Search

The N-Boc O-tert-butyldimethysilyl-substituted hexa-β-peptide methyl ester 18 was constructed from the O-TBS ether of (−)-(2R, 3S)-phenylisoserine. By NMR, it was determined that this homo β-peptide adopts a highly stable β-strand-type secondary structure in chloroform solution, which is stabilized by both hydrophobic interactions involving the OTBS methyl groups of residues i and i + 2, and inter-(five-membered)/intra (six-membered)-residue H-bonding interactions. These interactions are systematically repeated along the peptide chain and, thereby, operate in concert to stabilize the observed conformation of 18.

A peptide of 17 amino acid residues Ac-L-K-W-K-K-L-L-K-L-L-K-K-L-L-K-L-G-NH2, designed to form an amphiphilic basic alpha-helix [DeGrado, W.F., Prendergast, F. G., Wolfe, H. R., Jr., & Cox, J. A. (1985) J. Cell. Biochem. 29, 83-93], was labeled with 15N at positions 1, 7, 9, and 10. Homo- and heteronuclear NMR techniques were used to characterize the conformational changes of the peptide when it binds to calmodulin in the presence of Ca2+ ions. The spectrum of the free peptide in aqueous solution at pH 6.3 and 298 K was completely assigned by a combined application of several two-dimensional proton NMR methods. Analysis of the short- and medium-range NOE connectivities and of the secondary chemical shifts indicated that the peptide populates, to a significant extent, an alpha-helix conformational state, in agreement with circular dichroism measurements under similar physicochemical conditions. 15N-edited 1D spectra and 15N(omega 2)-half-filtered two-dimensional NMR experiments on the peptide in a 1:1 complex with calmodulin allowed assignment of half of the amide proton resonances and three C alpha H resonances of the bound peptide. The observed NOE connectivities between the peptide backbone protons are indicative of a stable helical secondary structure spanning at least the fragment L1-K11. The equilibrium and dynamic NMR parameters of the bound peptide are discussed in terms of a molecular interaction model.

The sequence-specific resonance assignment of apo-neocarzinostatin from Streptomyces carzinostaticus was carried out from two-dimensional proton-NMR spectra. The assignments were obtained for the backbone protons of 111 of the 113 residues of the protein, missing the two C alpha H of one glycine but including 3 of the 4 prolines. The majority of side chain protons were also assigned. The secondary structure derived from the analysis of sequential connections corresponds to ten beta-strands separated by clearly identified loops and turns. Inter-strand connectivities and slowly exchanging amide protons confirm the presence of the two disulfide bridges from Cys37 to Cys47 and from Cys88 to Cys93 and indicate a global folding similar to that of the similar proteins, actinoxanthin and macromomycin, for which crystallographic data are available.

Hemoglobin Saint Mandé (beta N102Y) is a low-affinity mutant with the substitution site situated in the quaternary-sensitive alpha 1 beta 2 interface. In adult hemoglobin the Asn102 beta contributes to the stability of the liganded (R) state, forming a hydrogen bond with Asp94 alpha. The quaternary and tertiary perturbations subsequent to the Tyr for Asn substitution in monocarboxylated hemoglobin Saint Mandé have been investigated by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Analysis of the one-dimensional NMR spectra of the liganded and unliganded samples in 1H2O provides evidence that both R and T quaternary structures of Hb Saint Mandé are different from the corresponding ones in HbA. In the monocarboxylated form of the mutant hemoglobin, at acid pH, we have observed the disappearance of an R-type hydrogen bond and the appearance of a new one whose proton resonates like a deoxy T marker. Using two-dimensional NMR methods and on the basis of previous results on the monocarboxylated HbA, we have obtained a significant number of resonance assignments in the spectra of monocarboxylated Hb Saint Mandé at pH 5.6 in the presence or absence of a strong allosteric effector, inositol hexaphosphate. This enabled us to characterize the tertiary conformational changes (relative to the liganded normal hemoglobin) triggered by the quaternary-state modification. The observed structural variations are confined within the heme pocket regions but concern both the alpha and beta subunits. Most of them, localized in the C, F, G, and FG segments, could result directly from the side-chain substitution, while others, such as Leu141 beta, could be explained only by long-range interactions.

This paper reports the solution conformation and calmodulin binding of a 43-residue peptide from the calmodulin-binding domain of Bordetella pertussis adenylate cyclase. The peptide (P225-267) was synthesized and 15N-labeled at specific amino acids. It binds calmodulin with an equilibrium dissociation constant of 25 nM. Assignment of the NMR spectrum of the free peptide and analysis of the NOE connectivities and secondary shifts of C alpha protons allowed us to identify a 10-amino acid fragment (Arg237 to Arg246) which is in rapid equilibrium between alpha-helical and irregular structures. Titration experiments showed that at substoichiometric molar ratios the two molecules are in intermediate exchange between free and bound conformations. Using 15N-edited methods we assigned a large part of resonances of the labeled residues in the bound peptide. Analysis of the chemical shift differences between free and bound states shows that the fragment Leu240-Ala257 is the most affected by the interaction. The proton spectra of the calmodulin, in the free and complexed states were extensively assigned using homonuclear experiments. Medium- and long-range NOE patterns are consistent with a largely conserved secondary and tertiary structure. The main changes in chemical shift of calmodulin resonances are grouped in six structural regions both in NH2- and COOH-terminal domains. Intermolecular NOE connectivities indicate that the NH2-terminal of the bound peptide fragment is engulfed in the COOH-terminal domain of calmodulin. The interaction geometry appears to be similar to those previously described for myosin light chain kinase or calmodulin kinase II fragments.

The three-dimensional structure of apo-NCS, as revealed by proton NMR, is based on an antiparallel seven-stranded beta-barrel. This fold is frequently encountered in protein structures, especially for immunoglobulin domains. The strands forming the barrel are joined by flexible loops of which three are implicated in the ligand binding site of these proteins. In this paper a preliminary comparison is given with respect to the static and dynamic properties of both the constant beta-barrel and the active loops for apo-NCS and the variable VH domain of an immunoglobulin Fab' fragment.

The structural identification of nineteen metabolites of dibenzo[a,e]fluoranthene (DBF) obtained by incubation in rat and mouse liver microsomes, allows one to establish a qualitative and semi-quantitative metabolic chart, involving up to three distinct oxidative attacks. The primary steps lead to dihydrodiols on rings A and D and phenols on rings A and E. Secondary vicinal epoxidation of dihydrodiols is a minor route as compared to attack at a second peripheral ring. Even after a third oxidation, one of the peripheral rings A, D and E remains unsubstituted. A model for cytochrome P-450 enzymatic activity which takes into account most of the observations is proposed. It requires that the catalytic site for monooxygenation is 0.6 nm apart from the center of an hydrophobic protein site accommodating one of the unsubstituted peripheral benzenoid rings. both trans diequatorial dihydrodiols of ring A and D corresponding to the 'bay' and 'pseudo bay region'; of DBF appear in the activation pathways for the in vivo carcinogenesis. The ultimate metabolite reacting with DNA is thus, most probably, a vicinal dihydrodiol epoxide of ring A or D. The great complexity of the metabolic chart of DBF as compared to other carcinogenic polycyclic aromatic hydrocarbons leaves also the possibility of sequential reactions at these two distinct sites of the molecule.

Preparation des complexes cites dans le titre a partir de la tetraphenylporphyrine, de la tetratolylporphyrine et de la tetra-para-chlorophenylporphyrine de fer(II). Obtention de ces complexes a l'etat cristallin. Structure du complexe avec (TpclPP) par diffraction RX et susceptibilite magnetique. Spectre de masse, spectre UV-visible, spectre IR, spectre Mossbauer, spectre RMN de H 1 et C 13 du complexe avec (TPP)

The hydrogen exchange kinetics of the N delta H proton in His F8 of iodoacetamide- and N-ethylmaleimide-treated human deoxyhemoglobins were studied using a NMR method. Comparison with unmodified hemoglobin shows that the reagents, covalently bound to Cys beta 93, significantly increase (about one order of magnitude) the exchange kinetics in beta chains only. This effect was partially reversed by the strong allosteric effector inositol hexaphosphate. Study of the high resolution 400-MHz NMR spectra of modified oxy- and deoxy-hemoglobins permitted localization of the extent of chemically induced structural perturbations. The resonances corresponding to hydrogen bonds specific to the deoxy conformation are not changed, in accord with the preserved cooperativity. Under the experimental conditions (0.1 M bis-Tris, 10 mM Cl-, pH 7.2), the salt bridge at the C terminus of the beta chain in the deoxy state (His beta 146-Asp beta 94) is perturbed by both modifications. The His beta 146 appears to be rendered more immobilized by the reagents in the oxy conformation. From the resonances corresponding to heme pocket protons of oxyhemoglobin it is deduced that the perturbations do not extend over the distal side of the heme pocket but are limited to the FG, F, and HC segments of the beta chain.

For almost a decade heme model compounds have been designed to test the influence of proximal base restraint or of distal steric hindrance upon the ligand affinity of hemoglobins. Despite the variety of molecular structures which have been successively proposed, the evaluation of the reported data is rendered difficult because of the small number of examples available within each series. In this paper we report on the kinetics of binding of oxygen and carbon monoxide with a series of nine closely related heme models. The 'basket-handle porphyrins' allow one to modify the constraints exerted upon a chelated proximal base as well as the chemical environment of the distal side of the heme. One salient feature of these models is the possibility of introducing a hydrogen-bond stabilization of the oxygen by incorporating an amide group in the vicinity of the iron centre. The structural changes among models are sufficiently 'soft' to cause an almost continuous variation of the binding constants and rate parameters. The latter are found to obey a definite linear free energy relationship which proves that the series is homogenous from a thermodynamic viewpoint. This suggests an alternative way for comparing the trends in ligand binding in different heme model families with those of heme proteins, which is developed in the discussion using literature data.

The aromatic region of the proton NMR spectrum of human adult hemoglobin (HbA) contains resonances from at least 11 titratable histidine residues. Assignments for five beta chain histidines have previously been proposed. In order to further characterize the aromatic spectra of HbA we studied 11 histidine-substituted and -perturbed hemoglobin variants in oxy and deoxy states and at different pH values by 400 MHz NMR spectroscopy. We propose assignments for the resonances corresponding to the C2 protons of His alpha 20, His alpha 72, His alpha 112, and His beta 77 in oxy and deoxy spectra and of His beta 97 and His beta 117 in deoxy spectra. Our assignments for His beta 2 and His beta 117 in the oxy state agree with those previously reported for the CO form, but in the deoxy state our spectra suggest a different assignment. Studies with Hb variants in which a histidine is perturbed by a neighboring substitution suggest additional assignments for His alpha 50 and His alpha 89 and demonstrate a strong dependence of the imidazole ring pK on hydrogen bond interactions and on the net charge of neighboring residues. Some of the newly proposed assignments of histidine resonances are used to discuss specific intermolecular interactions implicating His alpha 20, His beta 77, and His beta 117 in deoxy HbS polymers.

The synthesis from 3-thiochromanone of indole and quinoline derivatives is reported. The structures of the compounds obtained, which are analogs of carcinogenic nitrogen polycycles, were determined by means of mass spectrometry and high resolution NMR spectrometry. In the latter case, Nuclear Overhauser Enhancements (NOE) allow the unequivocal determination of structures. Furthermore, direct (or“through space”) 1 H- 1 H couplings are reported and related to the NOE.

The preparation of the title compound, a model for the active site of oxygen carrier hemoproteins, is described and the kinetic parameters of the CO and O2 adducts show the importance of the co-ordinated imidazole in stabilization.

Two-dimensional nuclear magnetic resonance techniques were used to assign resonances corresponding to heme pocket residues of the isolated alpha(CO) subunits of the human adult hemoglobin (HbA). The assignment procedure was based on the partial identification of the amino acid spin system from the J-correlated (COSY) spectrum and on the nuclear Overhauser effect connectivities (from NOSEY spectra) with the heme substituents. We present here partial assignments corresponding to five amino acid residues: Leu86, Leu-91, Val-93, Leu-101 and Leu-136. Starting from the known crystallographic structure of the alpha subunit in the hemoglobin tetramer, we applied a dipolar model to compute the ring-current shift of the protons from fifteen amino acid residues in the heme pocket. Comparison of the predicted and observed chemical shifts suggests that there is a very close similarity between the heme pocket tertiary structure of the alpha(CO) subunits in crystals of HbA(CO) and of the free alpha(CO) chains. The one-dimensional NMR spectra were used to monitor the pH-induced structural changes, the effects of chemical modification and of ligand substitution. Upon increasing the pH from 5.6 to 9.0 the structure of the heme environment appears to be invariant with the exception of some residues in the CD corner. The structure is also largely conserved when p-chloromercuribenzoate is bound to Cys-104. In contrast, the substitution of CO by O2 as ligand induces many large changes in the heme cavity which can be partially characterized by NMR spectroscopy.

Isolated beta chains from human adult hemoglobin at millimolar concentration are mainly associated to form beta 4 tetramers. We were able to obtain relevant two-dimensional proton nuclear magnetic resonance (NMR) spectra of such supermolecular complexes (Mr approximately 66,000) in the carboxylated state. Analysis of the spectra enabled us to assign the major part of the proton resonances corresponding to the heme substituents. We also report assignments of proton resonances originating from 12 amino acid side chains mainly situated in the heme pocket. These results provide a basis for a comparative analysis of the tertiary heme structure in isolated beta(CO) chains in solution and in beta(CO) subunits of hemoglobin crystals. The two structures are generally similar. A significantly different position, closer to the heme center, is predicted by the NMR for Leu-141 (H19) in isolated beta chains. Comparison of the assigned resonances of conserved amino acids in alpha chains, beta chains and sperm whale myoglobin indicates a close similarity of the tertiary heme pocket structure in the three homologous proteins. Significant differences were noted on the distal heme side, at the position of Val-E11, and on Leu-H19 and Phe-G5 position on the proximal side.

The preparation and properties of molecules modeling the active site of natural oxygen carriers have received considerable attention in recent years for the elucidation of the factors that control the binding reactions of molecular oxygen and carbon monoxide in some hemoproteins. Such compounds have been synthesized following three requirements which seem necessary in the biological functions of natural systems: 1. pentacoordination of the iron(II) ion by a nitrogenous base on the proximal site; 2. steric protection of the heme in order to prevent it from irreversible oxidation into μ-oxo iron(III) dimers; 3. control of the dioxygen environment on the distal site. Several groups have investigated these three problems separately [1]. As part of our studies of the stereochemical influences on the formation and stability of oxygenated model compounds, we have prepared two series of porphyrin derivatives which correspond to the three structural conditions mentioned above. All the compounds were synthesized following the concept of both face hindered tetraphenylporphyrins (‘basket handle’ porphyrins) in which two opposite mesophenyl rings are bridged by a convenient chain [2,3]. The proximal base (pyridine or imidazole) was inserted into one of the handles. The size and polarity of the cage on the distal side can be modified to some extent by suitable chemical changes of the second handle. The newly ‘hanging base’ porphyrins were prepared either from 5,10,15,20-tetrakis(o-hydroxyphenyl)porphyrin (series A, compounds 1–4) or from 5,10,15,20-tetrakis(o-aminophenyl)porphyrin (series B, compounds 5–8). Thus the main structural difference between the two series lies in the presence of non polar ether groups in the former and of polar amide linkage in the latter [4]. Their iron(II) complexes have a magnetic high spin state (S = 2) characteristic of deoxymyoglobins and hemoglobins. Addition of O2 and CO to these complexes results in the formation of hexacoordinated diamagnetic species (S = O), as in oxy and carbooxyhemoproteins, without any paramagnetic perturbations by low lying thermally excited states. A systematic study of the O2 and CO rates and equilibrium binding constants has been carried out in toluene by laser flash photolysis using a modified exchange rate law [5]. This clearly establishes the important role of factors such as the distal steric hindrance, the proximal base constraint and the polarity of the dioxygen environment in the control of the O2 binding and the stability of oxygenated complexes. The association rate constants of O2 and CO are of the same order of magnitude in the two series, but a large variation in the dissociation rate constants is observed. The smaller dissociation rate constants are observed for the compounds having amide linkages as compared with their analogous ether bearings linkages. This results in an increase of the intrinsic stability of the oxygenated derivatives up to the values observed in the natural compounds. The proton magnetic resonance spectra of the oxygenated complexes indicate that the bound dioxygen molecule lies preferentially in a plane oriented toward the distal amide groups in the B series, in contrast with series A in which four nearly equivalent orientations are observed. This preferential orientation of the oxygen molecule results from an hydrogen bonding interaction with the amide groups as shown by the chemical shift of the corresponding protons. The role of this bond in the stability and the bent geometry of the oxygenated complex is of the same type as those proposed for hemoproteins involving distal histidine [6, 7].

The synthesis of a series of so-called amide ‘hanging base’ porphyrins (10), (14) and (18). derived from 5,10,15,20-tetrakis(o-aminophenyl)porphyrin (αβαβ-atropisomer)(3), in which one of the faces is hindered by an alkylene or an arylene-p-bisalkylene bridge and the other face is bridged by a pyridine-3,5-diyl-bisalkyleneoran imidazolyl-alkylene chain, is described. The structural assignment of the various compounds is based on the 1H n.m.r. spectra of the free bases, and of their zinc(II) and iron(II) complexes. Unlike the ether ‘hanging base’ metalloporphyrins, the metallic ion of amide ‘hanging base’ porphyrins is actually five-co-ordinated by the proximal base. Furthermore, not only is the equilibrium pyridine plane orientation dependent on the length of its linking chain, but the bridging forces the amide protons to point toward the centre of the macrocyle core. These structural properties are potential factors which may affect the binding of dioxygen. The synthesis of a bis-pyridine ‘basket handle’ porphyrin is also reported.