15N isotopic labelling for in-cell protein studies by NMR spectroscopy and single-cell IR synchrotron radiation FTIR microscopy: a correlative study

The ultimate goal of modern structural biology is to probe protein structures and dynamics in their physiological microenvironment. In-cell NMR spectroscopy is an ideal technique for achieving this goal, being able to investigate proteins at atomic-resolution in living cells. The reliability of the results provided by incell NMR relies on the selectivity of the labelling methodology coupled with the filtering capabilities offered by heteronuclear NMR experiments. However, solution NMR is not well-suited either for measuring to what extent the non-specific labelling occurs, or to evaluate how it is affected by cell-to-cell variability and, eventually, whether the labelling procedure affects the cellular macromolecular content in general. To answer these questions, we correlated in-cell 1D 1H and 2D 1H–15N NMR experiments on HEK293T cells overexpressing superoxide dismutase 1 (SOD1) with single-cell Synchrotron Radiation FTIR Microscopy (FTIRM) experiments on the same samples. We verified that SOD1 overexpression in 15Nenriched media does not induce modifications in the overall cellular profile, and that the cell-to-cell labelling variability is independent of SOD1 overexpression and is likely cell cycle-related. We concluded that the non-specific incorporation of 15N into cellular components other than the protein of interest is one of the main factors that hinder the possibility of in-cell conformational studies by FTIRM at the single-cell level. Improving labelling selectivity by employing protein insertion approaches, and increasing FTIRM sensitivity by plasmonic enhancement, would open new perspectives for in-cell ultra-sensitive singleprotein conformational studies complementing NMR and vibrational analyses.