Abstract
Transient, site-specific, or so-called quinary, interactions are omnipresent in live cells and modulate protein stability and activity. Quinary intreactions are readily detected by in-cell NMR spectroscopy as severe broadening of the NMR signals. Intact ribosome particles were shown to be necessary for the interactions that give rise to the NMR protein signal broadening observed in cell lysates and sufficient to mimic quinary interactions present in the crowded cytosol. Recovery of target protein NMR spectra that were broadened in lysates, in vitro and in the presence of purified ribosomes was achieved by RNase A digestion only after the structure of the ribosome was destabilized by removing magnesium ions from the system. Identifying intact ribosomal particles as the major protein-binding component of quinary interactions and consequent spectral peak broadening will facilitate quantitative characterization of macromolecular crowding effects in live cells and streamline models of metabolic activity.
Highlights
Broadening of target protein signals due to the formation of large slowly tumbling species that exhibit megadalton apparent molecular weights in-cell [1,2,3] during NMR spectroscopic experiments [4] and in concentrated cellular lysates is a common occurrence and regarded as evidence for specific transient complexes or protein quinary structure. [5,6,7,8,9,10,11] The source of quinary interactions has been investigated from the perspective of increased intracellular viscosity, [2, 12] volume exclusion, and the presence of neutral and charged polymeric and proteinaceous crowding agents. [12, 13] these non-specific physical phenomena do contribute to some degree to signal broadening, they fail to recapitulate the extent to which it is observed during in-cell NMR experiments except under non-physiological conditions. [14]
Recent work has advocated for ribosomes as the major target protein binding complement that give rise to quinary interactions and consequent spectral peak broadening [1, 15,16,17] and suggested that the ribosome may function as an electrostatic sponge that binds to a wide range of proteins and metabolites. In those studies in-cell NMR spectra of target proteins were compared to spectra obtained in vitro in the presence of total cellular RNA [16] and purified ribosomal preparations. [1, 15, 18] The work left open the question of whether intact ribosome particles per se, or unidentified proteins or free rRNA mediate these interactions
To demonstrate that intact ribosomes are a critical component of quinary interactions, the NMR spectrum of purified uniformly labeled [U- 15N] γD-crystallin was examined in the presence of stable and destabilized ribosomes in E. coli cell lysate. γD-crystallin is a small, 21 kDa, eukaryotic protein found in the eye lens of vertebrates
Summary
Recent work has advocated for ribosomes as the major target protein binding complement that give rise to quinary interactions and consequent spectral peak broadening [1, 15,16,17] and suggested that the ribosome may function as an electrostatic sponge that binds to a wide range of proteins and metabolites. In those studies in-cell NMR spectra of target proteins were compared to spectra obtained in vitro in the presence of total cellular RNA [16] and purified ribosomal preparations. In those studies in-cell NMR spectra of target proteins were compared to spectra obtained in vitro in the presence of total cellular RNA [16] and purified ribosomal preparations. [1, 15, 18] The work left open the question of whether intact ribosome particles per se, or unidentified proteins or free rRNA mediate these interactions.
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