Effect of amino acid at the beta 6 position on surface hydrophobicity, stability, solubility, and the kinetics of polymerization of hemoglobin. Comparisons among Hb A (Glu beta 6), Hb C (Lys beta 6), Hb Machida (Gln beta 6), and Hb S (Val beta 6)

Surface hydrophobicity, stability, solubility, and kinetics of polymerization were studied using hemoglobins with four different amino acids at the beta 6 position: Hb A (Glu beta 6), Hb C (Lys beta 6), Hb Machida (Gln beta 6), and Hb S (Val beta 6). The surface hydrophobicity increased in the order of Hb C, Hb A, Hb Machida, and Hb S, coinciding with the hydrophobicity of the amino acid at the beta 6 position. Solubility of the oxy-form of these hemoglobins decreased in relation to increases in their surface hydrophobicity, suggesting that the solubility is controlled by the strength of hydrophobicity of the amino acid at the beta 6 position. The solubility of the oxy-form of these hemoglobins is always higher than that of the deoxy-form. There is a similar linear relationship between the solubility and surface hydrophobicity among deoxyhemoglobins A, C, and Machida. However, the solubility of deoxy-Hb S deviated significantly from the expected value, indicating that the extremely low solubility of deoxy-Hb S is not directly related to the hydrophobicity of the beta 6 valine. Kinetic studies on the polymerization of deoxy-Hb Machida revealed a distinct delay time prior to polymerization. This confirms our previous hypothesis that beta 6 valine is not responsible for the delay time prior to gelation. The kinetics of the polymerization of 1:1 mixtures of sickle and non-sickle hemoglobins were similar to those of pure Hb S, suggesting that only one of the two beta 6 valines is involved in an intermolecular contact. In mixtures of equal amounts of Hb S and Hb A, Hb C, or Hb Machida, half of the asymmetrical AS, SC, and S-Machida hybrid hemoglobins behaved like Hb S during nucleation, while the other half behaved like the non-sickle hemoglobin.     

The Hb A variant (beta73 Asp-->Leu) disrupts Hb S polymerization by a novel mechanism

Polymerization of a 1:1 mixture of hemoglobin S (Hb S) and the artificial mutant HbAbeta73Leu produces a dramatic morphological change in the polymer domains in 1.0 M phosphate buffer that are a characteristic feature of polymer formation. Instead of feathery domains with quasi 2-fold symmetry that characterize polymerization of Hb S and all previously known mixtures such as Hb A/S and Hb F/S mixtures, these domains are compact structures of quasi-spherical symmetry. Solubility of Hb S/Abeta73Leu mixtures was similar to that of Hb S/F mixtures. Kinetics of polymerization indicated that homogeneous nucleation rates of Hb S/Abeta73Leu mixtures were the same as those of Hb S/F mixtures, while exponential polymer growth (B) of Hb S/Abeta73Leu mixtures were about three times slower than those of Hb S/F mixtures. Differential interference contrast (DIC) image analysis also showed that fibers in the mixture appear to elongate between three and five times more slowly than in equivalent Hb S/F mixtures by direct measurements of exponential growth of mass of polymer in a domain. We propose that these results of Hb S/Abeta73Leu mixtures arise from a non-productive binding of the hybrid species of this mixture to the end of the growing polymer. This "cap" prohibits growth of polymers, but by nature is temporary, so that the net effect is a lowered growth rate of polymers. Such a cap is consistent with known features of the structure of the Hb S polymer. Domains would be more spherulitic because slower growth provides more opportunity for fiber bending to spread domains from their initial 2-fold symmetry. Moreover, since monomer depletion proceeds more slowly in this mixture, more homogeneous nucleation events occur, and the resulting gel has a far more granular character than normally seen in mixtures of non-polymerizing hemoglobins with Hb S. This mixture is likely to be less stiff than polymerized mixtures of other hybrids such as Hb S with HbF, potentially providing a novel approach to therapy.     

Electron paramagnetic resonance of Hb St Louis beta28 (B10) Leu replaced by Gln.

Hemoglobin St Louis beta28 (B10) Leu replaced by Gln is a new mutant which occurs as a natural valency hybrid (alpha2beta+2), or hemoglobin M (Cohen-Solal, M., Seligmann, M., Thillet, J. and Rosa, J. (1973) FEBS Lett. 33, 37-41). The electron paramagnetic resonance (EPR) spectrum of native Hb St Louis at pH 6.2 shows a mixture of three species. Two are high spin, one with tetragonal symmetry, like Hb+ A, the other with rhombic distortion. The third is a low-spin form corresponding to a hemichrome with the distal (E7) histidine as the sixth ligand of the ferric iron. The hemichrome is also found in red blood cells. After oxidation to the alpha+2beta+2 form, three EPR species are seen. Surprisingly, there remains only one high-spin signal, with almost tetragonal symmetry. Besides the low-spin hemichrome, a hydroxy signal is observed even at pH 6.2. These observations imply interactions between the alpha and beta hemes.     

A maximal effort trial in obese women carrying the beta2-adrenoceptor Gln27Glu polymorphism

The aim of this study was to observe the relationship of the beta2-adrenoceptor (ADRB2) gene polymorphism Gln27Glu in a group of obese female subjects submitted to an acute physical activity test. Six Glu27/Glu27 obese women were compared with six Gln27/Gln27 obese women (coupled by age, BMI, waist circumference, percentage of fat mass and absence of smoking). These groups were selected after having genotyped 91 obese subjects (BMI>30 kg/m2, age 20-60). Polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) were used to genotype the obese population. The physical activity challenge consisted of a Maximal effort trial (VO2 max) following the Bruce protocol on a walking treadmill. The frequency for the Glu allele was 0.42, which is similar to other Caucasian populations. In basal conditions, subjects with the Glu27/Glu27 genotype showed a higher value of plasma insulin, while circulating glucose levels were similar in both groups. Also, diastolic arterial pressure was higher in the Glu27/Glu27 obese women. After the VO2 max trial, the Glu27 group had a significantly higher respiratory exchange ratio than the Gln27 group suggesting a lower post- exercise fat oxidation. These results provide evidence about a possible impact of the Gln27Glu polymorphism in the ADRB2 gene concerning a better response to exercise in obese Gln27 female subjects, in which basal insulinemia may play a role.     

Mouse alpha chains inhibit polymerization of hemoglobin induced by human beta S or beta S Antilles chains

A murine model of sickle cell disease was tested by studying the polymerization of hybrid hemoglobin tetramers between alpha mouse and human beta S or beta S Antilles chains were prepared from Hb S Antilles, which was a new sickling hemoglobin inducing a sickle cell syndrome more severe than Hb S. The hybrid molecules did not polymerize in solution, indicating that the mouse alpha chains inhibited fiber formation. Consequently, a mouse model for sickle cell disease requires the transfer and expression of both alpha and beta S or beta S Antilles genes.     

Genetic and clinical study of a family with abnormal hemoglobin Hb D Punjab 121 beta Glu----Gln

A second case of discovery of HbD Punjab in Russian population is reported. Abnormal hemoglobin was found in women with recurrent fetal wastage. The possibility of casual relationship between the presence of HbD Punjab and this type of pathology was rejected on the basis of detailed clinical and genetical investigation.     

Effect of the beta6 Glu replaced by Val mutation on the optical activity of hemoglobin S and of its beta subunits

The carbomonoxy derivatives of hemoglobin A and S showed a different optical activity in the Soret region of the spectrum as measured by circular dichroism. Different optical activity was also measured in the carbomonoxy derivatives of the beta subunits of hemoglobin A and S, the respective deoxy derivatives showed different circular dichroism spectra only in the presence of inositol hexaphosphate. Sedimentation velocity experiments showed that the differences in optical activity are not due to a different state of aggregation of the subunits. Modification of the tertiary structure of the beta subunits seems to be responsible for the phenomenon. Speculation based on the work of Hsu and Woody (Hsu, M.C., and Woody, R.W. (1971) J. Am. Chem. Soc. 93, 3515-3525) suggests the involvement of the beta15 tryptophan in the conformational changes produced by the beta6 Glu-Val mutation in hemoglobin S.     

The role of beta93 Cys in the inhibition of Hb S fiber formation

Recent studies have suggested that nitric oxide (NO) binding to hemoglobin (Hb) may lead to the inhibition of sickle cell fiber formation and the dissolution of sickle cell fibers. NO can react with Hb in at least 3 ways: 1) formation of Hb(II)NO, 2) formation of methemoglobin, and 3) formation of S-nitrosohemoglobin, through nitrosylation of the beta93 Cys residue. In this study, the role of beta93 Cys in the mechanism of sickle cell fiber inhibition is investigated through chemical modification with N-ethylmaleimide. UV resonance Raman, FT-IR and electrospray ionization mass spectroscopic methods in conjunction with equilibrium solubility and kinetic studies are used to characterize the effect of beta93 Cys modification on Hb S fiber formation. Both FT-IR spectroscopy and electrospray mass spectrometry results demonstrate that modification can occur at both the beta93 and alpha104 Cys residues under relatively mild reaction conditions. Equilibrium solubility measurements reveal that singly-modified Hb at the beta93 position leads to increased amounts of fiber formation relative to unmodified or doubly-modified Hb S. Kinetic studies confirm that modification of only the beta93 residue leads to a faster onset of polymerization. UV resonance Raman results indicate that modification of the alpha104 residue in addition to the beta93 residue significantly perturbs the alpha(1)beta(2) interface, while modification of only beta93 does not. These results in conjunction with the equilibrium solubility and kinetic measurements are suggestive that modification of the alpha104 Cys residue and not the beta93 Cys residue leads to T-state destabilization and inhibition of fiber formation. These findings have implications for understanding the mechanism of NO binding to Hb and NO inhibition of Hb S fiber formation.     

Stabilization of apoflavodoxin by replacing hydrogen-bonded charged Asp or Glu residues by the neutral isosteric Asn or Gln

Knowledge of protein stability principles provides a means to increase protein stability in a rational way. Here we explore the feasibility of stabilizing proteins by replacing solvent-exposed hydrogen-bonded charged Asp or Glu residues by the neutral isosteric Asn or GLN: The rationale behind this is a previous observation that, in some cases, neutral hydrogen bonds may be more stable that charged ones. We identified, in the apoflavodoxin from Anabaena PCC 7119, three surface-exposed aspartate or glutamate residues involved in hydrogen bonding with a single partner and we mutated them to asparagine or glutamine, respectively. The effect of the mutations on apoflavodoxin stability was measured by both urea and temperature denaturation. We observed that the three mutant proteins are more stable than wild-type (on average 0.43 kcal/mol from urea denaturation and 2.8 degrees C from a two-state analysis of fluorescence thermal unfolding data). At high ionic strength, where potential electrostatic repulsions in the acidic apoflavodoxin should be masked, the three mutants are similarly more stable (on average 0.46 kcal/mol). To rule out further that the stabilization observed is due to removal of electrostatic repulsions in apoflavodoxin upon mutation, we analysed three control mutants and showed that, when the charged residue mutated to a neutral one is not hydrogen bonded, there is no general stabilizing effect. Replacing hydrogen-bonded charged Asp or Glu residues by Asn or Gln, respectively, could be a straightforward strategy to increase protein stability.     

Effect of zeta-globin substitution on the O2-transport properties of Hb S in vitro and in vivo

Hemoglobin zeta(2)beta(2)(S) is generated by substituting embryonic zeta-globin subunits for the normal alpha-globin components of Hb S (alpha(2)beta(2)(S)). This novel hemoglobin has recently been shown to inhibit polymerization of Hb S in vitro and to normalize the pathological phenotype of mouse models of sickle cell disease in vivo. Despite its promise as a therapeutic tool in human disease, however, the basic O(2)-transport properties of Hb zeta(2)beta(2)(S) have not yet been described. Using human hemoglobins purified from complex transgenic-knockout mice, we show that Hb zeta(2)beta(2)(S) exhibits an O(2) affinity as well as a Hill coefficient, Bohr response, and allosteric properties in vitro that are suboptimally suited for physiological O(2) transport in vivo. These data are substantiated by in situ analyses demonstrating an increase in the O(2) affinity of intact erythrocytes from mice that express Hb zeta(2)beta(2)(S). Surprisingly, though, co-expression of Hb zeta(2)beta(2)(S) leads to a substantial improvement in the tissue oxygenation of mice that model sickle cell disease. These analyses suggest that, in the context of sickle cell disease, the beneficial antisickling effects of Hb zeta(2)beta(2)(S) outweigh its O(2)-transport liabilities. The potential structural bases for the antisickling properties of Hb zeta(2)beta(2)(S) are discussed in the context of these new observations.     

Effects of different beta73 amino acids on formation of 14-stranded fibers of Hb S versus double-stranded crystals of Hb C-Harlem

Hb S (alpha(2)beta(2)(6Glu-->Val)) forms polymers, while Hb C-Harlem (alpha(2)beta(2)(6Glu-->Val,73Asp-->Asn)) forms crystals upon oversaturation. Since the only difference between the two is the beta73 amino acid, it follows that this site is a critical determinant in promoting either polymerization or crystallization. Beta73 Asp in Hb S forms a hydrogen bond with beta4 Thr, while beta73 Asn in Hb C-Harlem may inhibit this interaction as well as increase the hydrophobicity at the EF helix beta6 Val acceptor sites. Two new beta73 Hb S variants (beta73 His and Leu) were constructed and analyzed to define other amino acids facilitating formation of Hb S-like polymers versus Hb C-Harlem-like crystals. The two variants that were chosen were expected to either (1) enhance formation of the beta73-beta4 hydrogen bond (beta73 His) or (2) inhibit it and increase the hydrophobicity of the EF helix beta6 Val acceptor sites (beta73 Leu). beta73 His Hb S formed fibers but at a lower concentration than Hb S, while beta73 Leu Hb S formed crystals but at a higher concentration than Hb C-Harlem. The solubility of beta73 His Hb S was (1)/(7) of that of Hb S, while the solubility of beta73 Leu Hb S was similar to that of Hb C-Harlem. The delay time prior to polymer or crystal formation depended on Hb concentration. The delay time for beta73 His Hb S was 10(5)-fold shorter than that for Hb S, while that for beta73 Leu Hb S was 10(5)-fold longer in 1.0 M phosphate buffer. NMR results indicate beta73 amino acid changes induce alteration in the beta-chain heme pocket region, while CD results indicate no change in the helical content of the variants. These results suggest that enhancing the beta73-beta4 hydrogen bond and/or induced changes in the heme pocket by the beta73 Asp to His change facilitate formation of Hb S-like fibers. Our results also suggest that removal of the beta73-beta4 hydrogen bond and enhancing the hydrophobicity of the EF helix beta6 Val acceptor sites by the beta73 Asp to Leu or Asn changes delay nuclei formation and facilitate formation of Hb C-Harlem-like crystals.     

RNase T1 mutant Glu46Gln binds the inhibitors 2'GMP and 2'AMP at the 3' subsite.

On the basis of molecular dynamics and free-energy perturbation approaches, the Glu46Gln (E46Q) mutation in the guanine-specific ribonuclease T1 (RNase T1) was predicted to render the enzyme specific for adenine. The E46Q mutant was genetically engineered and characterized biochemically and crystallographically by investigating the structures of its two complexes with 2'AMP and 2'GMP. The ribonuclease E46Q mutant is nearly inactive towards dinucleoside phosphate substrates but shows 17% residual activity towards RNA. It binds 2'AMP and 2'GMP equally well with dissociation constants of 49 microM and 37 microM, in contrast to the wild-type enzyme, which strongly discriminates between these two nucleotides, yielding dissociation constants of 36 microM and 0.6 microM. These data suggest that the E46Q mutant binds the nucleotides not to the specific recognition site but to the subsite at His92. This was confirmed by the crystal structures, which also showed that the Gln46 amide is hydrogen bonded to the Phe100 N and O atoms, and tightly anchored in this position. This interaction may either have locked the guanine recognition site so that 2'AMP and 2'GMP are unable to insert, or the contribution to guanine recognition of Glu46 is so important that the E46Q mutant is unable to function in recognition of either guanine and adenine.     

Pre-steady-state kinetic studies on the Glu171Gln active site mutant of adenosylcobalamin-dependent glutamate mutase

Glutamate-171 is involved in recognizing the amino group of the substrate in glutamate mutase. The effect of mutating this residue to glutamine on the ability of the enzyme to catalyze the homolysis of adenosylcobalamin has been investigated using UV-visible stopped-flow spectroscopy. Although Glu171 does not contact the coenzyme, the mutation results in the apparent rate constants for substrate-induced homolysis of the coenzyme that are slower by 7-fold and 13-fold with glutamate and methylaspartate, respectively, than those measured for the wild-type enzyme; furthermore, it weakens the binding of these substrates by approximately 50-fold and approximately 400-fold, respectively. These observations lend support to the idea that the enzyme may use substrate binding energy to accelerate homolysis of the coenzyme. The mutation also results in isotope effects on coenzyme homolysis that are much smaller than the very large effects observed when the wild-type enzyme is reacted with deuterated substrates. This observation is consistent with adenosylcobalamin homolysis being slowed relative to hydrogen abstraction from the substrate.     

Mutational analysis of phenylalanine beta 85 in the valine beta 6 acceptor pocket during hemoglobin S polymerization.

Hemoglobin (Hb) S containing Leu, Ala, Thr, or Trp substitutions at beta 85 were made and expressed in yeast in an effort to evaluate the role of Phe-beta 85 in the acceptor pocket during polymerization of deoxy Hb S. The four Hb S variants have the same electrophoretic mobility as Hb S, and these beta 85 substitutions do not significantly affect heme-globin interactions and tetramer helix content. Hb S containing Trp-beta 85 had decreased oxygen affinity, whereas those with Leu-, Ala-, and Thr-beta 85 had increased oxygen affinity. All four supersaturated beta 85 variants polymerized with a delay time as does deoxy Hb S. This is in contrast to deoxy Hb S containing Phe-beta 88, Ala-beta 88, Glu-beta 88, or Glu-beta 85, which polymerized with no clear delay time (Adachi K, Konitzer P, Paulraj CG, Surrey S, 1994, J Biol Chem 269:17477-17480; Adachi K, Reddy LR, Surrey S, 1994, J Biol Chem 269:31563-31566). Leu substitution at beta 85 accelerated deoxy Hb S polymerization, whereas Ala, Thr, or Trp substitution inhibited polymerization. The length of the delay time and total polymer formed for these beta 85 Hb S variants depended on hemoglobin concentration in the same fashion as for deoxy Hb S: the higher the concentration, the shorter the delay time and the more polymer formed. Critical concentrations required for polymerization of deoxy Hb SF veta 85L, Hb SF beta 85A, Hb SF beta 85T, and Hb SF beta 85W are 0.65-, 2.2-, 2.5- and 3-fold higher, respectively, than Hb S.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzymatic hydrolysis of nitriles by an engineered nitrile hydratase (papain Gln19Glu) in aqueous-organic media

A mutant of the cysteine protease papain, displaying nitrile hydratase and amidase activities, was expressed in Pichia pastoris and used for the hydrolysis of peptide nitriles in aqueous-organic media. The rate of hydrolysis of these nitriles is lowered by increasing acetone concentration. This is caused by an increase of the Michaelis constant, and a variation of Vmax proportional to the amount of water in the mixture. The hydrolysis of the amide is less affected by the increase in co-solvent, which results in lower accumulation of this intermediate product. With the peptide nitrile tested, high nitrile concentrations could be used to promote the production of the amide and prevent its hydrolysis to the acid by diminishing the relative rate of amide hydrolysis. A number of non-peptidyl nitriles were also tested as potential substrates but activity was detected for only one compound with structural resemblance to peptide nitriles.     

Preparation of antibodies capable of recognizing the Glu/Lys amino acid substitution at position 129 of the survivin sequence

Survivin is an oncofetal protein involved in the inhibition of apoptosis and the regulation of cell division. The functions of survivin are determined by its structural state (monomer or dimer). Owing to the natural polymorphism, either the Glu or the Lys residue can be at position 129 of the amino acid sequence of survivin. Lys has the capability for acetylation, and only the protein containing the acetylated residue Lys¹²⁹ tends to form a dimer. Thus, antibodies recognizing the amino acid substitution Glu129Lys can serve as a tool in the structural and functional investigations of survivin. For preparing the target antibodies, survivin fragments containing residue 129 were synthesized, rabbits were immunized with synthetic peptides, and the antibodies were purified by affinity chromatography on Sepharose conjugated with the corresponding peptides. It was shown by ELISA and immunoblotting that the affinity-purified antibodies are capable of recognizing the amino acid substitution Glu129Lys in the sequence of recombinant and endogenous survivin.     

Early intermediates in the photocycle of the Glu46Gln mutant of photoactive yellow protein: femtosecond spectroscopy

Transient absorption spectroscopy in the time range from -1 ps to 4 ns, and over the wavelength range from 420 to 550 nm, was applied to the Glu46Gln mutant of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila. This has allowed us to elucidate the kinetic constants of excited state formation and decay and photochemical product formation, and the spectral characteristics of stimulated emission and the early photocycle intermediates. Both the quantum efficiency ( approximately 0.5) and the rate constants for excited state decay and the formation of the initial photochemical intermediate (I(0)) were found to be quite similar to those obtained for wild-type PYP. In contrast, the rate constants for the formation of the subsequent photocycle intermediates (I(0)(double dagger) and I(1)), as well as for I(2) and for ground state regeneration as determined in earlier studies, were found to be from 3- to 30-fold larger. The structural implications of these results are discussed.     

Inhibition of hemoglobin S polymerization in vitro by a novel 15-mer EF-helix beta73 histidine-containing peptide

Our mutational studies on Hb S showed that the Hb S beta73His variant (beta6Val and beta73His) promoted polymerization, while Hb S beta73Leu (beta6Val and beta73Leu) inhibited polymerization. On the basis of these results, we speculated that EF-helix peptides containing beta73His interact with beta4Thr in Hb S and compete with Hb S, resulting in inhibition of Hb S polymerization. We, therefore, studied inhibitory effects of 15-, 11-, 7-, and 3-mer EF-helix peptides containing beta73His on Hb S polymerization. The delay time prior to Hb S polymerization increased only in the presence of the 15-mer His peptide; the higher the amount, the longer the delay time. DIC image analysis also showed that the fiber elongation rate for Hb S polymers decreased with increasing concentration of the 15-mer His peptide. In contrast, the same 15-mer peptide containing beta73Leu instead of His and peptides shorter than 11 amino acids containing beta73His including His alone showed little effect on the kinetics of polymerization and elongation of polymers. Analysis by protein-chip arrays showed that only the 15-mer beta73His peptide interacted with Hb S. CD spectra of the 15-mer beta73His peptide did not show a specific helical structure; however, computer docking analysis suggested a lower energy for interaction of Hb S with the 15-mer beta73His peptide compared to peptides containing other amino acids at this position. These results suggest that the 15-mer beta73His peptide interacts with Hb S via the beta4Thr in the betaS-globin chain in Hb S. This interaction may influence hydrogen bond interaction between beta73Asp and beta4Thr in Hb S polymers and interfere in hydrophobic interactions of beta6Val, leading to inhibition of Hb S polymerization.