Short Arginine Motifs Drive Protein Stickiness in the <i>Escherichia coli</i> Cytoplasm

Abstract

Although essential to numerous biotech applications, knowledge of molecular recognition by arginine-rich motifs in live cells remains limited. 1 H, 15 N HSQC and 19 F NMR spectroscopies were used to investigate the effects of C-terminal −GR n ( n = 1–5) motifs on GB1 interactions in Escherichia coli cells and cell extracts. While the “biologically inert” GB1 yields high-quality in-cell spectra, the −GR n fusions with n = 4 or 5 were undetectable. This result suggests that a tetra-arginine motif is sufficient to drive interactions between a test protein and macromolecules in the E. coli cytoplasm. The inclusion of a 12 residue flexible linker between GB1 and the −GR 5 motif did not improve detection of the “inert” domain. In contrast, all of the constructs were detectable in cell lysates and extracts, suggesting that the arginine-mediated complexes were weak. Together these data reveal the significance of weak interactions between short arginine-rich motifs and the E. coli cytoplasm and demonstrate the potential of such motifs to modify protein interactions in living cells. These interactions must be considered in the design of ( in vivo ) nanoscale assemblies that rely on arginine-rich sequences.