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I M Russu

Publications and source records attributed to I M Russu.

31 records · Page 2Linked to original sources
PubMed1987

A proton nuclear Overhauser effect investigation of the subunit interfaces in human normal adult hemoglobin.

High-resolution proton nuclear magnetic resonance spectroscopy and nuclear Overhauser effects for the low-field exchangeable proton resonances of human normal adult hemoglobin in aqueous solvents are being used to confirm and extend the assignments of these resonances to specific protons at the intersubunit interfaces of the molecule. Most of these exchangeable proton resonances of human normal adult hemoglobin have been found to be absent in the spectra of isolated alpha or beta subunits. This finding indicates that they are specific spectral markers for the quaternary structure of the hemoglobin tetramer. Based on the nuclear Overhauser effect results, we have assigned the exchangeable proton resonance at +7.4 ppm downfield from H2O to the hydrogen-bonded proton between alpha 103(G10)His and beta 108(G10)Asn at the alpha 1 beta 1 interface. The nuclear Overhauser effect results have also confirmed the assignments of the exchangeable proton resonances at +9.4 and +8.2 ppm downfield from H2O previously proposed by workers in this laboratory based on a comparison of human normal adult hemoglobin and appropriate mutant hemoglobins. This independent confirmation of previously proposed assignments is necessary in view of the possible long-range conformational effects of single amino-acid substitutions in mutant hemoglobin molecules.

Adult↗
PubMed1987

Proton NMR studies of the molecular basis for the anti-sickling activity of non-covalent antisickling compounds.

Two new techniques of nuclear magnetic resonance spectroscopy are proposed to identify and characterize the binding sites of antisickling compounds to Hb S: measurements of the longitudinal relaxation rates (T1(-1)) of the C2 protons of individual surface histidyl residues of the C2 protons of individual surface histidyl residues of Hb S and intermolecular transferred nuclear Overhauser effects from protons in the heme pockets of the Hb S molecule. Using these methods, we have investigated the binding sites of L-phenylalanine, L-tryptophan, L-valine, and p-bromobenzylalcohol to sickle hemoglobin. With the exception of L-valine, all of these molecules are known to inhibit the polymerization of deoxy Hb S. We have found that for the compounds with antisickling activity, there are at least two binding sites to the Hb S molecule, one at or near the heme pockets of the alpha and beta chains and the other one in the vicinity of the beta 6 mutation site.

Antisickling Agents↗
PubMed1986

Assessment of role of beta 146-histidyl and other histidyl residues in the Bohr effect of human normal adult hemoglobin.

The contribution of the carboxyl-terminal histidines of the beta chains, beta 146(HC3), to the alkaline Bohr effect of human normal adult hemoglobin has been shown by this laboratory to depend upon the solvent composition. Using high-resolution proton nuclear magnetic resonance spectroscopy, we have found that the pKa value of the beta 146-histidine is 8.0 in the deoxy form, while in the carbonmonoxy form it ranges from 7.1 to 7.85 depending upon the concentration of inorganic phosphate and chloride ions present. These conclusions have been questioned by Perutz and co-workers on the basis of biochemical, structural, and proton nuclear magnetic resonance studies of mutant and enzymatically or chemically modified hemoglobins [Perutz, M. F., Kilmartin, J. V., Nishikura, K., Fogg, J. H., Butler, P. J., & Rollema, H. S. (1980) J. Mol. Biol. 138, 649-670; Kilmartin, J. V., Fogg, J. H., & Perutz, M. F. (1980) Biochemistry 19, 3189-3193; Perutz, M. F., Gronenborn, A. M., Clore, G. M., Fogg, J. H., & Shih, D. T.-b. (1985) J. Mol. Biol. 183, 491-498]. In this work, we use proton nuclear magnetic resonance spectroscopy to assess the effects of structural modifications on the histidyl residues and on the overall conformation of the hemoglobin molecule in solution. The structural perturbations investigated all occur within the tertiary domains around the carboxyl-terminal region of the beta chain as follows: Hb Cowtown (beta 146His----Leu); Hb Wood (beta 97His----Leu); Hb Malmö (beta 97His----Gln); Hb Abruzzo (beta 143His----Arg). Our results demonstrate that the conformational effects of single-site structural modifications upon the conformation and dynamics of hemoglobin depend strongly on their location in the three-dimensional structure of the protein molecule and also on their chemical nature. Furthermore, in normal hemoglobin, the spectral properties of several surface histidyl residues are found to depend, in the ligated state, upon the nature of the ligand. Our present findings do not support the recent spectral assignments proposed by Perutz et al. (1985) for the proton resonances of the beta 146- and beta 97-histidines and their suggestion that the enzymatic removal of the carboxyl-terminal beta 146-histidyl residues induces a conformational equilibrium for the beta 97-histidines in the des-beta 146His hemoglobin molecule in the carbonmonoxy form.

Hemoglobin A↗
PubMed1986

Molecular basis for the anti-sickling activity of aromatic amino acids and related compounds: a proton nuclear magnetic resonance investigation.

High-resolution proton nuclear magnetic resonance spectroscopy and relaxation techniques have been used to investigate the interactions of sickle cell hemoglobin (Hb S) and human normal adult hemoglobin (Hb A) with p-bromobenzyl alcohol, L-phenylalanine, L-tryptophan, and L-valine. With the exception of valine, all these compounds inhibit the polymerization of deoxy-Hb S [Noguchi, C. T., & Schechter, A. N. (1978) Biochemistry 17, 5455)). Using transferred nuclear Overhauser effects among the proton resonances of the compound of interest and the corresponding longitudinal relaxation rates (T1(-1], we have shown that the binding of each of the compounds investigated to deoxy-Hb S is comparable to that to deoxy-Hb A. Intermolecular transferred nuclear Overhauser effects have been observed between proton resonances of the anti-sickling compounds and specific protons situated in the heme pockets of Hb. On the basis of these results, we suggest that one binding site, common to all compounds with anti-sickling activity, is at or near the heme pockets in the alpha and beta chains of both deoxy-HB S and deoxy-Hb A. The proton T1(-1) values of the histidyl residues situated over the surface of the hemoglobin molecule indicate that a second binding site is located at or near the beta 6 position, containing the mutation in Hb S (beta 6Glu----Val). The binding of the compounds investigated to the latter site induces conformational changes in the amino-terminal domains of the beta chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Antisickling Agents↗
PubMed1984

A proton nuclear magnetic resonance investigation of human hemoglobin A2. Implications on the intermolecular contacts in sickle hemoglobin fibers and on the Bohr effect of human normal adult hemoglobin.

High-resolution proton nuclear magnetic resonance spectroscopy at 300 and 600 MHz has been used to investigate the conformation of a minor hemoglobin component of human blood, hemoglobin A2 (alpha 2 delta 2), in solution. We have found that (i) the replacement of the beta chains by the delta chains in hemoglobin A2 conserves the alpha 1 delta 2 interface but slightly perturbs the alpha 1 delta 1 interface, and (ii) one surface histidine residue in the deoxy form and one in the carbonmonoxy form of hemoglobin A2 have local conformations and/or electrostatic environments which are different from the corresponding ones in human normal adult hemoglobin. By comparing the proton nuclear magnetic resonance titration of individual histidine residues in hemoglobin A2 and in human normal adult hemoglobin, we can conclude that in human normal adult hemoglobin, both beta 116 and beta 117 histidine residues are titratable in both the deoxy and the carbonmonoxy forms. Thus, these two histidine residues can contribute to the Bohr effect of human normal adult hemoglobin. The present nuclear magnetic resonance data on hemoglobin A2 and those previously obtained in our laboratory on sickle hemoglobin suggest that the antisickling property of hemoglobin A2 does not originate from an alteration of the intermolecular contact site at the beta 6 position, but involves additional amino-acid residues which are different in the beta and delta chains. We have found that the replacement of the beta 116 and beta 117 histidine residues in the delta chains does not play a significant role in the antisickling effect of hemoglobin A2 and, thus, these amino-acid residues do not participate in the intermolecular interactions responsible for the polymerization of sickle hemoglobin.

Carboxyhemoglobin↗
PubMed1983

Proton nuclear magnetic resonance studies of isonitrile-heme protein complexes.

Steric interactions between bound ligand molecules and the valine E11 methyl groups of human hemoglobin and sperm whale myoglobin have been examined directly by high resolution NMR techniques. The methyl proton resonances of this amino acid are shifted markedly upfield and away from the bulk of the protein resonances by the shielding effect of circulating pi electrons in the porphyrin ring. We have monitored the valine resonance in the presence of CO and a series of isonitriles and found considerable shifts in its position, both between the various protein complexes and among the different liganded states. The ring current shifts of the gamma 1-methyl group of Val E11 in the CO forms of isolated alpha and beta chains and myoglobin are -2.70, -2.91, and -3.30 ppm, respectively. In all the proteins, these positions show little change in going from bound CO to bound methyl and ethyl isocyanide. In alpha subunits and myoglobin, n-propyl and n-butyl isocyanide binding produces marked decreases in the magnitude of these shifts, indicating that the valine residue has been forced away from the center of ring by the presence of these large ligand molecules. In the case of beta subunits, however, only tert-butyl isocyanide produces a marked decrease (from -2.91 to -1.99 ppm) in the ring current shift of the valine methyl protons. New peaks were observed in the isonitrile-protein spectra and identified as ligand proton resonances by comparing the spectra of normal and totally deuterated isonitrile complexes. The magnitudes of the ring current shifts for the terminal methyl protons of ethyl isocyanide suggest a linear geometry for the Fe equal to C equal to N - C bonds in beta chains and a bent geometry for alpha chains. The bent geometry in alpha subunits appears to be dictated by the position of the Val E11 methyl group which is located further up from the heme plane but closer to the heme center than the corresponding position of the beta subunit residue. The free energy changes for ethyl isocyanide binding to the two chains are nearly identical, suggesting that the linear and bent geometries are energetically equivalent. Myoglobin ethyl isocyanide complexes exhibit ligand ring current shifts intermediate to those observed for the hemoglobin subunits. Assignment of resonances and positions to the alkyl protons of the longer isonitriles is more difficult.

Animals↗
PubMed1983

Magnetic and spectral properties of carp carbonmonoxyhemoglobin. Competitive effects of chloride ions and inositol hexakisphosphate.

We have extended our studies on the magnetic properties of carp carbonmonoxyhemoglobin and the dependence of these properties upon solution variables. Using an improved version of the superconducting magnetometer, we have found that the magnetic susceptibility of carp carbonmonoxyhemoglobin is sensitive to both inositol hexakisphosphate and chloride ion. The dependence upon chloride ion concentration is complex. At relatively low concentrations this anion reverses the effect of inositol hexakisphosphate, restoring paramagnetism. At higher chloride concentrations the protein is converted to a roughly diamagnetic state in the absence of inositol hexakisphosphate. Along with these susceptibility studies, we have examined the effects of these anions on other properties of carp carbonmonoxyhemoglobin. The positions of the Soret bands of human and carp methemoglobin derivatives are correlated with spin state; changes in the magnetic susceptibility of carbonmonoxyhemoglobin are similarly associated with alterations in this spectral band. We have also examined the effects of these anions on the proton nuclear magnetic resonance spectrum of carp carbonmonoxyhemoglobin. Both chloride and inositol hexakisphosphate alter the position of the proton resonances in the ring-current-shifted region of the spectrum.

Animals↗
PubMed1982

A proton nuclear magnetic resonance investigation of histidyl residues in human normal adult hemoglobin.

High-resolution proton nuclear magnetic resonance (NMR) spectroscopy at 250 MHz has been used to titrate 22 individual surface histidyl residues (11 per alpha beta dimer) of human normal adult hemoglobin in both the deoxy and the carbon monoxy forms. The proton resonances of beta 2, beta 143, and beta 146 histidyl residues are assigned by a parallel 1H NMR titration of appropriate mutant and chemically modified hemoglobins. The pK values of the 22 histidyl residues investigated are found to range from 6.35 to 8.07 in the deoxy form and from 6.20 to 7.87 in the carbon monoxy form, in the presence of 0.1 M Bis-Tris or 0.1 M Tris buffer in D2O with chloride ion concentrations varying from 5 to 60 mM at 27 degrees C. Four histidyl residues in the deoxy form and one histidyl residue in the carbon monoxy form are found to have proton nuclear magnetic resonance titration curves that deviate greatly from that predicted by the simple proton dissociation equilibrium of a single ionizable group. The proton nuclear magnetic resonance data are used to ascertain the role of several surface histidyl residues in the Bohr effect of hemoglobin under the above-mentioned experimental conditions. Under these experimental conditions, we have found that (i) the beta 146 histidyl residues do not change their electrostatic environments significantly upon binding of ligand to deoxyhemoglobin and, thus, their contribution to the Bohr effect is negligible, (ii) the beta 2 histidyl residues have a negative contribution to the Bohr effect, and (iii) the total contribution of the 22 histidyl residues investigated here to the Bohr effect is, in magnitude, comparable to the Bohr effect observed experimentally. These results suggest that the molecular mechanism of the Bohr effect proposed by Perutz [Perutz, M.F. (1970) Nature (London) 228, 726-739] is not unique and that the detailed mechanism depends on experimental conditions, such as the solvent composition.

Adult↗
PubMed1982

A proton nuclear magnetic resonance investigation of histidyl residues in sickle hemoglobin.

Using high-resolution proton nuclear magnetic resonance spectroscopy at 250 MHz, we have determined the individual pK values of 22 surface histidyl residues (11 per alpha beta dimer) of sickle hemoglobin in both deoxy and carbon monoxy forms. Seven histidyl residues in the deoxy form and three in the carbon monoxy form are found to have pK values and chemical shifts different from the corresponding ones in human normal adult hemoglobin. Two of these histidyl residues are the beta 2 histidine and the beta 146 histidine, indicating that the conformations of the amino- and carboxyl-terminal regions of the beta chain in sickle hemoglobin are altered compared to those in human normal adult hemoglobin. The differences in the pK values of the additional surface histidyl residues between sickle and normal hemoglobins suggest that the effect of the amino acid substitution at the sixth position of the beta chain in sickle hemoglobin, namely, glutamic acid replaced by valine, is not restricted to the region around the mutation site but can extend to other regions in the protein molecule. In the deoxy form, the histidyl residues of sickle hemoglobin that have altered pK values and chemical shifts compared to the corresponding ones in human normal adult hemoglobin have been found to be sensitive to the early stages of the polymerization process [Russu, I.M., & Ho, C. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6577-6581].

Adult↗
PubMed1982

Proton longitudinal relaxation investigation of histidyl residues in human normal adult hemoglobin.

The longitudinal relaxation of the C2 protons of surface histidyl residues as well as other aromatic protons of human normal adult deoxyhemoglobin investigated at 360 MHz is discussed in terms of the theory proposed by Kalk and Berendsen for the proton longitudinal relaxation in proteins (Kalk, A., and H.J.C. Berendsen. 1976. J. Magn. Reson. 24:343-366). The role of the four paramagnetic iron atoms of deoxyhemoglobin as fast-relaxing sinks for the overall proton longitudinal relaxation is evaluated according to the model proposed by Bloembergen for the relaxation of nuclei in crystals containing paramagnetic centers (Bloembergen, N. 1949. Physica. 15:386-426). The results suggest that the effectiveness of the paramagnetic iron atoms of deoxyhemoglobin for the overall proton longitudinal relaxation is reduced as a result of slower spin diffusion and wide distribution of methyl groups within the hemoglobin molecule. Thus, deoxyhemoglobin provides a good model for investigating the influence of cross relaxation on proton longitudinal relaxation in proteins at the slow motion limit and in the presence of paramagnetic centers. For the C2 protons of surface histidyl residues, we show that the cross relaxation resulting from the interresidue dipolar interaction makes an important contribution to their longitudinal relaxation.

Adult↗
PubMed1980

Role of the beta 146 histidyl residue in the alkaline Bohr effect of hemoglobin.

High-resolution proton nuclear magnetic resonance spectroscopy has been used to investigate the effects of inorganic anions, such as phosphate or chloride, on the alkaline Bohr effect of normal human adult hemoglobin. By monitoring the chemical shift of the C2 proton of the beta 146 histidyl residue as a function of pH, we have determined its pK values in both ligated and unligated forms. In the presence of 0.1 M Bis-Tris buffer (with chloride ion concentration ranging from 0.005 to 0.06 M) in D2O at 27 degrees C, the pK value of the beta 146 histidine of deoxyhemoglobin is 7.98 +/- 0.03 and that of (carbon monoxy)hemoglobin is 7.85 +/- 0.03. However, in the presence of 0.2 M phosphate and 0.2 M NaCl in D2O at 27 degrees C, the corresponding pK values are 8.08 and 7.14, as previously reported by this laboratory [Kilmartin, J. V., Breen, J. J., Roberts, G. C. K., & Ho, C. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 1246-1249]. This large difference in the pK value between the deoxy and carbon monoxy forms in the presence of 0.2 M phosphate and 0.2 M NaCl was interpreted as direct support for (1) the breaking of an intrasubunit salt bridge between beta 146 histidine and beta 94 aspartate when the hemoglobin molecule undergoes the quaternary structural transition as proposed by Perutz [Perutz, M. F. (1970) Nature (London) 228, 726-739] and (2) Perutz's suggestion that the beta 146 histidine is one of the amino acid residues responsible for the alkaline Bohr effect. The absence of a large change in the pK value of the beta 146 histidine in the presence of 0.1 M Bis-Tris buffer implies that (1) the above-mentioned intrasubunit salt bridge is not broken in going from the deoxy to the carbon monoxy form and (2) the beta 146 histidyl residue does not contribute significantly to the alkaline Bohr effect under these conditions. We have also found that in measuring the oxygen affinity of hemoglobin as a function of pH in the presence of 0.1 M Bis-Tris or 0.2 M phosphate plus 0.2 M NaCl (both in D2O), there is no significant difference in the alkaline Bohr effect in these two media. Hence, our results suggest that the detailed molecular mechanism for the Bohr effect depends on the experimental conditions.

Hemoglobin A↗
PubMed1980

Proton longitudinal relaxation investigation of histidyl residues of normal human adult and sickle deoxyhemoglobin: evidence for the existence of pregelation aggregates in sickle deoxyhemoglobin solutions.

Proton nuclear magnetic resonance longitudinal-relaxation-rate measurements have been used to investigate the molecular events that occur during the early stages of the polymerization process of sickle hemoglobin. The longitudinal relaxation rates (T1-1) of the C2 protons of 11 observable surface histidyl residues in normal human adult and sickle hemoglobin in the deoxy state were measured in 0.1 M bis[(2-hydroxyethyl)imino]tris(hydroxymethyl)methane (pH 6.8) in 2H2O. These proton resonances in hemoglobin occur at a position 1.5-5.0 ppm downfield from that of residual water in 2H2O. The T1-1 values for the C2 protons of several surface histidyl residues in sickle hemoglobin in the deoxy state were sensitive to the temperature and the concentration of hemoglobin, factors known to have a profound effect on the polymerization process of sickle hemoglobin. For hemoglobin concentrations of 13.5% or less and temperatures of 25 degrees C or less, the T1-1 values in sickle hemoglobin solutions were the same as the corresponding values in normal hemoglobin, except for the C2 proton of beta 2 histidine, which had a larger T1-1 value. When the temperature or the hemoglobin concentration was increased (i) several additional histidine resonances in sickle hemoglobin solutions had larger T1-1 values than the corresponding ones in normal hemoglobin and (ii) the differences between the T1-1 values (sickle versus normal hemoglobin) of these histidine resonances as well as that of the beta 2 histidine resonance gradually increased. It is proposed that these results reflect the formation of small aggregates in the deoxygenated sickle hemoglobin solutions before gelation. In this model, the histidyl residues for which the T1-1 values are greatly increased in sickle hemoglobin solutions as compared with those in normal hemoglobin are viewed as being located in or near the "contact" areas between sickle hemoglobin molecules within the pregelation aggregates. Thus, this magnetic resonance technique can also be used to identify the intermolecular contacts in the polymerization of sickle hemoglobin.

Hemoglobin A↗