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Investigating the structure, modification, interaction, and function of biomolecules in their native cellular environment leads to physiologically relevant knowledge about their mechanisms, which will benefit drug discovery and design. In recent years, nuclear and electron magnetic resonance (NMR) spectroscopy has emerged as a useful tool for elucidating the structure and function of biomacromolecules, including proteins, nucleic acids, and carbohydrates in living cells at atomic resolution. In this review, we summarize the progress and future of in-cell NMR as it is applied to proteins, nucleic acids, and carbohydrates.

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The mechanism of micelle formation of surfactants sodium dodecyl sulfate (SDS), n-hexyldecyltrimethylammonium bromide (CTAB) and Triton X-100 (TX-100) in heavy water solutions was studied by 1H NMR (chemical shift and line shape) and NMR self-diffusion experiments. 1H NMR and self-diffusion experiments of these three surfactants show that their chemical shifts (delta) begin to change and resonance peaks begins to broaden with the increase in concentration significantly below their critical micelle concentrations (cmc's). At the same time, self-diffusion coefficients ( D) of the surfactant molecules decrease simultaneously as their concentrations increase. These indicate that when the concentrations are near and lower than their cmc's, there are oligomers (premicelles) formed in these three surfactant systems. Carefully examining the dependence of chemical shift and self-diffusion coefficient on concentration in the region just slightly above their cmc's, one finds that the pseudophase transition model is not applicable to the variation of physical properties (chemical shift and self-diffusion coefficient) with concentration of these systems. This indicates that premicelles still exist in this concentration region along with the formation of micelles. The curved dependence of chemical shift and self-diffusion coefficient on the increase in concentration suggests that the premicelles grow as the concentration increases until a definite value when the size of the premicelle reaches that of the micelle, i.e., the system is likely dominated by the monomers and micelles. Additionally, the approximate values of premicelle coming forth concentration (pmc) and cmc were obtained by again fitting chemical shifts to reciprocals of concentrations at a different perspective, and are in good accordant with experimental results and literature values and prove the former conclusion.

Protein mobility in living cells is vital for cell function. Both cytosolic viscosity and weak protein-protein interactions affect mobility, but examining viscosity and weak interaction effects is challenging. Herein, we demonstrate the use of (19) F NMR spectroscopy to measure cytoplasmic viscosity and to characterize nonspecific protein-protein interactions in living Escherichia coli cells. The origins of resonance broadening in Escherichia coli cells were also investigated. We found that sample inhomogeneity has a negligible effect on resonance broadening, the cytoplasmic viscosity is only about 2-3 times that of water, and ubiquitous transient weak protein-protein interactions in the cytosol play a significant role in governing the detection of proteins by using in-cell NMR spectroscopy.

A new approach to the characterization of biomolecules in whole biological fluids is presented based on simplification of 1H NMR spectra by utilizing differences in molecular diffusion coefficients alone and combinations of relaxation and diffusion parameters. New NMR pulse sequences incorporating both spectral editing features together with solvent water resonance elimination are presented. The methods are exemplified using whole human blood plasma, and it is shown that it is possible to obtain NMR spectra of the slowly diffusing species (generally large molecules) by diffusion editing, the slowly relaxing species (generally small molecules) by spin relaxation editing, or spectra showing any range of molecular mobility using a combination of the two methods. The diffusion-based editing methods are also applicable to the selection of resonances in two-dimensional NMR spectroscopy of biofluids, and we show this for the first time by the production of 1H-1H diffusion-edited TOCSY spectra of human blood plasma where the resonance intensities are weighted according to the molecular diffusion coefficient. In this case, by measuring a diffusion-edited 1H-1H TOCSY NMR spectrum of plasma, it is possible to obtain signals from only the macromolecular components, and this may be of benefit in the analysis of blood lipoproteins. In complex biofluids, the combination of diffusion and relaxation editing brings about considerable spectral simplification leading to an easier resonance assignment process. We also demonstrate the production of 1H NMR spectra with intensities corresponding to diffusion coefficient rather than number of protons, and this opens up new possibilities for pattern recognition classification of samples based on altered molecular mobility features of biofluid components.

Another kind of dynamics: Ubiquitin noncovalently dimerizes with a dissociation constant of approximately 5 mM. The two subunits adopt an array of relative orientations, utilizing an interface also for binding to other proteins (see picture). Quaternary fluctuation among members of the dimer ensemble constitutes a different kind of dynamics that complements the tertiary dynamics of each ubiquitin subunit.

KDM5B is a histone H3K4me2/3 demethylase. The PHD1 domain of KDM5B is critical for demethylation, but the mechanism underlying the action of this domain is unclear. In this paper, we observed that PHD1KDM5B interacts with unmethylated H3K4me0. Our NMR structure of PHD1KDM5B in complex with H3K4me0 revealed that the binding mode is slightly different from that of other reported PHD fingers. The disruption of this interaction by double mutations on the residues in the interface (L325A/D328A) decreases the H3K4me2/3 demethylation activity of KDM5B in cells by approximately 50% and increases the transcriptional repression of tumor suppressor genes by approximately twofold. These findings imply that PHD1KDM5B may help maintain KDM5B at target genes to mediate the demethylation activities of KDM5B.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-014-0078-4) contains supplementary material, which is available to authorized users.