Showing papers in "Biochemistry in 1989"

Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease.

Abstract: This paper describes the use of novel two-dimensional nuclear magnetic resonance (NMR) pulse sequences to provide insight into protein dynamics. The sequences developed permit the measurement of the relaxation properties of individual nuclei in macromolecules, thereby providing a powerful experimental approach to the study of local protein mobility. For isotopically labeled macromolecules, the sequences enable measurements of heteronuclear nuclear Overhauser effects (NOE) and spin-lattice (T1) and spin-spin (T2) 15N or 13C relaxation times with a sensitivity similar to those of many homonuclear 1H experiments. Because T1 values and heteronuclear NOEs are sensitive to high-frequency motions (10(8)-10(12) s-1) while T2 values are also a function of much slower processes, it is possible to explore dynamic events occurring over a large time scale. We have applied these techniques to investigate the backbone dynamics of the protein staphylococcal nuclease (S. Nase) complexed with thymidine 3',5'-bisphosphate (pdTp) and Ca2+ and labeled uniformly with 15N. T1, T2, and NOE values were obtained for over 100 assigned backbone amide nitrogens in the protein. Values of the order parameter (S), characterizing the extent of rapid 1H-15N bond motions, have been determined. These results suggest that there is no correlation between these rapid small amplitude motions and secondary structure for S. Nase. In contrast, 15N line widths suggest a possible correlation between secondary structure and motions on the millisecond time scale. In particular, the loop region between residues 42 and 56 appears to be considerably more flexible on this slow time scale than the rest of the protein.

Implications of the three-dimensional structure of alpha 1-antitrypsin for structure and function of serpins.

Abstract: Article synthese sur l'α1-antitrypsine: caracteristiques structurales et fonctionnelles (capacite de liaison a des modulateurs); utilisation de l'α1-antitrypsine en tant que modele pour toutes les serpines

Overcoming the overlap problem in the assignment of 1H NMR spectra of larger proteins by use of three-dimensional heteronuclear 1H-15N Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1 beta

Abstract: The application of three-dimensional (3D) heteronuclear NMR spectroscopy to the sequential assignment of the 1H NMR spectra of larger proteins is presented, using uniformly labeled (approximately 95%) [15N]interleukin 1 beta, a protein of 153 residues and molecular mass of 17.4 kDa, as an example. The two-dimensional (2D) 600-MHz spectra of interleukin 1 beta are too complex for complete analysis, owing to extensive cross-peak overlap and chemical shift degeneracy. We show that the combined use of 3D 1H-15N Hartmann-Hahn-multiple quantum coherence (HOHAHA-HMQC) and nuclear Overhauser-multiple quantum coherence (NOESY-HMQC) spectroscopy, designed to provide the necessary through-bond and through-space correlations for sequential assignment, provides a practical general-purpose method for resolving ambiguities which severely limit the analysis of conventional 2D NMR spectra. The absence of overlapping cross-peaks in these 3D spectra allows the unambiguous identification of C alpha H(i)-NH(i+1) and NH(i)-NH(i+1) through-space nuclear Overhauser connectivities necessary for connecting a particular C alpha H(i)-NH(i) through-bond correlation with its associated through-space sequential cross-peak The problem of amide NH chemical shift degeneracy in the 1H NMR spectrum is therefore effectively removed, and the assignment procedure simply involves inspecting a series of 2D 1H-1H slices edited by the chemical shift of the directly bonded 15N atom. Connections between residues can be identified almost without any knowledge of the spin system types involved, though this type of information is clearly required for the eventual placement of the connected residues within the primary sequence.

An infrared spectroscopic study of the interactions of carbohydrates with dried proteins.

Abstract: Fourier-transform infrared spectroscopy was used to characterize the interaction of stabilizing carbohydrates with dried proteins. Freeze-drying of trehalose, lactose, and myo-inositol with lysozyme resulted in substantial alterations of the infrared spectra of the dried carbohydrates. In the fingerprint region (900-1500 cm-1), there were large shifts in the frequencies of bands, a decrease in absorbance, and a loss of band splitting. These effects mimic those of water on hydrated trehalose. Bands assigned to hydroxyl stretching modes (around 3350 cm-1) were decreased in intensity and shifted to higher frequencies in the presence of the protein. In complementary experiments, it was found that dehydration-induced shifts in the positions of amide I and amide II bands for lysozyme could be partially and fully reversed, respectively, when the protein was freeze-dried in the presence of either trehalose or lactose. In addition, the carboxylate band, which was not detectable in the protein dried without the sugar, was apparent when these sugars were present. myo-Inositol was less effective at shifting the amide bands, and the carboxylate band was not detected in the presence of this carbohydrate. Also tested was the concentration dependency of the carbohydrates' influence on the position of the amide II band for dried lysozyme. The results showed that the ability of a given concentration of a carbohydrate to shift this band back toward the position noted with the hydrated protein coincided, at least in the extreme cases, with the capacity of that same level of carbohydrate to preserve the activity of rabbit skeletal muscle phosphofructokinase during freeze-drying.(ABSTRACT TRUNCATED AT 250 WORDS)

Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules.

Abstract: Basic fibroblast growth factor (bFGF) exhibits specific binding to the extracellular matrix (ECM) produced by cultured endothelial cells. Binding was saturable as a function both of time and of concentration of 125I-bFGF. Scatchard analysis of FGF binding revealed the presence of about 1.5 X 10(12) binding sites/mm2 ECM with an apparent kD of 610nM. FGF binds to heparan sulfate (HS) in ECM as evidenced by (i) inhibition of binding in the presence of heparin or HS at 0.1-1 micrograms/mL, but not by chondroitin sulfate, keratan sulfate, or hyaluronic acid at 10 micrograms/mL, (ii) lack of binding to ECM pretreated with heparitinase, but not with chondroitinase ABC, and (iii) rapid release of up to 90% of ECM-bound FGF by exposure to heparin, HS, or heparitinase, but not to chondroitin sulfate, keratan sulfate, hyaluronic acid, or chondroitinase ABC. Oligosaccharides derived from depolymerized heparin, and as small as the tetrasaccharide, released the ECM-bound FGF, but there was little or no release of FGF by modified nonanticoagulant heparins such as totally desulfated heparin, N-desulfated heparin, and N-acetylated heparin. FGF released from ECM was biologically active, as indicated by its stimulation of cell proliferation and DNA synthesis in vascular endothelial cells and 3T3 fibroblasts. Similar results were obtained in studies on release of endogenous FGF-like mitogenic activity from Descemet's membranes of bovine corneas. It is suggested that ECM storage and release of bFGF provide a novel mechanism for regulation of capillary blood vessel growth. Whereas ECM-bound FGF may be prevented from acting on endothelial cells, its displacement by heparin-like molecules and/or HS-degrading enzymes may elicit a neovascular response.

Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling.

Abstract: Stereospecific 1H and 13C NMR assignments were made for the two diastereotopic methyl groups of the 14 valyl and leucyl residues in the DNA-binding domain 1-69 of the 434 repressor. These results were obtained with a novel method, biosynthetically directed fractional 13C labeling, which should be quite widely applicable for peptides and proteins. The method is based on the use of a mixture of fully 13C-labeled and unlabeled glucose as the sole carbon source for the biosynthetic production of the protein studied, knowledge of the independently established stereoselectivity of the pathways for valine and leucine biosynthesis, and analysis of the distribution of 13C labels in the valyl and leucyl residues of the product by two-dimensional heteronuclear NMR correlation experiments. Experience gained with the present project and a previous application of the same principles with the cyclic polypeptide cyclosporin A provides a basis for the selection of the optimal NMR experiments to be used in conjunction with biosynthetic fractional 13C labeling of proteins and peptides.

On the mechanism of topoisomerase I inhibition by camptothecin: evidence for binding to an enzyme-DNA complex

Abstract: Camptothecin, a cytotoxic antitumor compound, has been shown to produce protein-linked DNA breaks mediated by mammalian topoisomerase I. We have investigated the mechanism by which camptothecin disrupts DNA processing by topoisomerase I and have examined the effect of certain structurally related compounds on the formation of a DNA-topoisomerase I covalent complex. Enzyme-mediated cleavage of supercoiled plasmid DNA in the presence of camptothecin was completely reversed upon the addition of exogenous linear DNA or upon dilution of the reaction mixture. Camptothecin and topoisomerase I produced the same amount of cleavage from supercoiled DNA or relaxed DNA. In addition, the alkaloid decreased the initial velocity of supercoiled DNA relaxation mediated by catalytic quantities of topoisomerase I. Inhibition occurred under conditions favoring processive catalysis as well as under conditions favoring distributive catalysis. By use of [3H]camptothecin and an equilibrium dialysis assay, the alkaloid was shown to bind reversibly to a DNA-topoisomerase I complex, but not to isolated enzyme or isolated DNA. These results are consistent with a model in which camptothecin reversibly traps an intermediate involved in DNA unwinding by topoisomerase I and thereby perturbs a set of equilibria, resulting in increased DNA cleavage. By examining certain compounds that are structurally related to camptothecin, it was found that the 20-hydroxy group, which has been shown to be essential for antitumor activity, was also necessary for stabilization of the covalent complex between DNA and topoisomerase I. In contrast, no such correlation existed for UV-light-induced cleavage of DNA by Cu(II)-camptothecin derivatives.

Determination of the three-dimensional solution structure of the C-terminal domain of cellobiohydrolase I from Trichoderma reesei. A study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing.

Abstract: The solution structure of a synthetic 36-residue polypeptide comprising the C-terminal cellulose binding domain of cellobiohydrolase I (CT-CBH I) from Trichoderma reesei was investigated by nuclear magnetic resonance (NMR) spectroscopy. The 1H NMR spectrum was completely assigned in a sequential manner by two-dimensional NMR techniques. A large number of stereospecific assignments for beta-methylene protons, as well as ranges for the phi, psi, and chi 1 torsion angles, were obtained on the basis of sequential and intraresidue nuclear Overhauser enhancement (NOE) and coupling constant data in combination with a conformational data base search. The structure calculations were carried out in an iterative manner by using the hybrid distance geometry-dynamical simulated annealing method. This involved computing a series of initial structures from a subset of the experimental data in order to resolve ambiguities in the assignments of some NOE cross-peaks arising from chemical shift degeneracy. Additionally, this permitted us to extend the stereospecific assignments to the alpha-methylene protons of glycine using information on phi torsion angles derived from the initial structure calculations. The final experimental data set consisted of 554 interproton distance restraints, 24 restraints for 12 hydrogen bonds, and 33 phi, 24 psi, and 25 chi 1 torsion angle restraints. CT-CBH I has two disulfide bridges whose pairing was previously unknown. Analysis of structures calculated with all three possible combinations of disulfide bonds, as well as without disulfide bonds, indicated that the correct disulfide bridge pairing was 8-25 and 19-35. Forty-one structures were computed with the 8-25 and 19-35 disulfide bridges, and the average atomic rms difference between the individual structures and the mean structure obtained by averaging their coordinates was 0.33 +/- 0.04 A for the backbone atoms and 0.52 +/- 0.06 A for all atoms. The protein has a wedgelike shape with an amphiphilic character, one face being predominantly hydrophilic and the other mainly hydrophobic. The principal element of secondary structure is made up of an irregular triple-stranded antiparallel beta-sheet composed of residues 5-9 (beta 1), 24-28 (beta 2), and 33-36 (beta 3) in which strand beta 3 is hydrogen bonded to the other two strands.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of a partly folded protein by NMR methods: studies on the molten globule state of guinea pig alpha-lactalbumin.

Abstract: NMR spectroscopy has been used to investigate the structure of a partially folded state of a protein, the molten globule or A-state of {alpha}-lactalbumin. The {sup 1}H NMR spectrum of this species differs substantially from those of both the native and fully unfolded states, reflecting the intermediate level of order. The resolution in the spectrum is limited by the widespread overlap and substantial line widths of many of the resonances. Methods have therefore been developed that exploit the well-resolved spectrum of the native protein to probe indirectly the A-state. A number of resonances of the A-state have been found to be substantially shifted from their positions in the spectrum of the unfolded state and have been identified through magnetization transfer with the native state, under conditions where the two states are interconverting. The most strongly perturbed residues in the A-state were found to be among those that form a hydrophobic core to the native structure. A number of amides were found to be highly protected from solvent exchange in the A-state. These have been identified through pH-jump experiments, which label them in the spectrum of the native protein. They were found to occur mainly in segments that are helical inmore » the native structure. These results enable a model of the A-state to be proposed in which significant conformational freedom exists but where specific elements of native-like structure are preserved.« less

RNA catalytic properties of the minimum (-)sTRSV sequence.

Abstract: We have identified an RNA catalytic domain within the sequence of the 359 base long negative-strand satellite RNA of tobacco ringspot virus. The catalytic domain contains two minimal sequences of satellite RNA, a 50-base catalytic RNA sequence, and a 14-base substrate RNA sequence. The catalytic complex of catalytic RNA/substrate RNA represents a structure not previously found in any RNA catalytic reaction described to date. The reaction is truly catalytic since the catalytic RNA has multiple substrate cleavage events and is not consumed during the course of the reaction. A linear relationship is seen between reaction rate and catalytic RNA concentration. The reaction has a Km of 0.03 microM, a kcat of 2.1/min, a temperature optimum of near 37 degrees C, and an energy of activation of 19 kcal/mol.

The nature of the hydrophobic binding of small peptides at the bilayer interface: implications for the insertion of transbilayer helices.

Abstract: One method of obtaining useful information about the physical chemistry of peptide/bilayer interactions is to relate thermodynamic parameters of the interactions to structural parameters obtained by diffraction methods. We report here the results of the application of this approach to interactions of hydrophobic tripeptides of the form Ala-X-Ala-0-tert-butyl with lipid bilayers. The thermodynamic constants (ΔG_t> ΔH_1, and Δ1) for the transfer of the tripeptides from water into DMPC vesicles were determined for X = Leu, Phe, and Trp and found to be consistent with those expected for hydrophobic interactions above the phase transition of DMPC. Combining these results with the earlier ones of Jacobs and White [(1986) Biochemistry 25, 2605-2612], the favorable free energies of transfer with different amino acids in the -X- position increase in the order Gly

How do serine proteases really work

Abstract: Recent advances in genetic engineering have led to a growing acceptance of the fact that enzymes work like other catalysts by reducing the activation barriers of the corresponding reactions. However, the key question about the action of enzymes is not related to the fact that they stabilize transition states but to the question to how they accomplish this task. This work considers the catalytic reaction of serine proteases and demonstrates how one can use a combination of calculations and experimental information to elucidate the key contributions to the catalytic free energy. Recent reports about genetic modifications of the buried aspartic group in serine proteases, which established the large effect of this group (but could not determine its origin), are analyzed. Two independent methods indicate that the buried aspartic group in serine proteases stabilizes the transition state by electrostatic interactions rather than by alternative mechanisms. Simple free energy considerations are used to eliminate the double proton-transfer mechanism (which is depicted in many textbooks as the key catalytic factor in serine proteases). The electrostatic stabilization of the oxyanion side of the transition state is also considered. It is argued that serine proteases and other enzymes work by providing electrostatic complementarity to the changes in charge distribution occurring during the reactions they catalyze.

Mechanism of inhibition of DNA gyrase by quinolone antibacterials: a cooperative drug--DNA binding model.

Abstract: We have proposed a cooperative quinolone-DNA binding model for the inhibition of DNA gyrase. The essential feature of the model is that bound gyrase induces a specific quinolone binding site in the relaxed DNA substrate in the presence of ATP. The binding affinity and specificity are derived from two unique and equally important functional features: the specific conformation of the proposed single-stranded DNA pocket induced by the enzyme and the unique self-association phenomenon (from which the cooperativity is derived) of the drug molecules to fit the binding pocket with a high degree of flexibility. Supporting evidence for and implications of this model are provided.

Antimitotic natural products combretastatin A-4 and combretastatin A-2: studies on the mechanism of their inhibition of the binding of colchicine to tubulin

Abstract: Combretastatin A-4 (CS-A4), 3,4,5-trimethoxy-3'-hydroxy-4'-methoxy-(Z)-stilbene, and combretastatin A-2 (CS-A2), 3,4-(methylenedioxy)-5-methoxy-3'-hydroxy-4'-methoxy-(Z)-stilbene, are structurally simple natural products isolated from the South African tree Combretum caffrum. They inhibit mitosis and microtubule assembly and are competitive inhibitors of the binding of colchicine to tubulin [Lin et al. (1988) Mol. Pharmacol. 34, 200-208]. In contrast to colchicine, drug effects on tubulin were not enhanced by preincubating CS-A4 or CS-A2 with the protein. The mechanism of their binding to tubulin was examined indirectly by evaluating their effects on the binding of radiolabeled colchicine to the protein. These studies demonstrated rapid binding of both compounds to tubulin even at 0 degrees C (binding was complete at the earliest times examined), in contrast to the relatively slow and temperature-dependent binding of colchicine. Although the binding of the C. caffrum compounds to tubulin was quite tight, permitting ready isolation of near-stoichiometric amounts of drug-tubulin complex even in the absence of free drug, both CS-A4 and CS-A2 dissociated rapidly from tubulin in the presence of high concentrations of radiolabeled colchicine. Apparent rate constants for drug dissociation from tubulin at 37 degrees C were 3.2 x 10(-3) s-1 for CS-A4, 4.8 x 10(-3) s-1 for CS-A2, and 2.9 x 10(-5) s-1 for colchicine (half-lives of 3.6, 2.4, and 405 min, respectively). Thus, the effectiveness of the C. caffrum compounds as antimitotic agents appears to derive primarily from the rapidity of their binding to tubulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical and pharmacological properties of p170 and p180 forms of topoisomerase II

Abstract: The p170 and p180 forms of topoisomerase II have been compared. The concentration dependence of ATP for catalytic activity of the two forms of the enzyme was identical, and each was equally sensitive to novobiocin. Orthovanadate was found to be a potent inhibitor of catalytic activity of both p170 and p180, with an IC50 value of about 2 microM for each. Under standard reaction conditions, relaxation of supercoiled pBR322 by p180 was highly processive, while p170 performed the same reaction in a distributive manner. The optimal concentration of KCl for catalytic activity of p180 was 20-30 mM higher than that for p170. Comparison of their thermal stability showed that p180 was inactivated at twice the rate of p170. Teniposide and merbarone selectively inhibited catalytic activity of p170, requiring concentrations 3-fold and 8-fold lower, respectively, than those required for equivalent inhibition of p180. Similar selectivity for p170 was seen for teniposide-stimulated DNA cleavage or its inhibition by merbarone. Analysis of sites of DNA cleavage indicated a subset of sites that were either preferred or unique for each of the enzymes. A synthetic oligonucleotide representative of p170 sites selectively inhibited the p170 enzyme. Immunoblotting of p170 and p180 from U937 cells at different stages of proliferation showed that p170 levels declined as the cells reached the plateau phase of growth, while p180 levels were low during rapid proliferation and increased as the growth rate slowed. The data indicate that the p170 and p180 forms of topoisomerase II can be distinguished biochemically, pharmacologically, and by differential cellular regulation.

Conformational states in .beta.-lactamase: molten-globule states at acidic and alkaline pH with high salt

Abstract: We present evidence that beta-lactamase is close to fully unfolded (i.e., random coil conformation) at low ionic strength at the extremes of pH and that the presence of salt causes a cooperative transition to a conformation with the properties of a molten globule, namely, a compact state with native-like secondary structure but disordered side chains (tertiary structure). The conformation of beta-lactamase I from Bacillus cereus was examined over the pH 1.5-12.5 region by circular dichroism (CD), tryptophan fluorescence, dynamic light scattering, and 1-anilino-8-naphthalenesulfonate (ANS) binding. Under conditions of low ionic strength (I = 0.05) beta-lactamase was unfolded below pH 2.5 and above pH 11.5, on the basis of the far-UV and near-UV CD and tryptophan fluorescence. However, at high ionic strength and low pH an intermediate conformation (state A) was observed, with a secondary structure content similar to that of the native protein but a largely disordered tertiary structure. The transition from the unfolded state (U) to state A induced by KCl was cooperative and had a midpoint at 0.12 M KCl (I = 0.17 M) at pH 1.6. A similar conformation (state B) was observed at high pH and high ionic strength. The transition from the alkaline U state to state B induced by KCl at pH 12.2 was cooperative and had a midpoint at 0.6 M KCl (I = 0.65 M). Light scattering measurements showed that state B was compact although somewhat expanded compared to the N state. The compactness of state A could not be determined due to its strong propensity to aggregate.(ABSTRACT TRUNCATED AT 250 WORDS)

Human leukocyte and porcine pancreatic elastase: X-ray crystal structures, mechanism, substrate specificity, and mechanism-based inhibitors.

Abstract: The serine protease family of enzymes is one of the most widely studied group of enzymes, as evidenced by the fact that more crystal structures are available for individuals of this superfamily than for any other homologous group of enzymes. These enzymes contain a conserved triad of catalytic residues including Ser-195, His-57, and Asp-102. The active-site serine is very nucleophilic, and serine proteases are inhibited by specific serine protease reagents such as diisopropyl phosphorofluoridate (DFP), phenylmethanesulfonyl fluoride, and 3,4-dichloroisocoumarin. Elastases are a group of proteases that possess the ability to cleave the important connective tissue protein elastin. Elastin has the unique property of elastic recoil, is widely distributed in vertebrate tissue, and is particularly abundant in the lungs, arteries, skin, and ligaments. Human neutrophil elastase and pancreatic elastase are two major serine proteases that cleave elastin. Neutrophil elastase is found in the dense granules of polymorphonuclear leukycytes and is essential for phagocytosis and defense against infection by invading microorganisms. Pancreatic elastase is stored as an inactive zymogen in the pancreas and is secreted into the intestines where it becomes activated by trypsin and then participates in digestion. Both elastases cleave substrates at peptide bonds where the P{sub 1} residue ismore » an amino acid residue with a small alkyl side chain.« less

On the attribution of binding energy in antigen-antibody complexes McPC 603, D1.3, and HyHEL-5

Abstract: Using X-ray coordinates of antigen-antibody complexes McPC 603, D1.3, and HyHEL-5, we made semiquantitative estimates of Gibbs free energy changes (delta G) accompanying noncovalent complex formation of the McPC 603 Fv fragment with phosphocholine and the D1.3 or HyHEL-5 Fv fragments with hen egg white lysozyme. Our empirical delta G function, which implicitly incorporates solvent effects, has the following components: hydrophobic force, solvent-modified electrostatics, changes in side-chain conformational entropy, translational/overall rotational entropy changes, and the dilutional (cratic) entropy term. The calculated delta G ranges matched the experimentally determined delta G of McPC 603 and D1.3 complexes and overestimated it (i.e., gave a more negative value) in the case of HyHEL-5. Relative delta G contributions of selected antibody residues, calculated for HyHEL-5 complexes, agreed with those determined independently in site-directed mutagenesis experiments. Analysis of delta G attribution in all three complexes indicated that only a small number of amino acids probably contribute actively to binding energetics. These form a subset of the total antigen-antibody contact surface. In the antibodies, the bottom part of the antigen binding cavity dominated the energetics of binding whereas in lysozyme, the energetically most important residues defined small (2.5-3 nm2) "energetic" epitopes. Thus, a concept of protein antigenicity emerges that involves the active, attractive contributions mediated by the energetic antigenic epitopes and the passive surface complementarity contributed by the surrounding contact area. The D1.3 energetic epitope of lysozyme involved Gly 22, Gly 117, and Gln 121; the HyHEL-5 epitope consisted of Arg 45 and Arg 68. These are also the essential antigenic residues determined experimentally. The above positions belong to the most protruding parts of the lysozyme surface, and their backbones are not exceptionally flexible. Least-squares analysis of six different antibody binding regions indicated that the geometry of the VH-VL interface beta-barrel is well conserved, giving no indication of significant changes in domain-domain contacts upon complex formation.

Role of histidine 64 in the catalytic mechanism of human carbonic anhydrase II studied with a site-specific mutant

Abstract: To test the hypothesis that histidine 64 in the active site of human carbonic anhydrase II functions as a proton-transfer group in the catalysis of CO2 hydration, we have studied a site-specific mutant having histidine 64 replaced by alanine, which cannot transfer protons. The steady-state kinetics of CO2 hydration has been measured as well as the exchange of 18O between CO2 and water at chemical equilibrium. The results show that the rate of exchange between CO2 and HCO3- at chemical equilibrium is essentially unaffected by the amino acid substitution at pH greater than 7.0 and slightly decreased in the mutant at pH less than 7.0 (by a factor of 2 at pH 6.0). However, in the absence of buffer the rate of release from the active site of water bearing substrate oxygen is smaller by as much as 20-fold for the mutant as compared to unmodified enzyme. Furthermore, in the unmodified enzyme water release is inhibited by micromolar concentrations of Cu2+ ions, but no such inhibition is observed with the alanine 64 variant. These results suggest that the mutation has specifically affected the rate of proton transfer between the active site and the reaction medium. This kinetic defect in the mutant can be overcome by increasing the concentration of certain buffers, such as imidazole and 1-methylimidazole, but not by others buffers, such as MOPS or HEPES. Similarly, the maximal rate of CO2 hydration at steady state catalyzed by the alanine 64 variant is very low in the presence of MOPS or TAPS buffers but considerably higher in the presence of imidazole derivatives.(ABSTRACT TRUNCATED AT 250 WORDS)

Heteronuclear three-dimensional NMR spectroscopy of the inflammatory protein C5a

Abstract: The utility of three-dimensional heteronuclear NMR spectroscopy for the assignment of 1H and 15N resonances of the inflammatory protein C5a (MW 8500), uniformly labeled with 15N, is demonstrated at a protein concentration of 0.7 mM. It is shown that dramatic simplification of the 2D nuclear Overhauser effect spectrum (NOESY) is obtained by editing with respect to the frequency of the 15N heteronucleus in a third dimension. The improved resolution in the 3D experiment largely facilitates the assignment of protein NMR spectra and allows for the determination of distance constraints from otherwise overlapping NOE cross peaks for purposes of 3D structure determination. The results show that 15N heteronuclear 3D NMR can facilitate the structure determination of small proteins and promises to be a useful tool for the study of larger systems that cannot be studied by conventional 2D NMR techniques.

Energetics of complementary side-chain packing in a protein hydrophobic core.

Abstract: The energetics of complementary packing of nonpolar side chains in the hydrophobic core of a protein were analyzed by protein engineering experiments. We have made the mutations Ile----Val, Ile----Ala, and Leu----Ala in a region of the small bacterial ribonuclease barnase where the major alpha-helix packs onto the central beta-sheet. The destabilization resulting from the creation of cavities was determined by measuring the decrease in free energy of folding from reversible denaturation induced by urea, guanidinium chloride, or heat. The different methods give consistent and reproducible results. The loss in free energy of folding for the mutant proteins is 1.0-1.6 kcal/mol per methylene group removed. This exceeds by severalfold the values obtained from model experiments of the partitioning of relevant side chains between aqueous and nonpolar solvents. Much of this discrepancy arises because two surfaces are buried when a protein folds--both the amino acid side chain in question and the portions of the protein into which it packs. These experiments directly demonstrate that the interior packing of a protein is crucial in stabilizing its three-dimensional structure: the conversion of leucine or isoleucine to alanine in the hydrophobic core loses half the net free energy of folding of barnase with a concomitant decrease in yield of the expressed recombinant protein.

Hemagglutinins from Two Influenza Virus Variants Bind to Sialic Acid Derivatives with Millimolar Dissociation Constants: A 500 MHz Proton Nuclear Magnetic Resonance Study

Abstract: The equilibrium binding of influenza virus hemagglutinin to derivatives of its cell-surface ligand, sialic acid, was measured by nuclear magnetic resonance (NMR) spectroscopy. Binding was quantified by observing perturbations of sialic acid resonances in the presence of protein. The major perturbation observed was a chemical shift of the N-acetyl methyl resonance, presumably due to the proximity of the methyl group to tryptophan 153. X-31 hemagglutinin binds to the methyl alpha-glycoside of sialic acid with a dissociation constant of 2.8 mM and does not bind to the methyl beta-glycoside. Replacing the 4-hydroxyl group of sialic acid with an acetyl group has little effect, while replacing the 7-hydroxyl group with an acetyl prevents binding. Experiments with sialylated oligosaccharides confirm literature reports that mutations at amino acid 226 change the specificity of hemagglutinin for alpha(2,6) and alpha(2,3) glycosidic linkages. The NMR line broadening of sialyloligosaccharides suggests that sialic acid is the only component that contacts the protein. Saccharides containing two sialic acid residues appear to have two separate binding modes. Hemagglutinin that has undergone a low pH induced conformational change retains the ability to bind sialic acid.

Pervanadate [peroxide(s) of vanadate] mimics insulin action in rat adipocytes via activation of the insulin receptor tyrosine kinase.

Abstract: Both vanadate and hydrogen peroxide (H2O2) are known to have insulin-mimetic effects. We previously reported that the mixture of vanadate plus H2O2 results in the generation of a peroxide(s) of vanadate, which strongly enhances IGF-II binding to rat adipocytes (Kadota et al., 1987b). We now report that pervanadate mimics insulin in isolated rat adipocytes to (1) stimulate lipogenesis, (2) inhibit epinephrine-stimulated lipolysis, and (3) stimulate protein synthesis. The efficacy of pervanadate is comparable to that of insulin. However, it is 10(2)-10(3) times more potent than vanadate alone. Exposure of intact rat adipocytes to pervanadate was found to activate the WGA-purified insulin receptor tyrosine kinase assayed with the exogenous substrate poly(Glu80/Tyr20) in a dose-dependent manner to a maximum of 1464% of control at 10(-3) M compared with a maximum insulin effect of 1046% at 10(-6) M. In contrast, in vitro assayed autophosphorylation of the WGA-purified extract was increased 3-fold after exposure of intact cells to insulin but not significantly increased after pervanadate. Furthermore, high concentrations of pervanadate (10(-5) M) inhibited subsequent in vitro added insulin-stimulated autophosphorylation. In vitro addition of pervanadate to WGA-purified receptors could not stimulate autophosphorylation or exogenous tyrosine kinase activity and did not inhibit insulin-stimulated autophosphorylation. Labeling of intact adipocytes with [32P]orthophosphate followed by exposure to 10(-4) M pervanadate increased insulin receptor beta-subunit phosphorylation (7.9 +/- 3.0)-fold, while 10(-7) M insulin and 10(-4) vanadate increased labeling (5.3 +/- 1.8)- and (1.1 +/- 0.2)-fold, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane fusion and inverted phases.

Abstract: We have found a correlation between liposome fusion kinetics and lipid phase behavior for several inverted phase forming lipids. N-Methylated dioleoylphosphatidylethanolamine (DOPE-Me), or mixtures of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC), will form an inverted hexagonal phase (HII) at high temperatures (above TH), a lamellar phase (L alpha) at low temperatures, and an isotropic/inverted cubic phase at intermediate temperatures, which is defined by the appearance of narrow isotropic 31P NMR resonances. The phase behavior has been verified by using high-sensitivity DSC, 31P NMR, freeze-fracture electron microscopy, and X-ray diffraction. The temperature range over which the narrow isotropic resonances occur is defined as delta TI, and the range ends at TH. Extruded liposomes (approximately 0.2 microns in diameter) composed of these lipids show fusion and leakage kinetics which are strongly correlated with the temperatures of these phase transitions. At temperatures below delta TI, where the lipid phase is L alpha, there is little or no fusion, i.e., mixing of aqueous contents, or leakage. However, as the temperature reaches delta TI, there is a rapid increase in both fusion and leakage rates. At temperatures above TH, the liposomes show aggregation-dependent lysis, as the rapid formation of HII phase precursors disrupts the membranes. We show that the correspondence between the fusion and leakage kinetics and the observed phase behavior is easily rationalized in terms of a recent kinetic theory of L alpha/inverted phase transitions. In particular, it is likely that membrane fusion and the L alpha/inverted cubic phase transition proceed via a common set of intermembrane intermediates.

Diversity of oligosaccharide structures linked to asparagines of the scrapie prion protein.

Abstract: Prion proteins from humans and rodents contain two consensus sites for asparagine-linked glycosylation near their C-termini. The asparagine-linked oligosaccharides of the scrapie isoform of the hamster prion protein (PrP 27-30) were released quantitatively from the purified molecule by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. The radioactive oligosaccharides were fractionated into one neutral and three acidic oligosaccharide fractions by anion-exchange column chromatography. All oligosaccharides in the acidic fractions could be converted to neutral oligosaccharides by sialidase digestion. Structural studies on these oligosaccharides including sequential exoglycosidase digestion in combination with methylation analysis revealed that PrP 27-30 contains a mixture of bi-, tri-, and tetraantennary complex-type sugar chains with Man alpha 1----6(GlcNAc beta 1----4)(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4-(Fuc alpha 1----6)GlcNAc as their core. Variation is produced by the different combination of the oligosaccharides Gal beta 1----4GlcNAc beta 1----, Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----, GlcNAc beta 1----, Sia alpha 2----3Gal beta 1----4GlcNAc beta 1----, and Sia alpha 2----6Gal beta 1----4GlcNAc beta 1---- in their outer chain moieties. When both asparagine-linked consensus sites are glycosylated, the diversity of oligosaccharide structures yields over 400 different forms of the scrapie prion protein. Whether these diverse asparagine-linked oligosaccharides participate in scrapie prion infectivity or modify the function of the cellular prion protein remains to be established.

Potassium monopersulfate and a water-soluble manganese porphyrin complex, [Mn(TMPyP)](OAc)5, as an efficient reagent for the oxidative cleavage of DNA.

Abstract: Reported studies indicate that the association of potassium monopersulfate with [Mn(TMPyP)](OAc)5, a water-soluble manganese porphyrin complex, leads to an efficient reagent for the oxidative cleavage of DNA. Single-strand breaks (SSBs) are observed on double-stranded DNA at manganese porphyrin concentrations as low as 0.5 nM with a short incubation time of 1 min. The number of SSBs linearly varies with the concentration of the manganese complex, and potassium monopersulfate is at least 3 orders of magnitude more efficient as oxygen source than hydrogen peroxide. Cleavage efficiency is optimal in the pH range 7.5-9.0 for a NaCl concentration between 80 and 150 mM or for a MgCl2 concentration of 10 mM. At very low manganese porphyrin concentration and by increasing the incubation time a catalytic cleavage activity of the complex is evidenced: up to 5 SSBs per manganese porphyrin are observed. The high cleavage activity of the monopersulfate-manganese porphyrin system makes it a good candidate for DNA-footprinting experiments.

Directed alteration of the D1 polypeptide of photosystem II: evidence that tyrosine-161 is the redox component, Z, connecting the oxygen-evolving complex to the primary electron donor, P680.

Abstract: In photosystem II, electrons are sequentially extracted from water at a site containing Mn atoms and transferred through an intermediate carrier (Z) to the photooxidized reaction-center chlorophyll (P680+). Two polypeptides, D1 and D2, coordinate the primary photoreactants of the reaction center. Recently Debus et al. [Debus, R.J., Barry, B.A., Babcock, G.T., & McIntosh, L. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 427-430], have suggested that Z is a tyrosine residue located at position 161 of the D1 protein. To test this proposal, we have engineered a strain of the cyanobacterium Synechocystis PCC 6803 to produce a D1 polypeptide in which Tyr-161 has been replaced by phenylalanine. Wild-type Synechocystis PCC 6803 contains three nonidentical copies of the psbA gene which encode the D1 polypeptide. In the mutant strain, two copies were deleted by replacement with antibiotic-resistance genes, and site-directed mutations were constructed in a cloned portion of the remaining gene (psbA-3), carrying a third antibiotic-resistance gene downstream. Transformants were selected for antibiotic resistance and then screened for a photoautotrophy-minus phenotype. The mutant genotype was verified by complementation tests and by amplification and sequencing of genomic DNA. Cells of the mutant cannot evolve oxygen and, unlike the wild type, are unable to stabilize, with high efficiency, the charge-separated state in the presence of hydroxylamine and DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea]. Analyses by optical and EPR spectroscopy of reaction centers purified from this mutant indicate that Z can no longer be photooxidized and, instead, a chlorophyll cation radical, Chl+, is produced in the light. In the wild type, charge recombination between Z+ and the reduced primary quinone electron acceptor QA- occurs with a t1/2 of 80 ms. In the mutant, charge recombination between Chl+ and QA- occurs with a t1/2 of 1 ms. From these observations, we conclude that Z is indeed Tyr-161 of the D1 polypeptide.