Genetically-encoded probes to determine nonspecific hydrophobic and electrostatic binding in cells
Zuo, W.; Huang, M.-R.; Schmitz, F.; Boersma, A. J.
Show abstract
Proteins interact nonspecifically with other components in the crowded cell through associative interactions. This environmental stickiness alters for example folding stability, protein diffusion, and aggregation propensity. However, the magnitude and variation in nonspecific electrostatic and hydrophobic binding energies in the cell are unclear. Here, we develop genetically-encoded fluorescence excitation ratiometric probes to determine nonspecific binding interactions. We determine hydrophobic and electrostatic interactions by systematically varying a sensing peptide on the probe. The sensors are verified in vitro and tested in HEK293T, where the nonspecific binding is highest for highly cationic and hydrophobic domains. Perturbing the cell by energy depletion increases the dependence of binding strength on peptide electrostatics, showing that the cellular conditions tune the nonspecific interaction architecture in cells. The sensors will allow estimation of nonspecific interactions and how these interactions may change in response to stresses.
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