Tong-Jian Shen

Substitutions in woolly mammoth hemoglobin confer biochemical properties adaptive for cold tolerance

We have genetically retrieved, resurrected and performed detailed structure-function analyses on authentic woolly mammoth hemoglobin to reveal for the first time both the evolutionary origins and the structural underpinnings of a key adaptive physiochemical trait in an extinct species. Hemoglobin binds and carries O2; however, its ability to offload O2 to respiring cells is hampered at low temperatures, as heme deoxygenation is inherently endothermic (that is, hemoglobin-O2 affinity increases as temperature decreases). We identify amino acid substitutions with large phenotypic effect on the chimeric β/δ-globin subunit of mammoth hemoglobin that provide a unique solution to this problem and thereby minimize energetically costly heat loss. This biochemical specialization may have been involved in the exploitation of high-latitude environments by this African-derived elephantid lineage during the Pleistocene period. This powerful new approach to directly analyze the genetic and structural basis of physiological adaptations in an extinct species adds an important new dimension to the study of natural selection.

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)(β/δ)(1) and α(1)(β/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.

Autoxidation and Oxygen Binding Properties of Recombinant Hemoglobins with Substitutions at the αVal-62 or βVal-67 Position of the Distal Heme Pocket

Background: Tertiary structure of the ligand binding pocket influences oxygen binding and autoxidation of hemoglobin. Results: E11 mutants have increased autoxidation rate. βE11Phe increases, whereas βE11Ile decreases the oxygen binding affinity of hemoglobin. Conclusion: Bulky residues at βE11 affect ligand binding and cause noticeable tertiary structural changes at the heme pockets. Significance: Hemoglobin distal heme pocket mutations alter oxygen binding properties without changing the quaternary structure. 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. 1H NMR spectra were obtained for the CO and deoxy forms of Hb A and the mutants. The mutations did not disturb the α1β2 interface in either form, whereas the H-bond between αHis-103 and βGln-131 in the α1β1 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 (βV67I), and rHb (βV67F) compared with Hb A. In the deoxy form the proximal histidyl residue in the β-subunit of rHb (βV67F) has been altered. Furthermore, the interactions between the porphyrin ring and heme pocket residues have been perturbed in rHb (αV62I), rHb (αV62F), and rHb (βV67F). Functionally, the oxygen binding affinity (P50), cooperativity (n50), and the alkaline Bohr Effect of the three α-subunit mutants and rHb (βV67L) are similar to those of Hb A. rHb (βV67I) and rHb (βV67F) exhibit low and high oxygen affinity, respectively. rHb (βV67F) 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.

Site mutations disrupt inter-helical H-bonds (α14W–α67T and β15W–β72S) involved in kinetic steps in the hemoglobin R→T transition without altering the free energies of oxygenation

Abstract Three recombinant mutant hemoglobins (rHbs) of human normal adult hemoglobin (Hb A), rHb (αT67V), rHb (βS72A), and rHb (αT67V, βS72A), have been constructed to test the role of the tertiary intra-subunit H-bonds between α67T and α14W and between β72S and β15W in the cooperative oxygenation of Hb A. Oxygen-binding studies in 0.1 M sodium phosphate buffer at 29 °C show that rHb (αT67V), rHb (βS72A), and rHb (αT67V, βS72A) 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 (αT67V), rHb (βS72A), rHb (αT67V, βS72A), and Hb A have similar quaternary structures in the α 1 β 2 subunit interfaces. In particular, the inter-subunit H-bonds between α42Tyr and β99Asp and between β37Trp and α94Asp are maintained in the mutants in the deoxy form. There are slight perturbations in the distal heme pocket region of the α- and β-chains in the mutants. A comparison of the exchangeable 1 H resonances of Hb A with those of these three rHbs suggests that α67T and β72S are H-bonded to α14W and β15W, 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 αT67V and βS72A 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.

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. 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 β/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 β/δ101Gln in regulating the functional properties of these hemoglobins.

Recombinant Octameric Hemoglobins as Resuscitation Fluids in a Murine Model of Traumatic Brain Injury Plus Hemorrhagic Shock

Three recombinant octameric mutants of human normal adult hemoglobin (Hb A), rHb (αN78C), rHb (αN78C/L29F), and rHb (αN78C/L29W), were expressed in our Eschericia coli expression system and purified. They were used as resuscitation fluids in our unique mouse model of traumatic brain injury (TBI) combined with severe hemorrhagic shock (HS). A sulfhydryl group was introduced onto the surface of the α-subunits of rHb A by substituting Asn78 with cysteine. The rHb (αN78C) form octamers by linking the two tetramers with 2 intermolecular disulfide bonds. Nuclear magnetic resonance (NMR) spectroscopic studies indicate that rHb (αN78C) has the same quaternary and tertiary structures as those of Hb A. Furthermore, the oxygen-binding activity (as measured by P 50 ) and the cooperativity of the oxygenation process (as measured by the Hill coefficient) of this mutant have not been altered compared to Hb A. The Leu29 residue on the α-subunits of the octamers was then mutated into either phenylalanine (F) or tryptophan (W) to yield rHb (αN78C/L29F) and rHb (αN78C/L29W), respectively. Compared to Hb A, rHb (αN78C/L29F) has high- while rHb (αN78C/L29W) has low-oxygen affinity. Both mutants are cooperative in their oxygen-binding properties, but lower than that observed for Hb A. They maintain their quaternary structure as those of Hb A, but exhibit perturbation of their tertiary structure at or near the heme pockets as detected by NMR measurements. Although all octameric rHbs would be expected to have reduced nitric oxide (NO) binding based on their size, rHb (αN78C/L29F) and rHb (αN78C/L29W) may have a further reduction in NO binding as a result of introducing a bigger aromatic amino acid residue at the distal heme pocket of the α-chain. These three rHbs were used as resuscitation solutions in mice after TBI combined with HS. TBI was induced by a controlled cortical impact (CCI) to the left parietal cortex. Blood was then withdrawn (2.4 mL/100 g body weight) over 15 min to induce HS. A pressure-controlled model was used with the mean arterial pressure (MAP) maintained at 25–27 mm Hg for an additional 20 min. At the end of the HS Phase (35 min total), i.e., the beginning of Pre-Hosptial Phase, lactated Ringers (LR) or rHb (120 mg/mL) solutions were administered to the mice at 2 mL/100 g. Additional LR or rHb solution was given if needed at 1 mL/100 g to maintain the MAP at > 70 mm Hg for the next 90 min. The shed blood was then returned simulating definitive care with transfusion in a “Hospital Phase”, lasting 15 min. The mice were then recovered, returned to cages and observed for 24 h before sacrificing for neuropathology. A marked difference in the fluid requirements was observed between the LR and the rHb groups. At the end of the Pre-Hospital Phase, the LR group received >4 times more resuscitation fluid (21.5 ± 0.75 mL/100 g) than the rHb groups (5.0 ± 0 mL/100 g; P < 0.05), while the arterial Hb level in rHb groups were ~3 g/dL higher than that of the LR group. More importantly, the LR group exhibited persistent refractory hypotension during the Pre-Hospital Phase. The MAP of rHb groups stayed near baseline level over the entire Pre-Hospital Phase and Hospital Phase. However, an initial elevation in MAP above baseline followed by gradual diminution was observed only for the rHb (αN78C) group (P < 0.05). Brain tissue oxygen (PbtO2) levels in the hippocampus ipsilateral to the site of CCI did not differ significantly between groups, although the LR group showed deterioration in PbtO2 during late Pre-Hospital Phase. Numerically, the normal oxygen affinity rHb (αN78C) group and the high oxygen affinity rHb (αN78C/L29F) groups exhibited the highest and lowest PbtO2, respectively. Surprisingly, the high oxygen affinity rHb conferred a neuroprotective effect in the CA1 region of the selectively vulnerable region of the hippocampus vs. all other groups. No difference in neuronal survival was seen between groups in the CA3 hippocampus. Our present results suggest that these novel octameric rHbs have the potential to develop into a small-volume resuscitation fluid for treatment of TBI combined with HS.