title: Protein Structure-Function Correlation in Living Human Red Blood Cells Probed by Isotope Exchange-based Mass Spectrometry.
abstract: 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.
authors.affiliation: Clinical Proteomics Unit, Division of Molecular Medicine, St. John's Research Institute, St. John's National Academy of Health Sciences , 100 Feet Road, Koramangala, Bangalore 560034, India.
authors.forename: Sreekala
authors.initials: S
authors.lastname: Narayanan
authors.affiliation: Clinical Proteomics Unit, Division of Molecular Medicine, St. John's Research Institute, St. John's National Academy of Health Sciences , 100 Feet Road, Koramangala, Bangalore 560034, India.
authors.forename: Gopa
authors.initials: G
authors.lastname: Mitra
authors.affiliation: Clinical Proteomics Unit, Division of Molecular Medicine, St. John's Research Institute, St. John's National Academy of Health Sciences , 100 Feet Road, Koramangala, Bangalore 560034, India.
authors.forename: Monita
authors.initials: M
authors.lastname: Muralidharan
authors.affiliation: Clinical Proteomics Unit, Division of Molecular Medicine, St. John's Research Institute, St. John's National Academy of Health Sciences , 100 Feet Road, Koramangala, Bangalore 560034, India.
authors.forename: Boby
authors.initials: B
authors.lastname: Mathew
authors.affiliation: Clinical Proteomics Unit, Division of Molecular Medicine, St. John's Research Institute, St. John's National Academy of Health Sciences , 100 Feet Road, Koramangala, Bangalore 560034, India.
authors.forename: Amit K
authors.initials: AK
authors.lastname: Mandal
mesh_terms: D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D004912:Erythrocytes / Q000737:chemistry* / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D006454:Hemoglobins