Protein structure determination in living cells by in-cell NMR spectroscopy

Abstract

Investigating proteins 'at work' in a living environment at atomic resolution is a major goal of molecular biology, which has not been achieved even though methods for the three-dimensional (3D) structure determination of purified proteins in single crystals or in solution are widely used. Recent developments in NMR hardware and methodology have enabled the measurement of high-resolution heteronuclear multi-dimensional NMR spectra of macromolecules in living cells (in-cell NMR). Various intracellular events such as conformational changes, dynamics and binding events have been investigated by this method. However, the low sensitivity and the short lifetime of the samples have so far prevented the acquisition of sufficient structural information to determine protein structures by in-cell NMR. Here we show the first, to our knowledge, 3D protein structure calculated exclusively on the basis of information obtained in living cells. The structure of the putative heavy-metal binding protein TTHA1718 from Thermus thermophilus HB8 overexpressed in Escherichia coli cells was solved by in-cell NMR. Rapid measurement of the 3D NMR spectra by nonlinear sampling of the indirectly acquired dimensions was used to overcome problems caused by the instability and low sensitivity of living E. coli samples. Almost all of the expected backbone NMR resonances and most of the side-chain NMR resonances were observed and assigned, enabling high quality (0.96 ångström backbone root mean squared deviation) structures to be calculated that are very similar to the in vitro structure of TTHA1718 determined independently. The in-cell NMR approach can thus provide accurate high-resolution structures of proteins in living environments.

Residue level quantification of protein stability in living cells

“…Although the folding kinetics (5,6,8,51,63) and equilibrium thermodynamic stability (5-9) of globular proteins can be influenced by crowding, their tertiary structures should remain unchanged (51,54,64) because the packing densities of globular proteins approximate those for ideal packing of hard spheres (65). As discussed above, the ability to overlay the in-cell spectrum with that from dilute solution is consistent with this expectation.…”

Cellular solid-state nuclear magnetic resonance spectroscopy

“…Whereas advancements in molecular imaging have provided unprecedented insights into the macromolecular organization in the subnanometer range (1), studying atomic structure and motion in situ has been challenging for structural biology. NMR has provided insight into cellular processes (2-4) and can determine entire 3D molecular structures inside living cells (5) provided that molecular entities tumble rapidly in a cellular setting. In principle, solid-state NMR (ssNMR) spectroscopy offers a complementary spectroscopic tool to monitor molecular structure and dynamics at atomic resolution in a complex setting (see ref.…”

A Unique Tool for Cellular Structural Biology: In-cell NMR

“…Some important parameters were assessed, such as cell viability during the experiments, timing and type of isotopic labeling, and NMR line broadening. Uniform 15 N-labeling was found to be the ideal choice in most cases, whereas uniform 13 C labeling is often unsuitable for this type of experiment, due to the high natural occurrence of 13 C (1.1%) and to the high amount of carbon atoms in biological molecules. [methyl- 13 C]Methionine labeling was shown to be a viable strategy to detect side chain carbon atoms with good selectivity against the cellular 13 C background (3); other amino acid type-selective labeling strategies were also examined (2).…”

“…Uniform 15 N-labeling was found to be the ideal choice in most cases, whereas uniform 13 C labeling is often unsuitable for this type of experiment, due to the high natural occurrence of 13 C (1.1%) and to the high amount of carbon atoms in biological molecules. [methyl- 13 C]Methionine labeling was shown to be a viable strategy to detect side chain carbon atoms with good selectivity against the cellular 13 C background (3); other amino acid type-selective labeling strategies were also examined (2). Labeling of the protein of interest with non-natural amino acids containing 19 F was also demonstrated to be a useful approach to investigate protein dynamics in the cellular environment (4,5).…”