Danio rerio Oocytes for Eukaryotic In-Cell NMR

Abstract

Understanding how the crowded and complex cellular milieu affects protein stability and dynamics has only recently become possible by using techniques such as in-cell nuclear magnetic resonance. However, the combination of stabilizing and destabilizing interactions makes simple predictions difficult. Here we show the potential of Danio rerio oocytes as an in-cell nuclear magnetic resonance model that can be widely used to measure protein stability and dynamics. We demonstrate that in eukaryotic oocytes, which are 3–6-fold less crowded than other cell types, attractive chemical interactions still dominate effects on protein stability and slow tumbling times, compared to the effects of dilute buffer.

Radio Signals from Live Cells: The Coming of Age of In-Cell Solution NMR

“…150,151 In cells, where translational diffusion is further reduced, the approach has been pushed to its limits by Dobson and Christodoulou, who showed that 15 Nor 13 C-edited diffusion experiments allowed one to study the diffusion behavior of proteins in bacteria, even in the presence of interactions with cellular components, and could be employed as effective filters to distinguish intracellular from extracellular proteins. 152 In addition to the above approaches relying on 13 C and 15 N, 19 F NMR has also been applied to probe the conformational dynamics and diffusion of macromolecules in living cells. 19 F T 1 and T 2 , measured by inversion recovery and Carr-Purcell-Meiboom-Gill (CPMG), respectively, allow probing the nanosecond time-scale motions of a fluorinated protein both in the folded and in the unfolded state, as shown in bacterial cells by Pielak and Li.…”

“…Biomolecular NMR spectroscopy typically relies on spin-1/2 nuclides of biologically abundant atoms: 1 H, 13 C, and 15 N. Given the low natural abundance of 13 C (1.1%) and 15 N (0.4%) isotopes, the molecules of interest must be isotopically enriched for the NMR analysis. This poses further requirements when preparing samples for in-cell NMR.…”

“…Instead, the complex mixture of the cellular milieu gives rise to unwanted NMR signals that, in the case of 1 H (99.98% abundant), result in an extremely crowded NMR spectrum. Therefore, isotopic labeling also acts as a filter to eliminate background signals when investigating macromolecules in cells by exploiting the low natural background of 13 C and 15 N.…”

“…Thanks to its high gyromagnetic ratio, the 19 F isotope is highly sensitive, and it is 100% naturally abundant. Although fluorine atoms, unlike 13 C and 15 N, chemically alter the investigated macromolecules, with possible consequences to the conformation, stability, and activity, they are generally well-tolerated by proteins when introduced on single aromatic residues. 113 As fluorine is not naturally present in biological systems, in-cell 19 F NMR spectra are virtually background-free.…”

“…153,158 In X. laevis oocytes, the Li group reported a viscosity ∼1.2 times higher than that of water. 188 Furthermore, the same group measured 15 N and 19 F relaxation rates on concatenated GB1 constructs of increasing molecular weight, in bacteria and oocytes, and found that the transverse relaxation did not depend on the viscosity alone and had a different size dependence, likely due to the different molecular composition of the two environments. 155 Therefore, the above results indicate that the weak, transient interactions with the cellular environment, the same interactions that can affect protein folding thermodynamics (see above), are mainly responsible for the slow rotational diffusion of proteins.…”