15N isotopic labelling for in-cell protein studies by NMR spectroscopy and single-cell IR synchrotron radiation FTIR microscopy: a correlative study. Issue 5 (6th February 2018)

• Record Type: Journal Article
• Authors: Mitri, E.; Barbieri, L.; Vaccari, L.; Luchinat, E.
• Is Part Of: Analyst. Volume 143:Issue 5(2018)
• Page Start: 1171
• Page End: 1181
• Publication Date: 2018-02-06
• Subjects: Chemistry, Analytic -- Periodicals; 543
• DOI: 10.1039/c7an01464c
• Languages: English
• ISSNs: 0003-2654

Abstract

Abstract : The effect of 15 N-enrichment on human cells analyzed by correlative in-cell NMR and single-cell SR-FTIR experiments. Abstract : 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 in-cell 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 1 H and 2D 1 H– 15 N 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 15 N-enriched 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 15 N 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 single-protein conformational studies complementing NMR and vibrational analyses.