus integrase at least, its integrity appears not to be essential for

Interaction of the mammalian antibacterial peptide cecropin P1 with phospholipid vesicles.

Abstract: Cecropins are positively charged antibacterial polypeptides that were originally isolated from insects. Later on a mammalian homologue, cecropin P1 (CecP), was isolated from pig intestines. While insect cecropins are highly potent against both Gram-negative and Gram-positive bacteria, CecP is as active as insect cecropins against Gram-negative but has reduced activity against Gram-positive bacteria. To gain insight into the mechanism of action of CecP and the molecular basis of its antibacterial specificity, the peptide and its proline incorporated analogue (at the conserved position found in insect cecropins), P22-CecP, were synthesized and labeled on their N-terminal amino-acids with fluorescent probes, without significantly affecting their antibacterial activities. Fluorescence studies indicated that the N-terminal of CecP is located on the surface of phospholipid membranes. Binding experiments revealed that CecP binds acidic phosphatidylserine/phosphatidylcholine (PS/PC) vesicles better than zwitterionic PC vesicles, which correlates with its ability to permeate the former better than the latter. The shape of the binding isotherms suggest that CecP, like insect cecropin, binds phospholipids in a simple, noncooperative manner. However, resonance energy transfer (RET) measurements revealed that, unlike insect cecropins, CecP does not aggregate in the membrane even at relatively high peptide to lipid ratios. The stoichiometry of CecP binding to vesicles suggests that amount of CecP sufficient to form a monolayer causes vesicle permeation. In spite of the incorporation of the conserved proline in P22-CecP, the analogue has reduced antibacterial activity, which correlates with its reduced alpha-helical structure and its lower partitioning and membrane permeating activity with phospholipid vesicles. Taken together, our results support a mechanism in which CecP disrupts the structure of the bacterial membrane by (i) binding of peptide monomers to the acidic surface of the bacterial membrane and (ii) disintegrating the bacterial membrane by disrupting the lipid packing in the bilayers. These results, combined with data reported for other antibacterial polypeptides, suggest that the organization of peptide monomers within phospholipid membranes contributes to Gram-positive/Gram-negative antibacterial specificity.

Doubly-lipid-modified protein sequence motifs exhibit long-lived anchorage to lipid bilayer membranes.

Abstract: To understand better the potential functional importance of the dual-lipid modifications found in a number of intracellular proteins of eukaryotes, we have examined how "tenaciously" various doubly-lipid-modified peptides, with sequences and lipid modifications reflecting those found in intracellular proteins, are anchored to lipid bilayer membranes. Fluorescent-labeled peptides bearing dual-lipid modifications were incorporated into large unilamellar egg phosphatidylcholine/phosphatidylglycerol vesicles, and the kinetics of spontaneous intervesicle transfer of the lipopeptides were monitored by a fluorescence-dequenching assay. Lipopeptides incorporating the stable "dual-anchor" motif -C(geranylgeranyl)XC(geranylgeranyl)-OMe found in several rab and homologous proteins exhibit very slow rates of interbilayer transfer (t1/2 > 50 h), as do lipopeptides incorporating myristoyl-GC(palmitoyl)X- and -C(palmitoyl)XC(farnesyl)-OMe motifs found in various src-related intracellular tyrosine kinases and G-protein alpha-subunits and in p21H-ras, respectively. Lipopeptides terminating in an unmethylated -C(geranylgeranyl)C(geranylgeranyl)-OH motif show somewhat greater but still very slow rates of spontaneous interbilayer transfer (t1/2 = ca. 10 h). Extrapolating from these results, we estimate that the rate of spontaneous desorption of the corresponding doubly-anchored proteins from membranes should be much slower than that of regulated, protein-mediated release (effected by binding to an "escort" protein or by de-S-acylation). As a result the intracellular distributions of these species (and particularly their targeting to specific intracellular membranes) are likely to be governed (and regulated) primarily by kinetic rather than thermodynamic factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Translocation of a Channel-Forming Antimicrobial Peptide, Magainin 2, across Lipid Bilayers by Forming a Pore

Abstract: A channel-forming antimicrobial peptide, magainin 2, has been shown to translocate across phospholipid bilayers by forming a pore comprising multimeric peptides. The translocation was demonstrated by four sets of experiments by use of resonance energy transfer from tryptophan introduced into the peptide to a dansyl chromophore incorporated into the lipid membrane. The translocation was coupled to pore formation, as detected by the dye efflux from the lipid vesicles; about 30% of the total peptide molecules translocated into the inner leaflets over 10 min, while 80% of the dye molecules leaked out at a lipid to peptide ratio of 57. This novel model can explain the problems debated so far, i.e., the peptide forms an ion channel whereas the magainin helix essentially lies parallel to the membrane surface. Channel (pore) formation in the vesicles is a transient process observable mainly during the early stage of the peptide membrane interactions.

Nucleotide excision repair DNA synthesis by DNA polymerase epsilon in the presence of PCNA, RFC, and RPA.

Abstract: In eukaryotes, nucleotide excision repair of DNA is a complex process that requires many polypeptides to perform dual incision and remove a segment of about 30 nucleotides containing the damage, followed by repair DNA synthesis to replace the excised segment. Nucleotide excision repair DNA synthesis is dependent on proliferating cell nuclear antigen (PCNA). To study gap-filling DNA synthesis during DNA nucleotide excision repair, UV-damaged DNA was first incubated with PCNA-depleted human cell extracts to create repair incisions. Purified DNA polymerase delta or epsilon, with DNA ligase, was then used to form the repair patch. DNA polymerase delta could perform repair synthesis and was strictly dependent on the presence of both PCNA and replication factor C, but gave rise to a very low proportion of complete, ligated circles. The presence of replication protein A (which is also required for nucleotide excision repair) did not alter this result, while addition of DNase IV increased the fraction of ligated products. DNA polymerase epsilon, on the other hand, could fill the repair patch in the absence of PCNA and replication factor C, and most of the products were ligated circles. Addition of replication protein A changed the situation dramatically, and synthesis by polymerase epsilon became dependent on both PCNA and replication factor C. A combination of DNA polymerase epsilon, PCNA, replication factor C, replication protein A, and DNA ligase I appears to be well-suited to the task of creating nucleotide excision repair patches.