Probing the Conformation of Hemoglobin Presbyterian in the R-State

Abstract

The influence of allosteric effectors on the R-state (liganded) conformation of Tg-HbP (human hemoglobin Presbyterian expressed in transgenic pig) has been probed using a number of biophysical techniques, and the results have been compared with that of liganded of HbA (human normal adult hemoglobin) to gain insight into the molecular basis of Asn-108(beta)->Lys mutation-induced low-oxygen affinity of Hb. The nuclear magnetic resonance studies of Tg-HbP revealed that the conformation of the alpha1beta1 and alpha1beta1 interfaces of the protein in the deoxy state are indistinguishable from that of deoxy HbA, whereas the conformation of the microenvironment of His-103(alpha) of Tg-HbP, a residue of the alpha1beta1 interface, is distinct from that of HbA in the R-state. In addition, the Presbyterian mutation also influences the structure of oxy Hb in other regions of the molecule. First, it facilitates the generation of deoxy (T)-state marker at 14.2 ppm (from 2,2-dimethyl-s-silapentane-5-sulfonate) on the interaction of oxy Hb with inositol hexa-phosphate without changing the ligation state. Second, it increases the geminate yield of the 10 ns photoproduct of CO-Hb. Third, it enhances the propensity of phosphate to increase the geminate yield. Fourth, it potentiates the ability of phosphate to induce deoxy-like features at the heme environment in the R-state. Fifth, it induces T-state-like signatures at the switch and hinge regions of the alpha1beta2 interface. Finally, molecular modeling studies have indicated an increased affinity for the four anion binding sites mapped in the midcentral cavity of Hb caused by the presence of Lys-108(beta). In short, Lys-108(beta) in HbP induces a propensity for oxy Hb to access T-like conformational features in different regions of the oxy Hb molecule and also enhances the T-like signatures in the oxy state on interaction with allosteric effectors without changing its ligation. Interestingly, the intrinsic T-like conformational features of the R-state of HbP, in addition to those induced by the addition of allosteric effectors to liganded HbP, appear to be reminiscent of features of the B-state conformation of Hb found in rHb 1.1 (recombinant hemoglobin). We propose that the lowered oxygen affinity of Tg-HbP in the presence of allosteric effectors is a consequence of an altered R-state conformation of Hb, which reflects the facilitation of switching the R-state of HbP to the T-state compared with the normal R-state of HbA, thereby reducing HbA's affinity to oxygen.

Conformational Dependence of Hemoglobin Reactivity under High Viscosity Conditions: The Role of Solvent Slaved Dynamics

“…37,44 The highviscosity kinetic patterns from sol-gel-stabilized allosteric intermediates such as the low-affinity and high-affinity liganded T state species 37,44,45 are shown to also be obtainable from stable equilibrium populations of mutant forms of HbA. To that end, we used the Hb(βW37E) mutant that has been shown to undergo the LT-to-HT state conformational change upon ligand binding, 44,[46][47][48][49] as well as double and triple mutant (HbD and HbT) modifications of HbPresbyterian(βN108K), [50][51][52][53][54][55] i.e., HbRV96W/ βN108K 51 and HbRL29F/ RV96W/βN108K, 56,57 which are very low affinity hemoglobins that display T state NMR signatures even for the fully liganded derivatives. 56,57 Large R/β subunit differences in the high-viscosity kinetic patterns from halfliganded T state forms of HbA are exposed using encapsulated FeCO/Zn hybrid derivatives.…”

“…The LT samples can be allowed to evolve for a time duration that can vary between hours to weeks, depending on the gelation protocol, until spectroscopic signatures of high-affinity T (HT) are observed. , The high-viscosity kinetic patterns from sol−gel-stabilized allosteric intermediates such as the low-affinity and high-affinity liganded T state species ,, are shown to also be obtainable from stable equilibrium populations of mutant forms of HbA. To that end, we used the Hb(βW37E) mutant that has been shown to undergo the LT-to-HT state conformational change upon ligand binding, ,− as well as double and triple mutant (HbD and HbT) modifications of HbPresbyterian(βN108K), − i.e., HbαV96W/βN108K 51 and HbαL29F/ αV96W/βN108K, , which are very low affinity hemoglobins that display T state NMR signatures even for the fully liganded derivatives. , Large α / β subunit differences in the high-viscosity kinetic patterns from half-liganded T state forms of HbA are exposed using encapsulated FeCO/Zn hybrid derivatives. − The new protein dynamics-based model provides a coherent framework to explore the origin of variations in kinetic patterns as a function of solvent, conformation, and specific subunit.…”