Biological Regulation Studied in Vitro and in Cellulo with Modified Proteins

Conspectus Proteins and peptides occur ubiquitously in organisms and play key functional roles, as structural elements and catalysts. Their major natural source is ribosomal synthesis, which produces polypeptides from 20 amino acid building blocks. Peptides containing noncanonical amino acids have long been prepared by chemical synthesis, which has provided a wealth of physiologically active compounds. Comparatively, preparing modified proteins has been more challenging. Site-directed mutagenesis provided an important advance but was initially limited to canonical amino acids. New techniques for tRNA activation with noncanonical amino acids subsequently increased the scope of site-directed mutagenesis. Our report in 2012 demonstrated that modification of bacterial ribosomes at key positions enabled the selection of ribosomes capable of introducing β-amino acids into proteins in vitro. The generality of the selection procedure was tested further. Ribosomes capable of incorporating dipeptides, conformationally constrained dipeptides, dipeptidometics with embedded fluorophores, contiguous nucleobase amino acids, and phosphorylated amino acids were successfully identified. In this Account, we focus on the application of the new technology to dramatically alter protein structure in ways that enable new strategies for understanding and altering protein function. To illustrate the robustness of the technology we have provided examples studied in vitro and in cellulo. The first category involves the introduction of nucleobase amino acids into proteins in support of specific interactions with RNA and DNA. The energetic differences between potential protein-nucleic acid complexes formed from two binding partners are often quite small. It seems logical to think that selective binding can be achieved by using a nucleobase moiety in each of the binding partners by utilizing known interactions between nucleic acid bases (located in the protein and nucleic acid) to achieve energetically favorable interactions. We do so both in vitro and in cellulo. A second focus has involved the design of small fluorescent probes not much larger than amino acids that are genetically encodable and which can be incorporated during protein biosynthesis, serving as detectable probes of protein trafficking and interaction with other macromolecules. We provide an in vitro example of strongly fluorescent tryptophan analogues positioned at single sites within dihydrofolate reductase, permitting selective communication with a FRET acceptor at a known position, even in the presence of several tryptophans. An oxazole amino acid, weakly fluorescent in aqueous solution, fluoresced more strongly following incorporation into MreB, a scaffold protein produced in cellulo. Finally, we describe the introduction of a single phosphorylated tyrosine into the p50 subunit of NF-κB. When present at either of two key positions, the resulting NF-κB significantly enhanced binding in vitro to the promoter DNA as well as subsequent mRNA transcription of its client protein CD40 in cellulo. In a separate expression in activated Jurkat cells, an increased production of CD40 protein was observed.