UNASSIGNED: 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 @entity1 hemoglobin upon @entity26 binding in living red blood cells (RBCs), using @entity3884 / @entity6189 exchange-based mass spectrometry (H/DX-MS). Exploiting permeability of D2O across cell membrane, the isotope exchange of polypeptide backbone @entity6346 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.

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

@entity1 :: ('9606', 'Species') :: ['human'] @entity3884 :: ('MESH:D006859', 'Chemical') :: ['hydrogen'] @entity26 :: ('MESH:D010100', 'Chemical') :: ['oxygen'] @entity6189 :: ('MESH:D003903', 'Chemical') :: ['deuterium'] @entity6346 :: ('CHEBI:32988', 'Chemical') :: ['amide']
@entity1 :: (9606,Species) :: ['Human']
