Probing Protein Quinary Interactions by In-Cell Nuclear Magnetic Resonance Spectroscopy

Abstract

Historically introduced by McConkey to explain the slow mutation rate of highly abundant proteins, weak protein (quinary) interactions are an emergent property of living cells. The protein complexes that result from quinary interactions are transient and thus difficult to study biochemically in vitro. Cross-correlated relaxation-induced polarization transfer-based in-cell nuclear magnetic resonance allows the characterization of protein quinary interactions with atomic resolution inside live prokaryotic and eukaryotic cells. We show that RNAs are an important component of protein quinary interactions. Protein quinary interactions are unique to the target protein and may affect physicochemical properties, protein activity, and interactions with drugs.

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

“…It is therefore a powerful method for acquiring structural and functional information about biomolecules at atomic resolution in their natural, physiological environment [1][2][3][4][5] . In-cell NMR has been used successfully on bacterial cells to analyze protein structure 6 , dynamics 7,8 and interactions [9][10][11] . Although prokaryotic cells have some advantages, such as being easy to handle, growing fast and expressing exogenous proteins at high levels, in order for data on eukaryotic proteins to be meaningful, the proteins should be characterized in eukaryotic cells.…”

“…Microinjection, cell-penetrating peptides, pore-forming toxin and electroporation have been the most commonly used methods 3,10,[12][13][14] . The large size of Xenopus laevis oocytes allows microinjection of isotopically labeled proteins, produced in bacteria, into the cytosol 12 .…”

“…One advantage of this approach, in contrast to the CPP strategy, is that no modifications to the protein are required for intracellular sample delivery 19 . Recently, protein electroporation has been developed; this approach involves applying an electric pulse to cells to insert the purified protein of interest into the cytosol 10,14 .…”

Total Cellular RNA Modulates Protein Activity

“…We recently demonstrated 9 that specific low-affinity protein–RNA interactions, historically called quinary, 10,11 can be characterized in-cell by using cross-relaxation-induced polarization transfer (CRIPT) nuclear magnetic resonance (NMR) spectroscopy. 12 These interactions have also been detected in highly concentrated cell lysates.…”

“…12 These interactions have also been detected in highly concentrated cell lysates. 13–15 Quinary structures are large transient complexes that affect protein stability 16,17 and ligand binding 9,18 and can potentially modulate protein function. 9,17,19 Because the cellular content of RNA depends on growth conditions, 1,20,21 quinary structures have the potential to globally fine-tune diverse biological processes.…”