In-cell NMR: Why and how?

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

“…These are quite appealing systems for in-cell NMR studies: (i) only ∼170 of them are necessary to fill a 5 mm NMR tube; (ii) they can be manipulated and microinjected cell by cell in about 1 h; (iii) they do not require a specific wet-lab, even though a source of oocytes has to be found (commercial companies sell them at ∼$1–2 a piece); (iv) they can stand the injection of about 20–50 nL of high-concentration material, which generates ∼20–50-fold intracellular dilutions; (v) they can survive and maintain their integrity about 18 h in the NMR tube without any medium replenishment; and (vi) they are well-known model systems in biology . This strategy has been used for in-cell NMR investigations of both proteins − ,,,,, and nucleic acids. − We encourage the reader to refer to the published, detailed protocols. ,,− We will not present them extensively once again, but we must give a few words of caution: (i) Because of the cylindrical shape of NMR tubes, hundreds of oocytes must be stacked on each other and thus require high-density outer solvent (e.g., a buffer containing 20% w/w Ficoll) to not crush within a few hours; (ii) their cell-cycle stage is well-defined, which comes with certain advantages when studying cell-signaling and post-translational modifications, − , but it leaves little room for modifications of the cellular conditions; (iii) their high-lipid yolk compartment is problematic for molecules that would preferentially bind to lipids; (iv) they generate important peak broadening due to sample inhomogeneities (settled oocytes leave large intercellular spaces, and the intracellular compartments are also diverse enough to present distinct magnetic susceptibilities); , and (v) their cellular organization is clearly not that of more standard prokaryotic or eukaryotic cells. Oocyte cytosolic extracts represent a good proxy in many regards, e.g., to test the cellular stability, the NMR detectability or even to investigate post-translational modifications (PTMs). ,, Extracts are also easier to handle and allow a better control of concentrations, of the time origin for time-resolved NMR monitoring, and better NMR resolution. ,,,, Hence, while they enabled many seminal contributions in the field, oocytes tend to lose their attractivity in the very last years.…”

“…Most of them were actually proposed before the year 2000, in times where large diameter NMR probes were used to monitor organs and cells metabolism. We refer the reader to our detailed inventory spanning this wider range of in-cell NMR studies . Sadly enough, these flow-probe systems were developed in-house and were not transferred from lab to lab.…”

“…Adherent mammalian cells must still preferably be entrapped in hydrogels. In this regard, methyl cellulose has been shown to have improved impact on cell viability in two recent live-cell NMR studies. , An important aspect here is to avoid entrapping any air bubbles in the hydrogel because this would provoke important magnetic susceptibility inhomogeneities, which drastically affect NMR line width …”

“…We refer the reader to dedicated reviews. ,, It permits to obtain a footprint of the binding epitope, or more simply to detect weak-affinity interactions, with K d between 10 –7 and 10 –3 M and k off between 10 2 and 10 6 s –1 . , It has been applied to mammalian cells, even though one recent study focused on bacteria . STD-NMR has been used extensively to study drugs and glycans-binding of viral particles, which we will not detail here (see Theillet et al, ref ). Still, it is worth mentioning that these studies have revealed some important technical aspects of STD-NMR using very large particles or cells.…”

“…In-cell structural biology by NMR has emerged in the last 20 years, − building on the long-term practice of NMR spectroscopists, who manipulate and characterize live cells, tissues, and animals in their spectrometers for 70 years already. − Nuclei with a nonzero spin magnetic moment are indeed detectable using NMR spectroscopy in almost every material, in a nondestructive fashion and with a very good penetrance (see MRI, for example). The cellular context has nevertheless some inconvenient consequences for the manipulation and detection of nuclear spins.…”