A new carbamidemethyl-linked lanthanoid chelating tag for PCS NMR spectroscopy of proteins in living HeLa cells

Abstract

Structural analyses of proteins under macromolecular crowding inside human cultured cells by in-cell NMR spectroscopy are crucial not only for explicit understanding of their cellular functions but also for applications in medical and pharmaceutical sciences. In-cell NMR experiments using human cultured cells however suffer from low sensitivity, thus pseudocontact shifts from protein-tagged paramagnetic lanthanoid ions, analysed using sensitive heteronuclear two-dimensional correlation NMR spectra, offer huge potential advantage in obtaining structural information over conventional NOE-based approaches. We synthesised a new lanthanoid-chelating tag (M8-CAM-I), in which the eight-fold, stereospecifically methylated DOTA (M8) scaffold was retained, while a stable carbamidemethyl (CAM) group was introduced as the functional group connecting to proteins. M8-CAM-I successfully fulfilled the requirements for in-cell NMR: high-affinity to lanthanoid, low cytotoxicity and the stability under reducing condition inside cells. Large PCSs for backbone N-H resonances observed for M8-CAM-tagged human ubiquitin mutant proteins, which were introduced into HeLa cells by electroporation, demonstrated that this approach readily provides the useful information enabling the determination of protein structures, relative orientations of domains and protein complexes within human cultured cells.

Flow‐based in vivo NMR spectroscopy of small aquatic organisms

“…The ability to conduct in vivo NMR experiments hinges on a flow cell system that is capable of supplying food and oxygenated water to the organisms under investigation . The use of a flow cell system in NMR spectroscopy has been well documented . Therefore, only a brief overview will be given here.…”

“…In these systems, cells are often suspended in a culturing media, and nutrients are supplied mechanically to the system via liquid pumps . Many of these systems share a commonality that both inlet and outlet are separated, in which two different pumps are employed to achieve continuous flow . A two pump system can be problematic because the flow rate of both pumps must be matched to avoid formation of air bubbles.…”

“…As such, the living organism becomes the “ultimate biosensor” responding in real‐time to its environment, while the NMR spectrometer interprets the biochemical changes, providing information explaining sublethal toxicity at the molecular level. The reproducibility of NMR is second to none, making it a versatile profiling tool for the identification of stressed or diseased individuals . The manifestation of physiological responses to the exposure of toxins in the metabolic profile is rapid, compared with the genome or proteome, making the metabolome a key indicator of stress.…”

A Sterically Overcrowded, Isopropyl‐Substituted, Lanthanide‐Chelating Tag for Protein Pseudocontact Shift NMR Spectroscopy: Synthesis of its Macrocyclic Scaffold and Benchmarking on Ubiquitin S57 C and hCA II S166 C

“…Pseudocontact shifts (PCS) and residual dipolar couplings (RDC) generated by lanthanide‐chelating tags (LCT) yield valuable structural restraints for the analysis of structure, dynamics, and ligand‐binding of proteins in solution . In order to access valuable structural restraints by PCS measurements, stereospecifically methyl‐substituted 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA)‐based chelators present a suitable scaffold .…”

Efficient and Innocuous Live‐Cell Delivery: Making Membrane Barriers Disappear to Enable Cellular Biochemistry

“…In turn, this approach permits the determination of protein structure and protein dynamics in situ, as opposed to in vitro. Recently, Hikone et al have expanded on these ideas by introducing a ubiquitin labeled with a lanthanoid‐chelation tag into cells by electroporation. The authors successfully used this approach to perform pseudocontact chemical shifts measurements and assess the effects of intracellular crowding on the protein structure.…”