No abstract

Fluorine-containing amino acids are valuable probes for the biophysical characterization of proteins. Current methods for (19)F-labeled protein production involve time-consuming genetic manipulation, compromised expression systems and expensive reagents. We show that Escherichia coli BL21, the workhorse of protein production, can utilise fluoroindole for the biosynthesis of proteins containing (19)F-tryptophan.

Protein characterization in situ remains a major challenge for protein science. Here, the interactions of DTat-GB1 in Escherichia coli cell extracts were investigated by NMR spectroscopy and size exclusion chromatography (SEC). DTat-GB1 was found to participate in high molecular weight complexes that remain intact at physiologically-relevant ionic strength. This observation helps to explain why DTat-GB1 was not detected by in-cell NMR spectroscopy. Extracts pretreated with RNase A had a different SEC elution profile indicating that DTat-GB1 predominantly interacted with RNA. The roles of biological and laboratory ions in mediating macromolecular interactions were studied. Interestingly, the interactions of DTat-GB1 could be disrupted by biologicallyrelevant multivalent ions. The most effective shielding of interactions occurred in Mg 21 -containing buffers. Moreover, a combination of RNA digestion and Mg 21 greatly enhanced the NMR detection of DTat-GB1 in cell extracts.

Although essential to numerous biotech applications, knowledge of molecular recognition by arginine-rich motifs in live cells remains limited. H,N HSQC and F NMR spectroscopies were used to investigate the effects of C-terminal -GR (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 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 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.

Decades of dilute-solution studies have revealed the influence of charged residues on protein stability, solubility and stickiness. Similar characterizations are now required in physiological solutions to understand the effect of charge on protein behavior under native conditions. Toward this end, we used free boundary and native gel electrophoresis to explore the charge of cytochrome c in buffer and in Escherichia coli extracts. We find that the charge of cytochrome c was~2-fold lower than predicted from primary structure analysis. Cytochrome c charge was tuned by sulfate binding and was rendered anionic in E. coli extracts due to interactions with macroanions. Mutants in which three or four cationic residues were replaced with glutamate were chargeneutral and "inert" in extracts. A comparison of the interaction propensities of cytochrome c and the mutants emphasizes the role of negative charge in stabilizing physiological environments. Charge-charge repulsion and preferential hydration appear to prevent aggregation. The implications for molecular organization in vivo are discussed.