In-Cell NMR: Analysis of Protein-Small Molecule Interactions, Metabolic Processes, and Protein Phosphorylation

Abstract

Nuclear magnetic resonance (NMR) spectroscopy enables the non-invasive observation of biochemical processes, in living cells, at comparably high spectral and temporal resolution. Preferably, means of increasing the detection limit of this powerful analytical method need to be applied when observing cellular processes under physiological conditions, due to the low sensitivity inherent to the technique. In this review, a brief introduction to in-cell NMR, protein-small molecule interactions, posttranslational phosphorylation, and hyperpolarization NMR methods, used for the study of metabolites in cellulo, are presented. Recent examples of method development in all three fields are conceptually highlighted, and an outlook into future perspectives of this emerging area of NMR research is given.

Keywords: DNP; in-cell NMR; in-situ NMR; protein NMR; review.

Figure 1

Schematic overview of different known approaches in in-cell NMR. (Left) Endogenously expressed and isotopically labeled protein can be achieved by transferring the expression vector containing the gene of interest into (A) bacteria, (B) yeast, (C) insect cell lines, and (D) mammalian cells. (Right) An alternate way of in-cell NMR, where isotopically labeled protein is exogenously prepared followed by delivery into eukaryotic cells with different methods such as (E) electroporation, (F) attaching protein with cell-penetrating peptides (CPP), (G) protein transport via pore-forming toxins, and (H) microinjection-mediated delivery into Xenopus leavis oocytes.

Figure 2

In-cell NMR study of protein–small molecule interactions. Here, a “sensitive to lysis” (SlyD)-containing plasmid was transformed into Escherichia coli and protein expression was initiated by isopropyl β-d-1-thiogalactopyranoside (IPTG) induction. These cells were further incubated with a small molecule (Cu²⁺ complex). The interaction of this small molecule with SlyD could be observed using in-cell NMR, and correlated with corresponding in vitro NMR studies, revealing the binding site in SlyD for this Cu²⁺ complex (adopted according to Reference [4]).

Figure 3

Diagram of the para-hydrogen-induced hyperpolarization side-arm hydrogenation (PHIP-SAH) procedure. (1) Functionalization of the carboxylate group with the side-arm; (2) para-hydrogenation of the unsaturated alcohol; (3) transfer of para-hydrogen spin order to the ¹³C spin of the carboxylate group; (4) cleavage of the side-arm. The yellow background indicates reaction steps taking place in the organic phase, while the blue background indicates that the molecule is dissolved in the aqueous phase. This figure was adopted according to Reference [98].

Figure 4

Following cell lysate-induced protein phosphorylation using NMR spectroscopy. (A) Superposition of two-dimensional (2D) ¹H–¹⁵N HSQC spectra of phosphorylated p19^INK4d at Ser66 (red) and non-phosphorylated protein (black). (B) Superposition of 2D ¹H–¹⁵N HSQC spectra of doubly phosphorylated p19^INK4d at Ser66 and Ser76 (red) and the non-phosphorylated form (black). The bottom panels in (A) and (B) represent the backbone NMR chemical-shift mapping on the p19^INK4d structure. (C) Summary of the fate of p19^INK4d during the cell cycle controlled by phosphorylation and ubiquitination. This figure was adapted from Reference [24].

Figure 5

NMR signature of serine phosphorylation using a short peptide (A57-F86 selectively labeled with ¹⁵N-Ser66 and ¹⁵N-Ser76) of p19^INK4d modified by HeLa cell lysate. (A) Overlaid 2D ¹H–¹⁵N HSQC spectra of untreated (black) and treated (red) peptide showing the typical low-field shift of the serine amide proton upon side-chain phosphorylation. (B) ³¹P NMR spectra of the phosphorylated A57-F86 peptide. The asterisk in (B) shows the signal of the phosphate buffer.