Sickle Cell Hemoglobin with Mutation at αHis-50 Has Improved Solubility.

The unliganded tetrameric Hb S has axial and lateral contacts with neighbors and can polymerize in solution. Novel recombinants of Hb S with single amino acid substitutions at the putative axial (recombinant Hb (rHb) (βE6V/αH20R) and rHb (βE6V/αH20Q)) or lateral (rHb (βE6V/αH50Q)) or double amino acid substitutions at both the putative axial and lateral (rHb (βE6V/αH20R/αH50Q) and rHb (βE6V/αH20Q/αH50Q)) contact sites were expressed in Escherichia coli and purified for structural and functional studies. The (1)H NMR spectra of the CO and deoxy forms of these mutants indicate that substitutions at either αHis-20 or αHis-50 do not change the subunit interfaces or the heme pockets of the proteins.

Combining perfluorocarbon and superparamagnetic iron-oxide cell labeling for improved and expanded applications of cellular MRI.

Purpose: The ability to detect the migration of cells in living organisms is fundamental in understanding biological processes and important for the development of novel cell-based therapies to treat disease. MRI can be used to detect the migration of cells labeled with superparamagnetic iron-oxide (SPIO) or perfluorocarbon (PFC) agents. In this study, we explored combining these two cell-labeling approaches to overcome current limitations and enable new applications for cellular MRI.

Role of β/δ101Gln in regulating the effect of temperature and allosteric effectors on oxygen affinity in woolly mammoth hemoglobin.

The oxygen affinity of woolly mammoth hemoglobin (rHb WM) is less affected by temperature change than that of Asian elephant hemoglobin (rHb AE) or human normal adult hemoglobin (Hb A). We report here a biochemical-biophysical study of Hb A, rHb AE, rHb WM, and three rHb WM mutants with amino acid substitutions at β/δ101 (β/δ101Gln→Glu, Lys, or Asp) plus a double and a triple mutant, designed to clarify the role of the β/δ101 residue. The β/δ101Gln residue is important for responding to allosteric effectors, such as phosphate, inositol hexaphosphate (IHP), and chloride.

Solution structure and dynamics of human hemoglobin in the carbonmonoxy form.

The solution structure of human adult carbonmonoxy hemoglobin (HbCO A) was refined using stereospecifically assigned methyl groups and residual dipolar couplings based on our previous nuclear magnetic resonance structure. The tertiary structures of individual chains were found to be very similar to the X-ray structures, while the quaternary structures in solution at low salt concentrations resembled the X-ray R structure more than the R2 structure. On the basis of chemical shift perturbation by inositol hexaphosphate (IHP) titration and docking, we identified five possible IHP binding sites in HbCO A.

Autoxidation and oxygen binding properties of recombinant hemoglobins with substitutions at the αVal-62 or βVal-67 position of the distal heme pocket.

The E11 valine in the distal heme pocket of either the α- or β-subunit of human adult hemoglobin (Hb A) was replaced by leucine, isoleucine, or phenylalanine. Recombinant proteins were expressed in Escherichia coli and purified for structural and functional studies. (1)H NMR spectra were obtained for the CO and deoxy forms of Hb A and the mutants.

A biochemical--biophysical study of hemoglobins from woolly mammoth, Asian elephant, and humans.

This study is aimed at investigating the molecular basis of environmental adaptation of woolly mammoth hemoglobin (Hb) to the harsh thermal conditions of the Pleistocene ice ages. To this end, we have carried out a comparative biochemical-biophysical characterization of the structural and functional properties of recombinant hemoglobins (rHb) from woolly mammoth (rHb WM) and Asian elephant (rHb AE) in relation to human hemoglobins Hb A and Hb A(2) (a minor component of human blood). We have obtained oxygen equilibrium curves and calculated O(2) affinities, Bohr effects, and the apparent heat of oxygenation (ΔH) in the presence and absence of allosteric effectors [inorganic phosphate and inositol hexaphosphate (IHP)].

Human neuroglobin functions as a redox-regulated nitrite reductase.

Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved from a common ancestor to hemoglobin and myoglobin. It possesses a six-coordinate heme geometry with proximal and distal histidines directly bound to the heme iron, although coordination of the sixth ligand is reversible. We show that deoxygenated human neuroglobin reacts with nitrite to form nitric oxide (NO).

An investigation of the distal histidyl hydrogen bonds in oxyhemoglobin: effects of temperature, pH, and inositol hexaphosphate.

On the basis of X-ray crystal structures and electron paramagnetic resonance (EPR) measurements, it has been inferred that the O(2) binding to hemoglobin is stabilized by the hydrogen bonds between the oxygen ligands and the distal histidines. Our previous study by multinuclear nuclear magnetic resonance (NMR) spectroscopy has provided the first direct evidence of such H-bonds in human normal adult oxyhemoglobin (HbO(2) A) in solution. Here, the NMR spectra of uniformly (15)N-labeled recombinant human Hb A (rHb A) and five mutant rHbs in the oxy form have been studied under various experimental conditions of pH and temperature and also in the presence of an organic phosphate, inositol hexaphosphate (IHP).

Intravenous anesthetic diazepam does not induce amyloid-beta peptide oligomerization but diazepam co-administered with halothane oligomerizes amyloid-beta peptide: an NMR study.

Amyloid-beta peptide (Abeta) oligomerization has a profound role in Alzheimer's disease pathophysiology. Biophysical studies have shown that smaller sized inhaled anesthetics promote oligomerization by inducing perturbation of three critical amino acid residues (G29, A30, and I31) located in the helix-loop-helix domain of Abeta. In this present experimental study, using state-of-the-art nuclear magnetic resonance, we have monitored the influence of a larger sized intravenous anesthetic, diazepam, as well as diazepam co-administered with halothane, on Abeta.

Interfacial and distal-heme pocket mutations exhibit additive effects on the structure and function of hemoglobin.

Protein engineering strategies seek to develop a hemoglobin-based oxygen carrier with optimized functional properties, including (i) an appropriate O 2 affinity, (ii) high cooperativity, (iii) limited NO reactivity, and (iv) a diminished rate of auto-oxidation. The mutations alphaL29F, alphaL29W, alphaV96W and betaN108K individually impart some of these traits and in combinations produce hemoglobin molecules with interesting ligand-binding and allosteric properties. Studies of the ligand-binding properties and solution structures of single and multiple mutants have been performed.

Effector-induced structural fluctuation regulates the ligand affinity of an allosteric protein: binding of inositol hexaphosphate has distinct dynamic consequences for the T and R states of hemoglobin.

The present study reports distinct dynamic consequences for the T- and R-states of human normal adult hemoglobin (Hb A) due to the binding of a heterotropic allosteric effector, inositol hexaphosphate (IHP). A nuclear magnetic resonance (NMR) technique based on modified transverse relaxation optimized spectroscopy (TROSY) has been used to investigate the effect of conformational exchange of Hb A in both deoxy and CO forms, in the absence and presence of IHP, at 14.1 and 21.

Insights into the solution structure of human deoxyhemoglobin in the absence and presence of an allosteric effector.

We present a nuclear magnetic resonance (NMR) study in solution of the structures of human normal hemoglobin (Hb A) in the deoxy or unligated form in the absence and presence of an allosteric effector, inositol hexaphosphate (IHP), using 15N-1H residual dipolar coupling (RDC) measurements. There are several published crystal structures for deoxyhemoglobin A (deoxy-Hb A), and it has been reported that the functional properties of Hb A in single crystals are different from those in solution. Carbonmonoxyhemoglobin A (HbCO A) can also be crystallized in several structures.

A comparative NMR study of the polypeptide backbone dynamics of hemoglobin in the deoxy and carbonmonoxy forms.

Model-free-based NMR dynamics studies have been undertaken for polypeptide backbone amide N-H bond vectors for both the deoxy and carbonmonoxy forms of chain-specific, isotopically (15N and 2H) labeled tetrameric hemoglobin (Hb) using 15N-relaxation parameters [longitudinal relaxation rate (R1), transverse relaxation rate (R2), and heteronuclear nuclear Overhauser effect (NOE)] measured at two temperatures (29 and 34 degrees C) and two magnetic field strengths (11.7 and 14.1 T).

Molecular aspects of the high oxygen affinity of non-hypertensive hexa pegylated hemoglobin, [(SP-PEG5K)(6)-Hb].

The development of hexaPEGylated Hb, (SP-PEG5K)(6)-Hb, using the newly designed thiolation-mediated maleimide chemistry based PEGylation, has validated the concept that engineering 'plasma volume expander' -like properties to Hb neutralizes its vasoactivity. The high O(2) affinity of hexaPEGylated Hb has been attributed to the two PEG-5K chains on its two Cys-93(beta) residues. In an attempt to map the influence of the additional four PEG-5K chains of HexaPEGylated Hb on the O(2) affinity, we have now investigated the influence of PEGylation of the surface amino groups alone on the subunit interface interactions and O(2) affinity of Hb using rHb(betaC93A).

Orientation of deoxyhemoglobin at high magnetic fields: structural insights from RDCs in solution.

Human normal adult hemoglobin (Hb A) is a tetrameric protein molecule of ~64 kDa consisting of two identical -chains and two identical -chains of 141 and 146 amino acid residues each and four bound heme moieties. In the oxygen-free form of Hb A, also known as deoxyhemoglobin A (deoxy-Hb A), the hemes are paramagnetic with S = 2. We have measured the one-bond spin-spin couplings (1JNH + 1DNH) on (15N,2H)-labeled deoxy-Hb A in solution as a function of magnetic field strengths from 11.

Quaternary structure of carbonmonoxyhemoglobins in solution: structural changes induced by the allosteric effector inositol hexaphosphate.

We have applied the residual dipolar coupling (RDC) method to investigate the solution quaternary structures of (2)H- and (15)N-labeled human normal adult recombinant hemoglobin (rHb A) and a low-oxygen-affinity mutant recombinant hemoglobin, rHb(alpha96Val-->Trp), both in the carbonmonoxy form, in the absence and presence of an allosteric effector, inositol hexaphosphate (IHP), using a stretched polyacrylamide gel as the alignment medium. Our recent RDC results [Lukin, J. A.

Dynamics of allostery in hemoglobin: roles of the penultimate tyrosine H bonds.

The tyrosine residues adjacent to the C termini of the hemoglobin (Hb) subunits, alphaY140 and betaY145, are expected to play important structural roles, because the C termini are the loci of T-state quaternary salt-bridges, and because the tyrosine side-chains bridge the H and F helices via H bonds to the alphaV93 and betaV98 carbonyl groups. These roles have been investigated via measurements of oxygen binding, (1)H NMR spectra, resonance Raman (RR) spectra, and time-resolved resonance Raman (TR(3)) spectra on site mutants in which the Hcdots, three dots, centeredF H bonds are eliminated by replacing the tyrosine residues with phenylalanine. The TR(3) spectra confirm the hypothesis, based on TR(3) studies of wild-type Hb, that the Hcdots, three dots, centeredF H bonds break and then re-form during the sub-microsecond phase of the R-T quaternary transition.

Enhanced inhibition of polymerization of sickle cell hemoglobin in the presence of recombinant mutants of human fetal hemoglobin with substitutions at position 43 in the gamma-chain.

Four recombinant mutants of human fetal hemoglobin [Hb F (alpha2gamma2)] with amino acid substitutions at the position 43 of the gamma-chain, rHb (gammaD43L), rHb (gammaD43E), rHb (gammaD43W), and rHb (gammaD43R), have been expressed in our Escherichia coli expression system and used to investigate their inhibitory effect on the polymerization of deoxygenated sickle cell hemoglobin (Hb S). Oxygen-binding studies show that rHb (gammaD43E), rHb (gammaD43W), and rHb (gammaD43R) exhibit higher oxygen affinity than human normal adult hemoglobin (Hb A), Hb F, or rHb (gammaD43L), and all four rHbs are cooperative in binding O2. Proton nuclear magnetic resonance (NMR) studies of these four rHbs indicate that the quaternary and tertiary structures around the heme pockets are similar to those of Hb F in both deoxy (T) and liganded (R) states.

A biophysical investigation of recombinant hemoglobins with aromatic B10 mutations in the distal heme pockets.

This study examines the structural and functional effects of amino acid substitutions in the distal side of both the alpha- and beta-chain heme pockets of human normal adult hemoglobin (Hb A). Using our Escherichia coli expression system, we have constructed four recombinant hemoglobins: rHb(alphaL29F), rHb(alphaL29W), rHb(betaL28F), and rHb(betaL28W). The alpha29 and beta28 residues are located in the B10 helix of the alpha- and beta-chains of Hb A, respectively.

Site mutations disrupt inter-helical H-bonds (alpha14W-alpha67T and beta15W-beta72S) involved in kinetic steps in the hemoglobin R-->T transition without altering the free energies of oxygenation.

Three recombinant mutant hemoglobins (rHbs) of human normal adult hemoglobin (Hb A), rHb (alphaT67V), rHb (betaS72A), and rHb (alphaT67V, betaS72A), have been constructed to test the role of the tertiary intra-subunit H-bonds between alpha67T and alpha14W and between beta72S and beta15W in the cooperative oxygenation of Hb A. Oxygen-binding studies in 0.1 M sodium phosphate buffer at 29 degrees C show that rHb (alphaT67V), rHb (betaS72A), and rHb (alphaT67V, betaS72A) exhibit oxygen-binding properties similar to those of Hb A.

Quaternary structure of hemoglobin in solution.

Many important proteins perform their physiological functions under allosteric control, whereby the binding of a ligand at a specific site influences the binding affinity at a different site. Allosteric regulation usually involves a switch in protein conformation upon ligand binding. The energies of the corresponding structures are comparable, and, therefore, the possibility that a structure determined by x-ray diffraction in the crystalline state is influenced by its intermolecular contacts, and thus differs from the solution structure, cannot be excluded.