Effect of point mutation on structure-function correlation of hemoglobin variants, HbE and HbD Punjab.

Hemoglobinopathies are examples of autosomal recessive disorders of human hemoglobin. Hemoglobin E (HbE) and Hemoglobin D Punjab (HbD Punjab) are two of the most common hemoglobin variants geographically spread across Asian continent. These two variants differ from normal human hemoglobin (HbA) at a single amino acid residue caused by the point mutation of ß globin gene. The presence of the mutated amino acid residue causes perturbation in the function of both variants. However, the structure-function correlation of these variants has not been established yet. In the present study, we analyzed the conformational changes associated with oxygenation of hemoglobin variants using hydrogen/deuterium exchange-based mass spectrometry of backbone amide hydrogens of α and ß globin chains in the tetrameric hemoglobin molecule. We also performed the functional assay of these variants using oxygen dissociation equilibrium curve. Compared to HbA, both variants showed reduced oxygen affinity, as reported earlier. The functional perturbations exhibited by these variants were correlated well with their structural alterations with respect to the reported changes in the residue level interactions upon oxygenation of normal hemoglobin, monitored through the hydrogen/deuterium exchange kinetics of several peptic peptides originated from the isotopically exchanged oxy and deoxy forms of HbE and HbD Punjab.

Protein Structure-Function Correlation in Living Human Red Blood Cells Probed by Isotope Exchange-based Mass Spectrometry.

To gain insight into the underlying mechanisms of various biological events, it is important to study the structure-function correlation of proteins within cells. Structural probes used in spectroscopic tools to investigate protein conformation are similar across all proteins. Therefore, structural studies are restricted to purified proteins in vitro and these findings are extrapolated in cells to correlate their functions in vivo. However, due to cellular complexity, in vivo and in vitro environments are radically different. Here, we show a novel way to monitor the structural transition of human hemoglobin upon oxygen binding in living red blood cells (RBCs), using hydrogen/deuterium exchange-based mass spectrometry (H/DX-MS). Exploiting permeability of D2O across cell membrane, the isotope exchange of polypeptide backbone amide hydrogens of hemoglobin was carried out inside RBCs and monitored using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). To explore the conformational transition associated with oxygenation of hemoglobin in vivo, the isotope exchange kinetics was simplified using the method of initial rates. RBC might be considered as an in vivo system of pure hemoglobin. Thus, as a proof-of-concept, the observed results were correlated with structural transition of hemoglobin associated with its function established in vitro. This is the first report on structural changes of a protein upon ligand binding in its endogenous environment. The proposed method might be applicable to proteins in their native state, irrespective of location, concentration, and size. The present in-cell approach opens a new avenue to unravel a plethora of biological processes like ligand binding, folding, and post-translational modification of proteins in living cells.

Glutathionylation induced structural changes in oxy human hemoglobin analyzed by backbone amide hydrogen/deuterium exchange and MALDI-mass spectrometry.

Glutathionyl hemoglobin, a post-translationally modified form of hemoglobin, has been reported to serve as a marker of oxidative stress in several clinical conditions. This modification causes perturbations in the hemoglobin functionality by increasing oxygen affinity and reducing cooperativity. Moreover, glutathionylation of sickle hemoglobin was reported to lead to a significant reduction in the propensity of sickling of erythrocytes. The root cause of the above functional abnormality is not known in detail, as the crystal structure of the molecule is yet to be discovered. In this study, we investigated the effects of glutathionylation on quaternary structure of hemoglobin using hydrogen/deuterium exchange (H/DX) based mass spectrometry. H/DX kinetics of nine peptides from α and ß globin chains of hemoglobin were analyzed to understand the conformational change in deoxy to oxy transition of normal hemoglobin and structural perturbations associated with glutathionylation of oxy hemoglobin. Significant structural changes brought about by the glutathionylation of oxy hemoglobin were observed in the following regions of globin chains: ß86-102, ß1-14, α34-46, ß32-41, ß130-146, ß115-129, ß73-81. Isotope exchange kinetics monitored through mass spectrometry is a useful technique to understand structural perturbation on post-translational modification of proteins in solution phase.