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C T Craescu

Publications and source records attributed to C T Craescu.

At least 19 recordsLinked to original sources
PubMed1992

Sequential assignment of the proton NMR spectrum of isolated alpha(CO) chains from human adult hemoglobin.

In this paper we report proton two-dimensional NMR experiments on isolated alpha chains from human hemoglobin A (HbA) in the monocarboxylated state. Several J-correlated and NOE spectra in water or deuterium water and phosphate buffer (100 mM) at 310 K and pH 5.6 were acquired and analysed for the sequential assignment of the proton resonances. In addition, we used the topological data obtained from the crystal structure of alpha subunits in the monocarboxylated HbA tetramer. The assigned resonances correspond to 70% of the amino acid residues. The present results provide information on the tertiary structure of isolated alpha chains in solution, particularly in the heme region. This structure may be compared with that of the a subunits in the tetrameric HbA(CO) in crystal by comparison of observed chemical shifts and those calculated from the X-ray atomic coordinates. Overall, the global folding of the two forms are highly similar. However, this analysis points out several local conformational differences in the heme pocket and the neighboring of the unique Trp residue. Possible explanations of these differences are discussed.

Amino Acid Sequence↗
PubMed1992

1H and 15N NMR characterization of free and bound states of an amphiphilic peptide interacting with calmodulin.

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.

Amino Acid Sequence↗
PubMed1992

Structural modeling of the human erythrocyte bisphosphoglycerate mutase.

Using the crystallographic structure of yeast monophosphoglycerate mutase (MPGM) as a framework we constructed a three-dimensional model of the homologous human erythrocyte bisphosphoglycerate mutase (BPGM). The modeling procedure consisted of substituting 117 amino acid residues and positioning 19 C-terminal residues (unresolved in the X-ray structure) by empirical methods, followed by energy minimization. Among several differences in the active site region the most significant appears to be the replacement of Ser11 in MPGM by Gly in BPGM. The C-terminal segment, which contains mainly basic amino acids, lines the cavity of the active site. The seven amino acid residues, which have been shown to be essential for the three catalytic functions of the human BPGM, interact with the amino acids in the protein core, near the active site. In addition, a cluster of several positively charged residues, particularly arginines, has been identified at the entrance of the active site; this cluster may serve as a secondary binding site for polyanionic substrates or cofactors, as required by a two-binding-site model of the catalytic activities. This model is in agreement with recent studies of an inactive BPGM variant substituent at an Arg position situated in this positively charged cluster. The position of Cys20 in the model constructed suggests that this residue is responsible for inactivation of the enzyme by sulfhydryl reagents. Subunit interfaces have also been constructed for BPGM by analogy with MPGM and suggest that, in addition to the known dimerization of BPGM, tetramerization may occur under certain conditions.

Amino Acid Sequence↗
PubMed1991

NMR and circular dichroic studies on the solution conformation of a synthetic peptide derived from the calmodulin-binding domain of Bordetella pertussis adenylate cyclase.

The solution conformation of a synthetic peptide of 20 amino acids (P235-254) derived from the calmodulin-binding domain of Bordetella pertussis adenylate cyclase was studied by proton two-dimensional NMR spectroscopy and circular dichroism. Based on the standard techniques we have assigned all the resonances in the NMR spectrum to the corresponding protons of the peptide. Analysis of the secondary chemical shift distribution and of the nuclear Overhauser effect connectivities showed no evidence for a highly populated regular conformation but suggested the tendency to form an alpha-helix around the unique Trp residue. The propensity for a helical structure is in agreement with the results of circular dichroic spectroscopy showing a slight negative band at 222 nm which was cancelled by 6 M guanidine hydrochloride. Increasing amounts of 2,2,2-trifluoroethanol (up to 40%) increase considerably the helical population of the peptide as reflected in the circular dichroic spectra. Analysis of the present results shows that the free peptide P235-254 has the tendency to form a basic amphiphilic helix. The presence of two acid residues, Glu236 and Asp239, on the hydrophilic side of the alpha-helix, which is mainly composed by basic residues, may explain the lower affinity of this peptide for calmodulin as compared with other peptides derived from calmodulin-activated enzymes.

Adenylyl Cyclases↗
PubMed1990

Functional and NMR studies of Hb Sassari (Asp-126 alpha----His); role of the inter-subunit contacts in the affinity control of human hemoglobin.

The oxygen affinity of hemoglobin Sassari (Asp-126 alpha----His), a variant substituted in the alpha 1 beta 1 interface, was found to be 8-times greater relative to normal adult human hemoglobin. Study of the exchangeable hydrogen-bonded protons by NMR spectroscopy shows only minor changes at the alpha 1 beta 1 interface. In particular, the resonance previously assigned to the proton of the hydrogen bond Asp-126 alpha 1. . . Tyr-35 beta 1 in normal hemoglobin is still present in the variant spectrum, suggesting that His-126 alpha can also form a hydrogen bond with the Tyr-35 beta. The possible explanation of the increased affinity of hemoglobin Sassari and other variants substituted in the same structural region is discussed in terms of perturbations of the equilibrium between the two quaternary states.

Carboxyhemoglobin↗
PubMed1990

Changes of polymerization and conformation of hemoglobin S induced by thiol reagents.

Thiol reagents, covalently bound to cysteine beta 93, either inhibit or facilitate the polymerization process of hemoglobin S. The progelling effect of parahydroxymercurybenzoate or 2,2'-dithiodipyridine contrasted with the increased oxygen affinity and the destabilization of the T state of Hb shown by functional and NMR studies. Thiol reagents increased the oxygen affinity of Hb from 30 to 1000%. Such variability was also observed in the reduction (up to 50%) of the alkaline Bohr effect. We show that the antigelling or progelling activity of thiol reagents does not depend solely on the concentration of molecules present in the deoxy T state but that specific effects of the reagent affects molecular interactions of the hemoglobin S polymerization process.

2,2'-Dipyridyl↗
PubMed1990

Coupling of tertiary and quaternary changes in human hemoglobin: a 1D and 2D NMR study of hemoglobin Saint Mandé (beta N102Y).

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.

Hemoglobins↗
PubMed1989

Resonance assignments in the proton-NMR spectrum of carbonmonoxy hemoglobin by two-dimensional methods.

Proton-nuclear-magnetic-resonance spectroscopy is a powerful tool for investigating the solution structure of biopolymers provided that a substantial number of proton resonances are assigned in the spectrum. For large proteins the assignments have usually been made by the comparative one-dimensional NMR investigations of the parent and derivative proteins in different physicochemical conditions. In this paper, we show that the more powerful two-dimensional methods could be successfully applide to proteins of the size of human adult hemoglobin (Mr = 64,500). J-Correlated and NOE-correlated spectroscopy, together with topological relationships in the known crystalline structure, enabled us to assign a large number of resonances. The majority of the assigned resonances correspond to the heme substituents and to amino acids in the heme pockets of the two subunits. These results thus provide an extensive set of intrinsic probes for mapping the conformation of the ligand-binding site and its functional changes. Comparison of the observed ring-current shifts of the assigned resonances with those calculated from the known crystallographic coordinates suggests a close similarity between the heme-pocket tertiary conformation in solution and in the crystalline state. A significant difference was noted for Leu141 in beta subunits which, in solution, appears to have stronger contacts with the heme groups than in the crystalline form. The present results also demonstrate that two-dimensional-NMR methods could be successfully applied to the investigation of the structure of large biomolecules in solution (Mr less than or equal to 65,000).

Amino Acids↗
PubMed1989

Filterability of sickle cells as a function of pO2: role of physico-chemical factors.

A rigidity index (RI) related to red blood cell deformability was measured by using the hemorheometre. The RI for 13 patients homozygous for sickle cell disease was 109 +/- 44 at 37 degrees C and at atmospheric pO2. The filtration time curve as a function of pO2 is biphasic for sickle cell suspensions. The pO2 at which filtration time is maximum, pO2max., correlated with the rigidity index measured at atmospheric pO2. This pO2max. value was very sensitive to small changes in physico-chemical parameters such as osmolality, pH, temperature, hematocrit, and cell density. Conditions which reduced the Hb S polymerization induced a leftward shift of pO2max.. The experimental curves are in agreement with theoretical models based on the presence of two abnormal cell types: filtrable "slow cells" and infiltrable "sickled cells".

Anemia, Sickle Cell↗
PubMed1988

Assignment of proton resonances in the NMR spectrum of carbonmonoxy hemoglobin beta subunit tetramers.

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.

Amino Acids↗
PubMed1988

One- and two-dimensional NMR investigations of the heme pocket in free alpha(CO) chains from human hemoglobin.

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.

Amino Acids↗
PubMed1988

Proton NMR studies of human hemoglobin variants modified in the proximal side of beta heme pocket. Implications for the affinity control and cooperative mechanism.

Using high resolution proton NMR spectroscopy, we have investigated 10 human hemoglobin variants modified in the proximal side of the heme pocket in beta subunits. Comparative observation of several resonances in the spectra of liganded and unliganded hemoglobins allowed us to characterize the localization and nature of the structural perturbations induced by amino acid substitutions or chemical modification. The present data indicate that the structural perturbations are localized in the beta subunits, mainly in the tertiary domain surrounding the modification site. Analysis of the aromatic region of the liganded hemoglobin spectra gives substantial information for the assignment of the His-beta 97 C-2H resonance. Correlation of the spectroscopic observations with the functional characteristics of the studied hemoglobins demonstrates that structural factors localized in the proximal side of the heme pocket can control the ligand-iron interaction taking place on the other heme side. The structural perturbations induced by the modifications in the F or FG segments of the beta subunits do not extend to the distal side but rather to the alpha 1 beta 2 interface. This argues the existence of a gradient of tertiary structural stability, indicating a possible structural pattern of heme-heme interaction in the cooperativity control.

Amino Acids↗
PubMed1987

Phosphines as a new structural probe of hemoglobin. 1H-NMR evidence for perturbations in the beta heme pocket induced by a thiol reagent.

Binding of trimethylphosphine to myoglobins and hemoglobins from a variety of sources has been examined by 1H-nuclear magnetic resonance. The hemoglobins exhibit two resonances at high field (approx. -3.5 ppm) which have been assigned to PMe3 bound to alpha or to beta subunits. Perturbations in the beta heme pocket induced by a thiol reagent have been detected both in 1H and 31P spectra.

Animals↗
PubMed1986

Covalent binding of glutathione to hemoglobin. II. Functional consequences and structural changes reflected in NMR spectra.

Binding of glutathione by disulfide linkage to Cys-beta 93 of hemoglobin tetramers within sickle cells increases the oxygen affinity and significantly inhibits sickling at low partial oxygen pressure (Garel, M-C., Domenget, C., Caburi-Martin, J., Prehu, C., Galacteros, F., and Beuzard, Y. (1986) J. Biol. Chem. 261, 14704-14709). This article reports a characterization of the oxygen-binding properties of glutathionyl hemoglobin (G-Hb) in solution in the presence or absence of allosteric effectors. The studies reveal a nearly 6-fold increase in oxygen affinity compared to native HbA and a Hill coefficient at half-saturation (n50) of 1.50 compared to n50 of approximately 2.9 for HbA. The oxygen Bohr effect measured in the alkaline pH range is reduced by 38%. Addition of 2,3-diphosphoglycerate decreases the oxygen affinity of G-Hb and HbA to a similar extent and increases the Bohr effect, indicating that the binding sites for organic phosphates are not perturbed in G-Hb. The rate of autooxidation of G-HbO2 is slower than of HbAO2. Oxidation by ferricyanide of G-HbCO is also reduced and is biphasic, demonstrating a heterogeneous susceptibility of the hemes in G-Hb. Flash photolysis experiments indicate that the tetramer-dimer dissociation constant is 1 order of magnitude greater for G-HbCO than for HbACO. High resolution NMR spectra at 400 MHz show that in G-Hb: the tertiary structure of the beta heme pocket is significantly perturbed, particularly in the F helix and the EF corner; the formation of the salt bridge between His-beta 146 and Asp-beta 94, a feature of the deoxy state, is precluded; and a deoxy interchain (alpha 1 beta 2) contact between Asp beta 2 99 and Tyr alpha 1 42 is appreciably destabilized. The NMR data provide a structural basis for interpreting the high oxygen affinity, reduced cooperativity, and diminished polymerization of G-HbS.

Binding Sites↗
PubMed1986

High resolution NMR studies of histidine-substituted and histidine-perturbed hemoglobin variants. Histidine assignments, electrostatic interactions at the protein surface, and implications for hemoglobin S polymerization.

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.

Electrochemistry↗
PubMed1986

Hemoglobin J Iran alpha 2 beta 2 77 (EF1) his----Asp in a Russian-Armenian family.

A third case of Hb J Iran is reported. The propositus is of Russian-Armenian origin and was investigated for hematuria. The electrophoretic behavior and the characterization of primary structure are described. Hb J Iran is stable and has normal functional properties. High resolution Nuclear Magnetic Resonance spectra suggest the presence of structural perturbations in the heme pocket of the variant. Solubility studies of Hb S/Hb J Iran mixture indicated that His beta 77 belongs to a contact region of deoxy Hb S polymers.

Adolescent↗
PubMed1985

Structural and functional studies of hemoglobin Poissy alpha 2 beta 2(56) (D7) Gly----Arg and 86 (F2) Ala----Pro.

Hemoglobin Poissy alpha 2 beta 2(56) (D7) Gly----Arg and 86 (F2) Ala----Pro, is a new variant of the beta chain with two substitutions within the second exon of the corresponding gene. The electrophoretic mobilities are identical to those of Hb Hamadan alpha 2 beta 2(56) (D7) Gly----Arg as is the fingerprint of the tryptic hydrolysate of the two abnormal beta chains. The second substitution beta 86 Ala----Pro was detected by high-pressure liquid chromatography. Hb Poissy has a threefold increase in oxygen affinity with low Hill coefficient and diminished Bohr effect, which are restored to normal upon addition of 2,3-bisphosphoglycerate. Since the functional properties of Hb Hamadan (beta 56 Gly----Arg) have been described as normal, the abnormal function of Hb Poissy may be attributed to the beta 86 (F2) Ala----Pro substitution. Hb Poissy exhibits a mild instability and a greater reactivity of the thiol groups of the beta 93 (F9) Cys residues in the deoxy form than does Hb A. The oxidation rate of Hb Poissy is biphasic indicating a large inequivalence between the alpha and beta hemes. Thereafter NMR studies demonstrated that the beta 86 Ala----Pro substitution produces a displacement of the F helix closer to the heme plane and a large increase in the dynamic fluctuations of the tertiary structure on the proximal side of the beta hemes. These results lead to the conclusion that the beta 86 Ala----Pro substitution produces a destabilization of the F helix extending downwards to the FG corner and altering both the beta hemes and the alpha 1 beta 2 contacts.

Amino Acids↗