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) (ßE6V/αH20R) and rHb (ßE6V/αH20Q)) or lateral (rHb (ßE6V/αH50Q)) or double amino acid substitutions at both the putative axial and lateral (rHb (ßE6V/αH20R/αH50Q) and rHb (ßE6V/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. The double mutants show only slight structural alteration in the ß-heme pockets. All mutants have similar cooperativity (n50), alkaline Bohr effect, and autoxidation rate as Hb S. The oxygen binding affinity (P50) of the single mutants is comparable with that of Hb S. The double mutants bind oxygen with slightly higher affinity than Hb S under the acidic conditions. In high salt, rHb (ßE6V/αH20R) is the only mutant that has a shorter delay time of polymerization and forms polymers more readily than Hb S with a dextran-Csat value of 1.86 ± 0.20 g/dl. Hb S, rHb (ßE6V/αH20Q), rHb (ßE6V/αH50Q), rHb (ßE6V/αH20R/αH50Q), and rHb (ßE6V/αH20Q/αH50Q) have dextran-Csat values of 2.95 ± 0.10, 3.04 ± 0.17, 11.78 ± 0.59, 7.11 ± 0.66, and 10.89 ± 0.83 g/dl, respectively. rHb (ßE6V/αH20Q/αH50Q) is even more stable than Hb S under elevated temperature (60 °C).

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. METHODS: We characterized (19)F-NMR relaxation properties of PFC-labeled cells in the presence of SPIO and imaged cells both ex vivo and in vivo in a rodent inflammation model to demonstrate selective visualization of cell populations. RESULTS: We show that with UTE3D, RARE, and FLASH (19) F images one can uniquely identify PFC-labeled cells, colocalized PFC- and SPIO-labeled cells, and PFC/SPIO-colabeled cells. CONCLUSION: This new methodology has the ability to improve and expand applications of MRI cell tracking. Combining PFC and SPIO strategies can potentially provide a method to quench PFC signal transferred from dead cells to macrophages, thereby eliminating false positives. In addition, combining these techniques could also be used to track two cell types simultaneously and probe cell-cell proximity in vivo with MRI.

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

The E11 valine in the distal heme pocket of either the α- or ß-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. The mutations did not disturb the α1ß2 interface in either form, whereas the H-bond between αHis-103 and ßGln-131 in the α1ß1 interfaces of the deoxy α-subunit mutants was weakened. Localized structural changes in the mutated heme pocket were detected for the CO form of recombinant Hb (rHb) (αV62F), rHb (ßV67I), and rHb (ßV67F) compared with Hb A. In the deoxy form the proximal histidyl residue in the ß-subunit of rHb (ßV67F) has been altered. Furthermore, the interactions between the porphyrin ring and heme pocket residues have been perturbed in rHb (αV62I), rHb (αV62F), and rHb (ßV67F). Functionally, the oxygen binding affinity (P50), cooperativity (n50), and the alkaline Bohr Effect of the three α-subunit mutants and rHb (ßV67L) are similar to those of Hb A. rHb (ßV67I) and rHb (ßV67F) exhibit low and high oxygen affinity, respectively. rHb (ßV67F) has P50 values lower that those reported for rHb (αL29F), a B10 mutant studied previously in our laboratory (Wiltrout, M. E., Giovannelli, J. L., Simplaceanu, V., Lukin, J. A., Ho, N. T., and Ho, C. (2005) Biochemistry 44, 7207-7217). These E11 mutations do not slow down the autoxidation and azide-induced oxidation rates of the recombinant proteins. Results from this study provide new insights into the roles of E11 mutants in the structure-function relationship in hemoglobin.

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. Amide-water proton exchange experiments demonstrated that αThr38 located in the α1ß2 interface and several loop regions in both α- and ß-chains were dynamic on the subsecond time scale. Side chain methyl dynamics revealed that methyl groups in the α1ß2 interface were dynamic, but those in the α1ß1 interface were quite rigid on the nanosecond to picosecond and millisecond to microsecond time scales. All the data strongly suggest a dynamic α1ß2 interface that allows conformational changes among different forms (like T, R, and R2) easily in solution. Binding of IHP to HbCO A induced small structural and dynamic changes in the α1ß2 interface and the regions around the hemes but did not increase the conformational entropy of HbCO A. The binding also caused conformational changes on the millisecond time scale, very likely arising from the relative motion of the α1ß1 dimer with respect to the α2ß2 dimer. Heterotropic effectors like IHP may change the oxygen affinity of Hb through modulating the relative motion of the two dimers and then further altering the structure of heme binding regions.

Role of ß/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 ß/δ101 (ß/δ101Gln→Glu, Lys, or Asp) plus a double and a triple mutant, designed to clarify the role of the ß/δ101 residue. The ß/δ101Gln residue is important for responding to allosteric effectors, such as phosphate, inositol hexaphosphate (IHP), and chloride. The rHb WM mutants studied generally have higher affinity for oxygen under various conditions of pH, temperature, and salt concentration, and in the presence or absence of organic phosphate, than do rHb WM, rHb AE, and Hb A. Titrations for the O2 affinity of these mutant rHbs as a function of chloride concentration indicate a lower heterotopic effect of this anion due to the replacement of ß/δ101Gln in rHb WM. The alkaline Bohr effect of rHb WM and its mutants is reduced by 20-50% compared to that of Hb A and is independent of changes in temperature, in contrast to what has been observed in the hemoglobins of most mammalian species, including human. The results of our study on the temperature dependence of the O2 affinity of rHb WM and its mutant rHbs illustrate the important role of ß/δ101Gln in regulating the functional properties of these hemoglobins.

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). This reaction is regulated by redox-sensitive surface thiols, cysteine 55 and 46, which regulate the fraction of the five-coordinated heme, nitrite binding, and NO formation. Replacement of the distal histidine by leucine or glutamine leads to a stable five-coordinated geometry; these neuroglobin mutants reduce nitrite to NO ∼2000 times faster than the wild type, whereas mutation of either Cys-55 or Cys-46 to alanine stabilizes the six-coordinate structure and slows the reaction. Using lentivirus expression systems, we show that the nitrite reductase activity of neuroglobin inhibits cellular respiration via NO binding to cytochrome c oxidase and confirm that the six-to-five-coordinate status of neuroglobin regulates intracellular hypoxic NO-signaling pathways. These studies suggest that neuroglobin may function as a physiological oxidative stress sensor and a post-translationally redox-regulated nitrite reductase that generates NO under six-to-five-coordinate heme pocket control. We hypothesize that the six-coordinate heme globin superfamily may subserve a function as primordial hypoxic and redox-regulated NO-signaling proteins.

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)]. Here, we show that the four Hbs exhibit distinct structural properties and respond differently to allosteric effectors. In addition, the apparent heat of oxygenation (ΔH) for rHb WM is less negative than that of rHb AE, especially in phosphate buffer and the presence of IHP, suggesting that the oxygen affinity of mammoth blood was also less sensitive to temperature change. Finally, (1)H NMR spectroscopy data indicates that both α(1)(ß/δ)(1) and α(1)(ß/δ)(2) interfaces in rHb WM and rHb AE are perturbed, whereas only the α(1)δ(1) interface in Hb A(2) is perturbed compared to that in Hb A. The distinct structural and functional features of rHb WM presumably facilitated woolly mammoth survival in the Arctic environment.

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). We have found significant effects of pH and temperature on the strength of the H-bond markers, i.e., the cross-peaks for the side chains of the two distal histidyl residues, α58His and ß63His, which form H-bonds with the O(2) ligands. At lower pH and/or higher temperature, the side chains of the distal histidines appear to be more mobile, and the exchange with water molecules in the distal heme pockets is faster. These changes in the stability of the H-bonds with pH and temperature are consistent with the changes in the O(2) affinity of Hb as a function of pH and temperature and are clearly illustrated by our NMR experiments. Our NMR results have also confirmed that this H-bond in the ß-chain is weaker than that in the α-chain and is more sensitive to changes in pH and temperature. IHP has only a minor effect on these H-bond markers compared to the effects of pH and temperature. These H-bonds are sensitive to mutations in the distal heme pockets but not affected directly by the mutations in the quaternary interfaces, i.e., α(1)ß(1) and/or α(1)ß(2) subunit interface. These findings provide new insights regarding the roles of temperature, hydrogen ion, and organic phosphate in modulating the structure and function of hemoglobin in solution.

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. The aromatic side chains placed in the distal-heme pocket environment affect the intrinsic ligand-binding properties of the mutated subunit itself, beyond what can be explained by allostery, and these changes are accompanied by local structural perturbations. In contrast, hemoglobins with mutations in the alpha 1beta 1 and alpha 1beta 2 interfaces display functional properties of both "R"- and "T"-state tetramers because the equilibrium between quaternary states is altered. These mutations are accompanied by global structural perturbations, suggesting an indirect, allostery-driven cause for their effects. Combinations of the distal-heme pocket and interfacial mutations exhibit additive effects in both structural and functional properties, contribute to our understanding of allostery, and advance protein-engineering methods for manipulating the O 2 binding properties of the hemoglobin molecule.

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. The TR(3) spectra support the inference from other mutational studies that the alphabeta dimers act as single dynamic units in this early phase, motions of the E and F helices being coupled tightly across the dimer interface. Formation of T quaternary contacts occurs at about the same rate in the mutants as in HbA. However, these contacts are weakened substantially by the Y/F substitutions. Equilibrium perturbations are apparent also, especially for the alpha-subunits, in which relaxation of the Fe-His bond, strengthening of the Acdots, three dots, centeredE interhelical H bond, and weakening of the "switch" quaternary contact in deoxyHb are all apparent. Structural effects are less marked for the beta-chain Y/F replacement, but the Bohr effect is reduced by 25%, indicating that the salt-bridge and H bond interactions of the adjacent C terminus are loosened. The alpha-chain replacement reduces the Bohr effect much more, consistent with the global perturbations detected by the structure probes.

NMR investigation of the dynamics of tryptophan side-chains in hemoglobins.

NMR relaxation measurements of 15N spin-lattice relaxation rate (R(1)), spin-spin relaxation rate (R(2)), and heteronuclear nuclear Overhauser effect (NOE) have been carried out at 11.7T and 14.1T as a function of temperature for the side-chains of the tryptophan residues of 15N-labeled and/or (2H,15N)-labeled recombinant human normal adult hemoglobin (Hb A) and three recombinant mutant hemoglobins, rHb Kempsey (betaD99N), rHb (alphaY42D/betaD99N), and rHb (alphaV96W), in the carbonmonoxy and the deoxy forms as well as in the presence and in the absence of an allosteric effector, inositol hexaphosphate (IHP). There are three Trp residues (alpha14, beta15, and beta37) in Hb A for each alphabeta dimer. These Trp residues are located in important regions of the Hb molecule, i.e. alpha14Trp and beta15Trp are located in the alpha(1)beta(1) subunit interface and beta37Trp is located in the alpha(1)beta(2) subunit interface. The relaxation experiments show that amino acid substitutions in the alpha(1)beta(2) subunit interface can alter the dynamics of beta37Trp. The transverse relaxation rate (R(2)) for beta37Trp can serve as a marker for the dynamics of the alpha(1)beta(2) subunit interface. The relaxation parameters of deoxy-rHb Kemspey (betaD99N), which is a naturally occurring abnormal human hemoglobin with high oxygen affinity and very low cooperativity, are quite different from those of deoxy-Hb A, even in the presence of IHP. The relaxation parameters for rHb (alphaY42D/betaD99N), which is a compensatory mutant of rHb Kempsey, are more similar to those of Hb A. In addition, TROSY-CPMG experiments have been used to investigate conformational exchange in the Trp residues of Hb A and the three mutant rHbs. Experimental results indicate that the side-chain of beta37Trp is involved in a relatively slow conformational exchange on the micro- to millisecond time-scale under certain experimental conditions. The present results provide new dynamic insights into the structure-function relationship in hemoglobin.

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. The binding of oxygen to these rHbs is highly cooperative, with a Hill coefficient of approximately 2.8, compared to approximately 3.1 for Hb A. Proton nuclear magnetic resonance (NMR) studies show that rHb (alphaT67V), rHb (betaS72A), rHb (alphaT67V, betaS72A), and Hb A have similar quaternary structures in the alpha(1)beta(2) subunit interfaces. In particular, the inter-subunit H-bonds between alpha42Tyr and beta99Asp and between beta37Trp and alpha94Asp are maintained in the mutants in the deoxy form. There are slight perturbations in the distal heme pocket region of the alpha- and beta-chains in the mutants. A comparison of the exchangeable 1H resonances of Hb A with those of these three rHbs suggests that alpha67T and beta72S are H-bonded to alpha14W and beta15W, respectively, in the CO and deoxy forms of Hb A. The absence of significant free energy changes for the oxygenation process of these three rHbs compared to those of Hb A, even though the inter-helical H-bonds are abolished, indicates that these two sets of H-bonds are of comparable strength in the ligated and unligated forms of Hb A. Thus, the mutations at alphaT67V and betaS72A do not affect the overall energetics of the oxygenation process. The preserved cooperativity in the binding of oxygen to these three mutants also implies that there are multiple interactions involved in the oxygenation process of Hb A.

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. It was concluded that diazepam (in isolation) does not interact with the G29, A30, and I31 residues, and no Abeta oligomerization occurs in the presence of 0.101 mM diazepam, even after 63 days. However, when diazepam was co-administered with halothane, profound Abeta oligomerization is observed. These results strengthen the hypothesis that the presence of smaller molecular sized anesthetic is instrumental in promoting Abeta oligomerization even when co-administered with a larger sized anesthetic, namely diazepam.

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 21.1 T, and at 37 degrees C. Our results show that the majority of the polypeptide backbone amino acid residues of deoxy- and carbonmonoxy-forms of Hb A in the absence of IHP is not mobile on the micros-ms time scale, with the exception of several amino acid residues, that is, beta109Val and beta132Lys in deoxy-Hb A, and alpha40Lys in HbCO A. The mobility of alpha40Lys in HbCO A can be explained by the crystallographic data showing that the H-bond between alpha40Lys and beta146His in deoxy-Hb A is absent in HbCO A. However, the conformational exchange of beta109Val, which is located in the intradimer (alpha 1beta 1 or alpha 2beta 2) interface, is not consistent with the crystallographic observations that show rigid packing at this site. IHP binding appears to rigidify alpha40Lys in HbCO A, but does not significantly affect the flexibility of beta109Val in deoxy-Hb A. In the presence of IHP, several amino acid residues, especially those at the interdimer (alpha 1beta 2 or alpha 2beta 1) interface of HbCO A, exhibit significant conformational exchange. The affected residues include the proximal beta92His in the beta-heme pocket, as well as some other residues located in the flexible joint (betaC helix-alphaFG corner) and switch (alphaC helix-betaFG corner) regions that play an important role in the dimer-dimer rotation of Hb during the oxygenation process. These findings suggest that, upon IHP binding, HbCO A undergoes a conformational fluctuation near the R-state but biased toward the T-state, apparently along the trajectory of its allosteric transition, accompanied by structural fluctuations in the heme pocket of the beta-chain. In contrast, no significant perturbation of the dynamic features on the ms-micros time scale has been observed upon IHP binding to deoxy-Hb A. We propose that the allosteric effector-induced quaternary structural fluctuation may contribute to the reduced ligand affinity of ligated hemoglobin. Conformational exchange mapping of the beta-chain of HbCO A observed at 21.1 T shows significantly increased scatter in the chemical exchange contribution to the transverse relaxation rate ( R ex) values, relative to those at lower fields, due to the enhanced effect of the local chemical shift anisotropy (CSA) fluctuation. A spring-on-scissors model is proposed to interpret the dynamic phenomena induced by the heterotropic effector, IHP.

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. Our recent RDC studies of HbCO A in the absence and presence of IHP have shown that the solution structure of this Hb molecule is distinctly different from its classical crystal structures (R and R2). To have a better understanding of the structure-function relationship of Hb A under physiological conditions, we need to evaluate its structures in both ligated and unligated states in solution. Here, the intrinsic paramagnetic property of deoxy-Hb A has been exploited for the measurement of RDCs using the magnetic-field dependence of the apparent one-bond 1H-15N J couplings. Our RDC analysis suggests that the quaternary and tertiary structures of deoxy-Hb A in solution differ from its recently determined high-resolution crystal structures. Upon binding of IHP, structural changes in deoxy-Hb A are also observed, and these changes are largely within the alpha1beta1 (or alpha2beta2) dimer itself. These new structural findings allow us to gain a deeper insight into the structure-function relationship of this interesting allosteric protein.

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). In both deoxy and carbonmonoxy forms of human normal adult hemoglobin (Hb A), the amide N-H bonds of most amino acid residues are rigid on the fast time scale (nanosecond to picosecond), except for the loop regions and certain helix-helix connections. Although rigid in deoxy-Hb A, beta146His has been found to be free from restriction of its backbone motions in the CO form, presumably due to the rupture of its hydrogen bond/salt bridge network. We now have direct dynamics evidence for this structural transition of Hb in solution. While remarkably flexible in the deoxy state, alpha31Arg and beta123Thr, neighbors in the intradimer (alpha1beta1) interface, exhibit stiffening upon CO binding. These findings imply a role for alpha31Arg and beta123Thr in the intradimer communication but contradict the results from X-ray crystallography. We have also found that there is considerable flexibility in the intradimer (alpha1beta1) interface (i.e., B, G, and H helices and the GH corner) and possible involvement of several amino acid residues (e.g., alpha31Arg, beta3Leu, beta41Phe, beta123Thr, and beta146His) in the allosteric pathway. Several amino acid residues at the intradimer interfaces, such as beta109Val, appear to be involved in possible conformational exchange processes. The dynamic picture derived from the present study provides new insights into the traditional description of the stereochemical mechanism for the cooperative oxygenation of Hb A based on X-ray crystallographic results.

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). The molecular radius of PEGylated rHb(betaC93A) was slightly smaller than that of (SP-PEG5K)(6)-Hb, and the overall site-selectivity of PEGylation in the PEGylated rHb(betaC93A) at Lys-residues was comparable to that of (SP-PEG5K)(6)-Hb. Proton NMR studies have shown that the conjugation of the protein with PEG-5K does not have any significant influence on its subunit interface interactions. Surprisingly, the influence of PEGylation on the O(2) affinity and Bohr effect of HbA and rHb(betaC93A) is also nearly the same. Apparently, conjugation of PEG-chains to Lys residues of Hb by the thiolation mediated PEGylation induces unique changes in the structure of the hydration shell of Hb (layer of tightly bound water molecules), which, in turn, induces constraints in its R to T conformational transition to favor the more hydrated R-state.

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., Kontaxis, G., Simplaceanu, V., Yuan, Y., Bax, A., and Ho, C. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 517-520] indicate that the quaternary structure of HbCO A in solution is a dynamic ensemble between two previously determined crystal structures, R (crystals grown under high-salt conditions) and R2 (crystals grown under low-salt conditions). On the basis of a comparison of the geometric coordinates of the T, R, and R2 structures, it has been suggested that the oxygenation of Hb A follows the transition pathway from T to R and then to R2, with R being the intermediate structure [Srinivasan, R., and Rose, G. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11113-11117]. The results presented here suggest that IHP can shift the solution quaternary structure of HbCO A slightly toward the R structure. The solution quaternary structure of rHbCO(alpha96Val-->Trp) in the absence of IHP is similar to that of HbCO A in the presence of IHP, consistent with rHbCO(alpha96Val-->Trp) having an affinity for oxygen lower than that of Hb A. Moreover, IHP has a much stronger effect in shifting the solution quaternary structure of rHbCO(alpha96Val-->Trp) toward the R structure and toward the T structure, consistent with IHP causing a more pronounced decrease in its oxygen affinity. The results presented in this work, as well as other results recently reported in the literature, clearly indicate that there are multiple quaternary structures for the ligated form of hemoglobin. These results also provide new insights regarding the roles of allosteric effectors in regulating the structure and function of hemoglobin. The classical two-state/two-structure allosteric mechanism for the cooperative oxygenation of hemoglobin cannot account for the structural and functional properties of this protein and needs to be revised.