Showing papers in "Biochemistry in 1992"

The chemical shift index: A fast and simple method for the assignment of protein secondary structure through NMR spectroscopy

Abstract: Previous studies by Wishart et al. [Wishart, D. S., Sykes, B. D., & Richards, F. M. (1991) J. Mol. Biol. (in press)] have demonstrated that 1H NMR chemical shifts are strongly dependent on the character and nature of protein secondary structure. In particular, it has been found that the 1H NMR chemical shift of the alpha-CH proton of all 20 naturally occurring amino acids experiences an upfield shift (with respect to the random coil value) when in a helical configuration and a comparable downfield shift when in a beta-strand extended configuration. On the basis of these observations, a technique is described for rapidly and quantitatively determining the identity, extent, and location of secondary structural elements in proteins based on the simple inspection of the alpha-CH 1H resonance assignments. A number of examples are provided to demonstrate both the simplicity and the accuracy of the technique. This new method is found to be almost as accurate as the more traditional NOE-based methods of determining secondary structure and could prove to be particularly useful in light of the recent development of sequential assignment techniques which are now almost NOE-independent [Ikura, M., Kay, L. E., & Bax, A. (1990) Biochemistry 29, 4659-4667]. We suggest that this new procedure should not necessarily be seen as a substitute to existing rigorous methods for secondary structure determination but, rather, should be viewed as a complement to these approaches.

Neither delta- nor lambda-tris(phenanthroline)ruthenium(II) binds to DNA by classical intercalation.

Abstract: Equilibrium binding studies and viscosity experiments are described that characterize the interaction of delta- and lambda-[Ru(o-phen)3]2+ with calf thymus DNA. The mode of binding of these compounds to DNA is a matter of controversy. Both isomers of [Ru(o-phen)3]2+ were found to bind but weakly to DNA, with binding constants of 4.9 (+/- 0.3) x 10(4) M-1 and 2.8 (+/- 0.2) x 10(4) M-1 determined for the delta and lambda isomers, respectively, at 20 degrees C in a solution containing 5 mM Tris-HCl (pH 7.1) and 10 mM NaCl. We determined that the quantity delta log K/delta log [Na+] equals 1.37 and 1.24 for the delta and lambda isomers, respectively. Application of polyelectrolyte theory allows us to use these values to show quantitatively that both the delta and lambda isomers are essentially electrostatically bound to DNA. Viscosity experiments show that binding the lambda isomer does not alter the relative viscosity of DNA to any appreciable extent, while binding of the delta isomer decreases the relative viscosity of DNA. From these viscosity results, we conclude that neither isomer of [Ru(o-phen)3]2+ binds to DNA by classical intercalation.

Backbone dynamics of calmodulin studied by 15N relaxation using inverse detected two-dimensional NMR spectroscopy: the central helix is flexible.

Abstract: The backbone dynamics of Ca(2+)-saturated recombinant Drosophila calmodulin has been studied by 15N longitudinal and transverse relaxation experiments, combined with 15N(1H) NOE measurements. Results indicate a high degree of mobility near the middle of the central helix of calmodulin, from residue K77 through S81, with order parameters (S2) in the 0.5-0.6 range. The anisotropy observed in the motion of the two globular calmodulin domains is much smaller than expected on the basis of hydrodynamic calculations for a rigid dumbbell type structure. This indicates that, for the purposes of 15N relaxation, the tumbling of the N-terminal (L4-K77) and C-terminal (E82-S147) lobes of calmodulin is effectively independent. A slightly shorter motional correlation time (tau c approximately 6.3 ns) is obtained for the C-terminal domain compared to the N-terminal domain (tau c approximately 7.1 ns), in agreement with the smaller size of the C-terminal domain. A high degree of mobility, with order parameters of approximately 0.5, is also observed in the loop that connects the first with the second EF-hand type calcium binding domain and in the loop connecting the third and fourth calcium binding domain.

Bile acid biosynthesis

Abstract: To conclude, the last several years have seen a resurgence of interest in the biosynthesis of bile acids This focus has come about due to the central roles that these molecules play in cholesterol and fat metabolism and due to recent advances in their chemistry, biochemistry, and molecular biology The application of probes generated by these methodologies has begun to generate novel insight into bile acid metabolism, regulation, and genetics The next several years should be equally exciting

Interaction of antimicrobial dermaseptin and its fluorescently labeled analogues with phospholipid membranes.

Abstract: Dermaseptin, a 34 amino-acid residue antimicrobial polypeptide [Mor, A., Nguyen, V. H., Delfour, A., Migliore-Samour, D., & Nicolas, P. (1991) Biochemistry 30, 8824-8830] was synthesized and selectively labeled at its N-terminal amino acid with either 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD), rhodamine, or fluorescein. The fluorescent emission spectra of the NBD-labeled dermaseptin displayed a blue-shift upon binding to small unilamellar vesicles (SUV), reflecting the relocation of the fluorescent probe to an environment of increased apolarity. Titrations of solutions containing NBD-labeled dermaseptin with SUV composed of zwitterionic or acidic phospholipids were used to generate binding isotherms, from which were derived surface partition constants of (0.66 +/- 0.06) x 10(4) M-1 and (2.8 +/- 0.3) x 10(4) M-1, respectively. The shape of the binding isotherms, as well as fluorescence energy transfer measurements, suggests that some aggregation of membrane-bound peptide monomers occurs in acidic but not in zwitterionic vesicles. The preferential susceptibility of the peptide to proteolysis when bound to zwitterionic but not to acidic SUV suggests that these aggregates might then penetrate a relatively short distance into the hydrophobic region of the acidic membrane. Furthermore, the results provide good correlation between the peptide's strong binding and its ability to permeate membranes composed of acidic phospholipids, as revealed by a dissipation of diffusion potential and a release of entrapped calcein from SUV.

Structure and novel exons of the human tau gene.

Abstract: The microtubule-binding protein tau is important in establishing and maintaining neuronal morphology and is a major component of the neurofibrillary tangles (NFTs) characteristic of Alzheimer's brain The neuron-specific tau transcript undergoes complex alternative splicing The human tau gene has been cloned and mapped The restriction analysis and partial sequencing of the gene shows that it contains (1) four alternatively spliced exons previously described in rodent and bovine but not in human tau cDNAs and (2) two CpG islands, one associated with the promoter region, the other with exon 9 Examination of human tau mRNA indicates that the human cerebrocortical splicing pattern differs from that previously reported for the murine and bovine tau mRNAs, despite conserved exon organization in all three genes

Nitric oxide synthase is a cytochrome P-450 type hemoprotein

Abstract: Nitric oxide has emerged as an important mammalian metabolic intermediate involved in critical physiological functions such as vasodilation, neuronal transmission, and cytostasis. Nitric oxide synthase (NOS) catalyzes the five-electron oxidation of L-arginine to citrulline and nitric oxide. Cosubstrates for the reaction include molecular oxygen and NADPH. In addition, there is a requirement for tetrahydrobiopterin. NOS also contains the coenzymes FAD and FMN and demonstrates significant amino acid sequence homology to NADPH-cytochrome P-450 reductase. Herein we report the identification of the inducible macrophage NOS as a cytochrome P-450 type hemoprotein. The pyridine hemochrome assay showed that the NOS contained a bound protoporphyrin IX heme. The reduced carbon monoxide binding spectrum shows an absorption maximum at 447 nm indicative of a cytochrome P-450 hemoprotein. A mixture of carbon monoxide and oxygen (80%/20%) potently inhibited the reaction (73-79%), showing that the heme functions directly in the oxidative conversion of L-arginine to nitric oxide and citrulline. Additionally, partially purified NOS from rat cerebellum was inhibited by CO, suggesting that this isoform may also contain a P-450-type heme. NOS is the first example of a soluble cytochrome P-450 in eukaryotes. In addition, the presence of FAD and FMN indicates that this is the first catalytically self-sufficient mammalian P-450 enzyme, containing both a reductase and a heme domain on the same polypeptide.

Substrate specificity of the Escherichia coli Fpg protein (formamidopyrimidine-DNA glycosylase): excision of purine lesions in DNA produced by ionizing radiation or photosensitization.

Abstract: We have investigated the excision of a variety of modified bases from DNA by the Escherichia coli Fpg protein (formamidopyrimidine-DNA glycosylase) [Boiteux, S., O'Connor, T. R., Lederer, F., Gouyette, A., & Laval, J. (1990) J. Biol. Chem. 265, 3916-3922]. DNA used as a substrate was modified either by exposure to ionizing radiation or by photosensitization using visible light in the presence of methylene blue (MB). The technique of gas chromatography/mass spectrometry, which can unambiguously identify and quantitate pyrimidine- and purine-derived lesions in DNA, was used for analysis of hydrolyzed and derivatized DNA samples. Thirteen products resulting from pyrimidines and purines were detected in gamma-irradiated DNA, whereas only the formation of 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 8-hydroxyguanine (8-OH-Gua) was observed in visible light/MB-treated DNA. Analysis of gamma-irradiated DNA after incubation with the Fpg protein followed by precipitation revealed that the Fpg protein significantly excised 4,6-diamino-5-formamidopyrimidine (FapyAde), FapyGua, and 8-OH-Gua. The excision of a small but detectable amount of 8-hydroxyadenine was also observed. The detection of these products in the supernatant fractions of the same samples confirmed their excision by the enzyme. Nine pyrimidine-derived lesions were not excised. The Fpg protein also excised FapyGua and 8-OH-Gua from visible light/MB-treated DNA. The presence of these products in the supernatant fractions confirmed their excision.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of trifluoroethanol on protein secondary structure: an NMR and CD study using a synthetic actin peptide.

Abstract: The structure of a synthetic peptide comprising the 28 amino-terminal residues of actin has been examined by 1H-NMR and CD spectroscopy. The peptide is largely unstructured and flexible in solution but becomes increasingly structured at higher trifluoroethanol (TFE) concentrations. As judged by CD with the use of two additional peptides (actin 1-20 and actin 18-28), TFE induces formation of up to 48% helical content within residues 1-20, while residues 21-28 exhibit no helical propensity. Similar results were obtained by using NMR-derived distance information in restrained molecular dynamics calculations. The calculated structure of actin 1-28 peptide in 80% TFE is well defined for the first 23 residues with a backbone root mean square deviation of 0.5 A. Two helices are formed from residues 4-13 and 16-20, and a beta-turn is formed from residues 13-16. The N-terminal residues 1-3 exhibit increased flexibility and a helix-like conformation while the C-terminal residues 21-28 show no regular secondary structure. These results are compared with the predicted secondary structure and the structure of the corresponding sequence in the crystal structure of actin [Kabsch et al. (1990) Nature 347, 37-44]. The significance of the TFE-induced peptide structure is discussed.

Reconstitution of functional water channels in liposomes containing purified red cell CHIP28 protein.

Abstract: Water rapidly crosses the plasma membranes of red blood cells (RBCs) and renal tubules through highly specialized channels. CHIP28 is an abundant integral membrane protein in RBCs and renal tubules, and Xenopus laevis oocytes injected with CHIP28 RNA exhibit high osmotic water permeability, Pf [Preston et al. (1992) Science 256, 385-387]. Purified CHIP28 from human RBCs was reconstituted into proteoliposomes in order to establish if CHIP28 is itself the functional unit of water channels and to characterize its physiological behavior. CHIP28 proteoliposomes exhibit Pf which is up to 50-fold above that of control liposomes, but permeability to urea and protons is not increased. Like intact RBC, the Pf of CHIP28 proteoliposomes is reversibly inhibited by mercurial sulfhydryl reagents and exhibits a low Arrhenius activation energy. The magnitude of CHIP28-mediated water flux (11.7 x 10(-14) cm3/s per CHIP28) corresponds to the known Pf of intact RBCs. These results demonstrate that CHIP28 protein functions as a molecular water channel and also indicate that CHIP28 is responsible for most transmembrane water movement in RBCs.

Lateral diffusion in the liquid phases of dimyristoylphosphatidylcholine/cholesterol lipid bilayers: a free volume analysis.

Abstract: The technique of fluorescence recovery after photobleaching is used to perform an extensive study of the lateral diffusion of a phospholipid probe in the binary mixture dimyristoylphosphatidylcholine/cholesterol, above the melting temperature of the phospholipid In the regions of the phase diagram where a single liquid phase exists, diffusion can be quantitatively described by free volume theory, using a modified Macedo-Litovitz hybrid equation In the liquid-liquid immiscibility region, the temperature dependence of the diffusion coefficient is in excellent agreement with current theories of generalized diffusivities in composite two-phase media A consistent interpretation of the diffusion data can be provided based essentially on the idea that the primary effect of cholesterol addition to the bilayer is to occupy free volume On this basis, a general interpretation of the phase behavior of this mixture is also proposed

Inhibition of T cell signaling by immunophilin-ligand complexes correlates with loss of calcineurin phosphatase activity.

Abstract: Calcineurin, a Ca2+, calmodulin-dependent protein phosphatase, was recently found to bind with high affinity to two different immunosuppressant binding proteins (immunophilins) with absolute dependence on the presence of the immunosuppressants FK506 or cyclosporin A (CsA) [Liu et al. (1991) Cell 66, 807-815]. The binding affinities of the immunophilin-drug complexes toward calcineurin and the stoichiometry of the resultant multimeric complexes have now been determined, and structural elements of FK506, CsA, and calcineurin that are critical for mediating their interactions have been identified. Analogues of FK506 (FK520, FK523, 15-O-demethyl-FK520) and CsA (MeBm2t1-CsA and MeAla6-CsA) whose affinities for their cognate immunophilins do not correlate with their immunosuppressive activities have been prepared and evaluated in biochemical and cellular assays. We demonstrate a strong correlation between the ability of these analogues, when bound to their immunophilins, to inhibit the phosphatase activity of calcineurin and their ability to inhibit transcriptional activation by NF-AT, a T cell specific transcription factor that regulates IL-2 gene synthesis in human T cells. In addition, FKBP-FK506 and CyP-CsA do not inhibit members of the PP1, PP2A, and PP2C classes of serine/threonine phosphatases. These data suggest that calcineurin is the relevant cellular target of these immunosuppressive agents and is involved in Ca(2+)-dependent signal transduction pathways in, among others, T cells and mast cells.

Use of liquid hydrocarbon and amide transfer data to estimate contributions to thermodynamic functions of protein folding from the removal of nonpolar and polar surface from water.

Abstract: This extension of the liquid hydrocarbon model seeks to quantify the thermodynamic contributions to protein stability from the removal of nonpolar and polar surface from water. Thermodynamic data for the transfer of hydrocarbons and organic amides from water to the pure liquid phase are analyzed to obtain contributions to the thermodynamics of folding from the reduction in water-accessible surface area. Although the removal of nonpolar surface makes the dominant contribution to the standard heat capacity change of folding (delta C0fold), here we show that inclusion of the contribution from removal of polar surface allows a quantitative prediction of delta C0fold within the uncertainty of the calorimetrically determined value. Moreover, analysis of the contribution of polar surface area to the enthalpy of transfer of liquid amides provides a means of estimating the contributions from changes in nonpolar and polar surface area as well as other factors to the enthalpy of folding (delta H0fold). In addition to estimates of delta H0fold, this extension of the liquid hydrocarbon model provides a thermodynamic explanation for the observation [Privalov, P. L., & Khechinashvili, N. N. (1974) J. Mol. Biol. 86, 665-684] that the specific enthalpy of folding (cal g-1) of a number of globular proteins converges to a common value at approximately 383 K. Because amounts of nonpolar and polar surface area buried by these proteins upon folding are found to be linear functions of molar mass, estimates of both delta C0fold and delta H0fold may be obtained given only the molar mass of the protein of interest.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial denaturation of transthyretin is sufficient for amyloid fibril formation in vitro

Abstract: Amyloid diseases are caused by the self-assembly of a given protein into an insoluble cross-beta-sheet quaternary structural form which is pathogenic. An understanding of the biochemical mechanism of amyloid fibril formation should prove useful in understanding amyloid disease. Toward this end, a procedure for the conversion of the amyloidogenic protein transthyretin into amyloid fibrils under conditions which mimic the acidic environment of a lysosome has been developed. Association of a structured transthyretin denaturation intermediate is sufficient for amyloid fibril formation in vitro. The rate of fibril formation is pH dependent with significant rates being observed at pHs accessible within the lysosome (3.6-4.8). Far-UV CD spectroscopic studies suggest that transthyretin retains its secondary structural features at pHs where fibrils are formed. Near-UV CD studies demonstrate that transthyretin has retained the majority of its tertiary structure during fibril formation as well. Near-UV CD analysis in combination with glutaraldehyde cross-linking studies suggests that a pH-mediated tetramer to monomer transition is operative in the pH range where fibril formation occurs. The rate of fibril formation decreases markedly at pHs below pH 3.6, consistent with denaturation to a monomeric TTR intermediate which has lost its native tertiary structure and capability to form fibrils. It is difficult to specify with certainty which quaternary structural form of transthyretin is the amyloidogenic intermediate at this time. These difficulties arise because the maximal rate of fibril formation occurs at pH 3.6 where tetramer, traces of dimer, and significant amounts of monomer are observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence specificity in the dimerization of transmembrane alpha-helices.

Abstract: While several reports have suggested a role for helix-helix interactions in membrane protein oligomerization, there are few direct biochemical data bearing on this subject. Here, using mutational analysis, we show that dimerization of the transmembrane alpha-helix of glycophorin A in a detergent environment is spontaneous and highly specific. Very subtle changes in the side-chain structure at certain sensitive positions disrupt the helix-helix association. These sensitive positions occur at approximately every 3.9 residues along the helix, consistent with their comprising the interface of a closely fit transmembranous supercoil of alpha-helices. By contrast with other reported cases of interactions between transmembrane helices, the set of interfacial residues in this case contains no highly polar groups. Amino acids with aliphatic side chains define much of the interface, indicating that precise packing interactions between the helices may provide much of the energy for association. These data highlight the potential general importance of specific interactions between the hydrophobic anchors of integral membrane proteins.

Catalysis at the interface: the anatomy of a conformational change in a triglyceride lipase.

Abstract: The crystal structure of an extracellular triglyceride lipase (from a fungus Rhizomucor miehei) inhibited irreversibly by diethyl p-nitrophenyl phosphate (E600) was solved by X-ray crystallographic methods and refined to a resolution of 2.65 A. The crystals are isomorphous with those of n-hexylphosphonate ethyl ester/lipase complex [Brzozowski, A. M., Derewenda, U., Derewenda, Z. S., Dodson, G. G., Lawson, D. M., Turkenburg, J. P., Bjorkling, F., Huge-Jensen, B., Patkar, S. A., & Thim, L. (1991) Nature 351, 491-494], where the conformational change was originally observed. The higher resolution of the present study allowed for a detailed analysis of the stereochemistry of the change observed in the inhibited enzyme. The movement of a 15 amino acid long "lid" (residues 82-96) is a hinge-type rigid-body motion which transports some of the atoms of a short alpha-helix (residues 85-91) by over 12 A. There are two hinge regions (residues 83-84 and 91-95) within which pronounced transitions of secondary structure between alpha and beta conformations are caused by dramatic changes of specific conformational dihedral angles (phi and psi). As a result of this change a hydrophobic area of ca. 800 A2 (8% of the total molecule surface) becomes exposed. Other triglyceride lipases are also known to have "lids" similar to the one observed in the R. miehei enzyme, and it is possible that the general stereochemistry of lipase activation at the oil-water interfaces inferred from the present X-ray study is likely to apply to the entire family of lipases.

Crystallographic evidence of a large ligand-induced hinge-twist motion between the two domains of the maltodextrin binding protein involved in active transport and chemotaxis.

Abstract: The periplasmic maltodextrin binding protein of Escherichia coli serves as an initial receptor for the active transport of and chemotaxis toward maltooligosaccharides. The three-dimensional structure of the binding protein complexed with maltose has been previously reported [Spurlino, J. C., Lu, G.-Y., & Quiocho, F. A. (1991) J. Biol. Chem. 266, 5202-5219]. Here we report the structure of the unliganded form of the binding protein refined to 1.8-A resolution. This structure, combined with that for the liganded form, provides the first crystallographic evidence that a major ligand-induced conformational change occurs in a periplasmic binding protein. The unliganded structure shows a rigid-body "hinge-bending" between the two globular domains by approximately 35 degrees, relative to the maltose-bound structure, opening the sugar binding site groove located between the two domains. In addition, there is an 8 degrees twist of one domain relative to the other domain. The conformational changes observed between this structure and the maltose-bound structure are consistent with current models of maltose/maltodextrin transport and maltose chemotaxis and solidify a mechanism for receptor differentiation between the ligand-free and ligand-bound forms in signal transduction.

Characterization of magnetically orientable bilayers in mixtures of dihexanoylphosphatidylcholine and dimyristoylphosphatidylcholine by solid-state NMR.

Abstract: Mixtures of long-chain and short-chain phosphatidylcholine (PC) were characterized by multinuclear (13C, 31P, 2H) solid-state nuclear magnetic resonance. This work complements and extends previous characterization of such mixtures by focusing on concentrated mixtures at temperatures above the gel to liquid crystalline phase transition temperature (Tm) of the long-chain PC component. Above Tm it was observed that highly oriented, bilayer-like assemblies could be formed of mixtures of dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC) in molar ratios ranging from approximately 1:3.5 to 1:2 (DHPC:DMPC) over a considerable range of lipid concentrations (at least 3-40% w/v total lipid, for a 1:2.5 sample). Orientation was observed to occur only in an L alpha-like phase. The NMR data can be accounted for by a general model of the DHPC-DMPC aggregates in which DHPC can be found in two distinct populations (one highly ordered, one not). The averaged conformations of the glycerol backbone/headgroup regions of the long- and short-chain PC composing the assemblies were judged by solid-state 13C NMR to be similar to each other. The information gleaned about these mixtures and the quality of the oriented NMR spectra obtained suggest that DHPC-DMPC mixtures may prove to be useful as model membrane media in solid-state NMR studies of biomembranes.

Increased thermal stability of proteins in the presence of naturally occurring osmolytes.

Abstract: Organisms and cellular systems which have adapted to stresses such as high temperature, desiccation, and urea-concentrating environments have responded by concentrating particular organic solutes known as osmolytes. These osmolytes are believed to confer protection to enzyme and other macromolecular systems against such denaturing stresses. Differential scanning calorimetric (DSC) experiments were performed on ribonuclease A and hen egg white lysozyme in the presence of varying concentrations of the osmolytes glycine, sarcosine, N,N-dimethylglycine, and betaine. Solutions containing up to several molar concentrations of these solutes were found to result in considerable increases in the thermal unfolding transition temperature (Tm) for these proteins. DSC scans of ribonuclease A in the presence of up to 8.2 M sarcosine resulted in reversible two-state unfolding transitions with Tm increases of up to 22 degrees C and unfolding enthalpy changes which were independent of Tm. On the basis of the thermodynamic parameters observed, 8.2 M sarcosine results in a stabilization free energy increase of 7.2 kcal/mol for ribonuclease A at 65 degrees C. This translates into more than a 45,000-fold increase in stability of the native form of ribonuclease A over that in the absence of sarcosine at this temperature. Catalytic activity measurements in the presence of 4 M sarcosine give kcat and Km values that are largely unchanged from those in the absence of sarcosine. DSC of lysozyme unfolding in the presence of these osmolytes also results in Tm increases of up to 23 degrees C; however, significant irreversibly occurs with this protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular DNA molecules imaged in air by scanning force microscopy.

Abstract: Routine and reproducible imaging of DNA molecules in air with the scanning force microscope (SFM) has been accomplished. Circular molecules of plasmid DNA were deposited onto red mica and imaged under various relative humidities. In related experiments, the first images of the Escherichia coli RNA polymerase-DNA complex have also been obtained. This has been possible by (1) the use of specially modified SFM tips with a consistent radius of curvature of 10 nm or less, to minimize the amount of image distortion introduced by the finite dimensions of commercially available tips, (2) the optimization of a method to deposit and bind DNA molecules to the mica surface in a stable fashion, and (3) careful control of the sample humidity, to prevent solvation of the molecules and detachment from the surface by the scanning tip or stylus. Contact forces in the range of a few nanonewtons are routinely possible in air and in the presence of residual humidity. The spatial resolution of the images appears determined by the radius of curvature of the modified styli, which can be estimated directly from the apparent widths of the DNA molecules in the images.

Characterization of dipyridophenazine complexes of ruthenium(II): the light switch effect as a function of nucleic acid sequence and conformation.

Abstract: Spectroscopic parameters for two novel ruthenium complexes on binding to nucleic acids of varying sequences and conformations have been determined. These complexes, Ru(bpy)_2dppz^(2+) and Ru(phen)_2dppz^(2+) (bpy = 2,2'-bipyridine; phen = 1,10-phenanthroline; dppz = dipyrido[3,2:ɑ-2',3':c]phenazine) serve as "molecular light switches" for DNA, displaying no photoluminescence in aqueous solution but luminescing intensely in the presence of DNA. The luminescent enhancement observed upon binding is attributed to the sensitivity of the excited state to quenching by water; in DNA, the metal complex, upon intercalation into the helix, is protected from the aqueous solvent, thereby preserving the luminescence. Correlations between the extent of protection (depending upon the DNA conformation) and the luminescence parameters are observed. Indeed, the strongest luminescent enhancement is observed for intercalation into DNA conformations which afford the greatest amount of overlap with access from the major groove, such as in triple helices. Differences are observed in the luminescent parameters between the two complexes which also correlate with the level of water protection. In the presence of nucleic acids, both complexes exhibit biexponential decays in emission. Quenching studies are consistent with two intercalative binding modes for the dppz ligand from the major groove: one in which the metal-phenazine axis lies along the DNA dyad axis and another where the metal-phenazine axis lies almost perpendicular to the DNA dyad axis. Ru(bpy)_2dppz^(2+) and Ru(phen)_2dppz^(2+) are shown here to be unique reporters of nucleic acid structures and may become valuable in the design of new diagnostics for DNA.

Miniantibodies: use of amphipathic helices to produce functional, flexibly linked dimeric FV fragments with high avidity in Escherichia coli.

Abstract: We have designed dimeric antibody fragments that assemble in Escherichia coli. They are based on single-chain FV fragments, with a flexible hinge region from mouse IgG3 and an amphiphilic helix fused to the C-terminus of the antibody fragment. The sequence of the helix was taken either from that of a previously reported four-helix bundle design or from a leucine zipper, optionally extended with a short cysteine-containing peptide. The bivalent fragments associate in vivo, either with covalent linkage or with a monomer-dimer equilibrium, and results from ultracentrifugation sedimentation studies and SDS-PAGE are consistent with dimers. All constructs are able to bind to surface-bound antigen under conditions in which only bivalent but not monovalent antibody fragments bind. The covalent bundle helix construct shows binding characteristics nearly identical to those of the much larger whole mouse antibody, resulting in substantially more stable immunoglobulin-antigen complexes than in the case of monovalent fragments. This modular design of natural and engineered protein domains directly leads to a boost of avidity, and it allows the construction of bispecific antibody fragments in functional form in E. coli.

Design of model amphipathic peptides having potent antimicrobial activities

Abstract: Induced amphipathic alpha-helical conformations play an important role in the biological activity of peptides. By using reversed-phase high-performance liquid chromatography (RP-HPLC) as a means to study the secondary structure of peptides at aqueous/lipid interfaces, a sequence (Ac-LKLLKKLLKKLKKLLKKL-NH2) was found to readily adopt an amphipathic alpha-helical conformation upon interacting with the lipid groups of the stationary phase during RP-HPLC. This peptide exhibited potent antimicrobial activities against both Gram-positive and Gram-negative bacteria. We have prepared a complete set of omission, as well as of leucine and lysine substitution, analogs of this sequence. These analogs were used to investigate the effects of such alterations on the parent sequence's antimicrobial and hemolytic activities relative to each analog's behavior during RP-HPLC. The potential for the formation of ion channels through cell membranes by this amphipathic model peptide was also evaluated through preparation of analogs which varied in length from 8 to 22 residues, while maintaining their amphipathicity.

The three-dimensional structure of a glutathione S-transferase from the mu gene class. Structural analysis of the binary complex of isoenzyme 3-3 and glutathione at 2.2-A resolution.

Abstract: The crystal structure of a mu class glutathione S-transferase (EC 2.5.1.18) from rat liver (isoenzyme 3-3) in complex with the physiological substrate glutathione (GSH) has been solved at 2.2-A resolution by multiple isomorphous replacement methods. The enzyme crystallized in the monoclinic space group C2 with unit cell dimensions of a = 87.98 A, b = 69.41 A, c = 81.34 A, and beta = 106.07 degrees. Oligonucleotide-directed site-specific mutagenesis played an important role in the solution of the structure in that the cysteine mutants C86S, C114S, and C173S were used to help locate the positions of mercuric ion sites in nonisomorphous derivatives with ethylmercuric phosphate and to align the sequence with the model derived from MIR phases. A complete model for the protein was not obtained until part of the solvent structure was interpreted. The dimer in the asymmetric unit refined to a crystallographic R = 0.171 for 19,298 data and I > or = 1.5 sigma (I). The final model consists of 4150 atoms, including all non-hydrogen atoms of 434 amino acid residues, two GSH molecules, and oxygen atoms of 474 water molecules. The dimeric enzyme is globular in shape with dimensions of 53 x 62 x 56 A. Crystal contacts are primarily responsible for conformational differences between the two subunits which are related by a noncrystallographic 2-fold axis. The structure of the type 3 subunit can be divided into two domains separated by a short linker, a smaller alpha/beta domain (domain I, residues 1-82), and a larger alpha domain (domain II, residues 90-217). Domain I contains four beta-strands which form a central mixed beta-sheet and three alpha-helices which are arranged in a beta alpha beta alpha beta beta alpha motif. Domain II is composed of five alpha-helices. Domain I can be considered the glutathione binding domain, while domain II seems to be primarily responsible for xenobiotic substrate binding. The active site is located in a deep (19-A) cavity which is composed of three relatively mobile structural elements: the long loop (residues 33-42) of domain I, the alpha 4/alpha 5 helix-turn-helix segment, and the C-terminal tail. GSH is bound at the active site in an extended conformation at one end of the beta-sheet of domain I with its backbone facing the cavity and the sulfur pointing toward the subunit to which it is bound.(ABSTRACT TRUNCATED AT 400 WORDS)

Three-dimensional solution structure of the B domain of staphylococcal protein A: comparisons of the solution and crystal structures.

Abstract: The three-dimensional solution structure of the recombinant B domain (FB) of staphylococcal protein A, which specifically binds to the Fc portion of immunoglobulin G, was determined by NMR spectroscopy and hybrid distance geometry-dynamical simulated annealing calculations. On the basis of 692 experimental constraints including 587 distance constraints obtained from the nuclear Overhauser effect (NOE), 57 torsion angle (phi, chi 1) constraints, and 48 constraints associated with 24 hydrogen bonds, a total of 10 converged structures of FB were obtained. The atomic root mean square difference among the 10 converged structures is 0.52 +/- 0.10 A for the backbone atoms and 0.98 +/- 0.08 A for all heavy atoms (excluding the N-terminal segment from Thr1 to Glu9 and the C-terminal segment from Gln56 to Ala60, which are partially disordered). FB is composed of a bundle of three alpha-helices, i.e., helix I (Gln10-His19), helix II (Glu25-Asp37), and helix III (Ser42-Ala55). Helix II and helix III are antiparallel to each other, whereas the long axis of helix I is tilted at an angle of about 30 degrees with respect to those of helix II and helix III. Most of the hydrophobic residues of FB are buried in the interior of the bundle of the three helices. It is suggested that the buried hydrophobic residues form a hydrophobic core, contributing to the stability of FB.(ABSTRACT TRUNCATED AT 250 WORDS)

Amyloid fibril formation requires a chemically discriminating nucleation event: studies of an amyloidogenic sequence from the bacterial protein OsmB

Abstract: The sequence of the Escherichia coli OsmB protein was found to resemble that of the C-terminal region of the beta amyloid protein of Alzheimer's disease, which seems to be the major determinant of its unusual structural and solubility properties. A peptide corresponding to residues 28-44 of the OsmB protein was synthesized, and its conformational properties and aggregation behavior were analyzed. The peptide OsmB(28-44) was shown to form amyloid fibrils, as did two sequence analogs designed to test the sequence specificity of fibril formation. These fibrils bound Congo red, and two of the peptides showed birefringence. The peptide fibrils were analyzed by electron microscopy and Fourier transform infrared spectroscopy. Subtle differences were observed which were not interpretable at the molecular level. The rate of fibril formation by each peptide was followed by monitoring the turbidity of supersaturated aqueous solutions. The kinetics of aggregation were characterized by a delay period during which the solution remained clear, followed by a nucleation event which led to a growth phase, during which the solution became viscous and turbid due to the presence of insoluble fibrils. The observation of a kinetic barrier to aggregation is typical of a crystallization event. The delay period could be eliminated by seeding the supersaturated solution with previously formed fibrils. Each peptide could be nucleated by fibrils formed from that same peptide, but not by fibrils from closely related sequences, suggesting that fibril growth requires specific hydrophobic interactions. It appears likely that this repeated sequence motif, which comprises most of the OsmB protein sequence, dictates the structure and possibly the function of that protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Antimicrobial Peptides From Amaranthus Caudatus Seeds With Sequence Homology to the Cysteine/Glycine-Rich Domain of Chitin-Binding Proteins

Abstract: Two antimicrobial peptides (Ac-AMP1 and Ac-AMP2) were isolated from seeds of amaranth (Amaranthus caudatus), and their physicochemical and biological properties were characterized. On the basis of fast atom bombardment mass spectroscopy, Ac-AMP1 and Ac-AMP2 have monoisotopic molecular masses of 3025 and 3181, respectively. Both proteins have pI values above 10. The amino acid sequence of Ac-AMP1 (29 residues) is identical to that of Ac-AMP2 (30 residues), except that the latter has 1 additional residue at the carboxyl terminus. The sequences are highly homologous to the cysteine/glycine-rich domain occurring in many chitin-binding proteins. Both Ac-AMP1 and Ac-AMP2 bind to chitin in a reversible way. Ac-AMP1 and Ac-AMP2 inhibit the growth of different plant pathogenic fungi at much lower doses than other known antifungal chitin-binding proteins. In addition, they show some activity on Gram-positive bacteria. The antimicrobial effect of Ac-AMP1 and Ac-AMP2 is strongly antagonized by cations.

A test of the linear extrapolation of unfolding free energy changes over an extended denaturant concentration range.

Abstract: Guanidine hydrochloride (GdnHCl) and thermally induced unfolding measurements on the oxidized form of Escherichia coli thioredoxin at pH 7 were combined for the purpose of assessing the functional dependence of unfolding free energy changes on denaturant concentration over an extended GdnHCl concentration range. Conventional analysis of GdnHCl unfolding exhibits a linear plot of unfolding delta G vs [GdnHCl] in the transition zone. In order to extend unfolding delta G measurements outside of that narrow concentration range, thermal unfolding measurements were performed using differential scanning calorimetry (DSC) in the presence of low to moderate concentrations of GdnHCl. The unfolding delta G values from the DSC measurements were corrected to 25 degrees C using the Gibbs-Helmholtz equation and mapped onto the delta G vs [GdnHCl] plot. The dependence of unfolding delta G on [GdnHCl] was found to be linear over the full denaturant concentration range, provided that the chloride ion concentration was kept at a threshold of greater than or equal to 1.5 M. In the DSC experiments performed in the presence of GdnHCl, chloride concentrations were maintained at 1.5 M by addition of appropriate amounts of NaCl. The linear extrapolation method (LEM) gives an unfolding free energy change in the absence of denaturant (delta G degrees N-U) in excellent agreement with the delta G determined by DSC measurement in 1.5 M NaCl. The various methods give a consensus unfolding delta G value of 8.0 kcal/mol at 25 degrees C in the absence of denaturant.(ABSTRACT TRUNCATED AT 250 WORDS)

Mapping of the spectral densities of N-H bond motions in eglin c using heteronuclear relaxation experiments.

Abstract: A new strategy is used for studying the internal motions of proteins based on measurements of NMR relaxation parameters. The strategy yields values of the so-called spectral density functions J(omega) for N-H bond vectors. The spectral density functions are related to the distribution of frequencies contained in the rotational (overall and internal) motions of these NH bond vectors. No a priori model assumptions about the dynamics are required in this approach. The method involves measurements of six relaxation parameters consisting of 15N longitudinal relaxation rates, transverse relaxation rates of in-phase and antiphase coherence, the relaxation rates of heteronuclear 1H-15N two-spin order, the heteronuclear 1H-15N nuclear Overhauser effects, and longitudinal relaxation rates of the amide protons. The values of the spectral density functions at the five frequencies 0, omega N, omega H + omega N, omega H, and omega H - omega N are determined from the relaxation parameters using analytical relations derived previously [Peng & Wagner (1992) J. Magn. Reson. 98, 308-332]. Here, the method is applied to characterize the backbone dynamics of the 15N-enriched proteinase inhibitor eglin c, a protein of 70 residues. The values for J(0) and J(omega N = 50 MHz) vary significantly with the amino acid sequence, whereas the spectral densities at higher frequencies, J(450 MHz), J(500 MHz), and J(550 MHz), are typically much smaller and show no significant variation with the sequence. The collective behavior of the J(omega) values indicate greater internal motion for the proteinase binding loop residues and the first eight N-terminal residues. The additional internal motion in these regions is in the rate range below 450 MHz. The values of J(omega) are also compared with root mean square deviations (rmsds) of backbone atoms as obtained in NMR structure determinations. Low values of J(0) and J(omega N) are correlated with high rmsds. Spectral densities at higher frequencies, J(450 MHz), J(500 MHz), and J(550 MHz), are small and show no correlation with rmsds. A comparison with the spectral density functions obtained by fitting the experimental data to the functional dependence of the Lipari and Szabo formalism [Lipari & Szabo (1982a) J. Am. Chem. Soc. 104, 4546-4559] is made.