Characterization of proteins by in-cell NMR spectroscopy in cultured mammalian cells

Abstract

In-cell NMR spectroscopy is a unique tool for characterizing biological macromolecules in their physiological environment at atomic resolution. Recent progress in NMR instruments and sample preparation methods allows functional processes, such as metal uptake, disulfide-bond formation and protein folding, to be analyzed by NMR in living, cultured human cells. This protocol describes the necessary steps to overexpress one or more proteins of interest inside human embryonic kidney 293T (HEK293T) cells, and it explains how to set up in-cell NMR experiments. The cDNA is transiently transfected as a complex with a cationic polymer (DNA:PEI (polyethylenimine)), and protein expression is carried on for 2-3 d, after which the NMR sample is prepared. (1)H and (1)H-(15)N correlation NMR experiments (for example, using band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence (SOFAST-HMQC)) can be carried out in <2 h, ensuring cell viability. Uniform (15)N labeling and amino-acid-specific (e.g., cysteine, methionine) labeling schemes are possible. The entire procedure takes 4 d from cell culture seeding to NMR data collection.

Longitudinal Relaxation Optimization Enhances 1H‐Detected HSQC in Solid‐State NMR Spectroscopy on Challenging Biological Systems

“…To date, the SOFAST‐HMQC and longitudinal relaxation optimized HSQC (LHSQC) methods have been used to accelerate solution‐state NMR spectroscopy experiments. Both schemes have been applied extensively in multidimensional spectroscopy and are used widely in protein, RNA, and in‐cell NMR spectroscopy studies . The flip‐back‐water trick has also been applied to enhance the spectral sensitivity of cross‐polarization (CP)‐based experiments in SSNMR spectroscopy, achieving about 30 % sensitivity enhancement compared with that of conventional CP‐type experiments.…”

“…Both schemesh ave been appliede xtensively in multidimensionals pectroscopy and are used widely in protein, [49] RNA, [50] and in-cell NMR spectroscopys tudies. [47,[51][52][53][54] The flip-backwater trick has also been appliedt oe nhance the spectrals ensitivity of cross-polarization (CP)-based experiments in SSNMR spectroscopy, [55][56][57] achievinga bout 30 %s ensitivitye nhancement compared with that of conventionalC P-typee xperiments. 1 H-detected HSQC experiments are widely used in SSNMR spectroscopy to study mobile segments of large complex systems, that is, flexible loopso fm embrane proteins, at am oderate magic angle spinning (MAS) rate.…”

Drug Screening in Human Cells by NMR Spectroscopy Allows the Early Assessment of Drug Potency

“…In humans there are 15 CA isoforms, many of which are relevant drug targets involved in several pathologies . To date, several CA inhibitors are routinely administered in the treatment of glaucoma, epilepsy, or as diuretics, with some of them in clinical development as antitumor agents, In order to detect CA2 by NMR, the protein was directly expressed and labelled in the cytosol of human cells (see the Experimental Methods section of the Supporting Information) . NMR signals arising from [ 15 N]‐CA2 were clearly detected in the 1 H‐ 15 N correlation spectra (Supporting Information, Figure S1), indicating that the intracellular protein is soluble and free from interactions with slow‐tumbling cellular components, which would otherwise increase transverse relaxation and cause signal broadening beyond detection .…”

A cell is more than the sum of its (dilute) parts: A brief history of quinary structure

“…injected the B1 domain of protein G (GB1), a model protein, into Xenopus laevis oocytes . The utility of in‐cell NMR has also been extended to proteins overexpressed in human cells . Majumder et al .…”