Mechanistic studies of non-canonical amino acid mutagenesis.

Over the past decade, harnessing the cellular protein synthesis machinery to incorporate non-canonical amino acids (ncAAs) into tailor-made peptides has significantly advanced many aspects of molecular science. More recently, groundbreaking progress in our ability to engineer this machinery for improved ncAA incorporation has led to significant enhancements of this powerful tool for biology and chemistry. By revealing the molecular basis for the poor or improved incorporation of ncAAs, mechanistic studies of ncAA incorporation by the protein synthesis machinery have tremendous potential for informing and directing such engineering efforts. In this chapter, we describe a set of complementary biochemical and single-molecule fluorescence assays that we have adapted for mechanistic studies of ncAA incorporation. Collectively, these assays provide data that can guide engineering of the protein synthesis machinery to expand the range of ncAAs that can be incorporated into peptides and increase the efficiency with which they can be incorporated, thereby enabling the full potential of ncAA mutagenesis technology to be realized.

Trimethylation of the R5 Silica-Precipitating Peptide Increases Silica Particle Size by Redirecting Orthosilicate Binding.

The unmodified R5 peptide from silaffin in the diatom Cylindrotheca fusiformis rapidly precipitates silica particles from neutral aqueous solutions of orthosilicic acid. A range of post-translational modifications found in R5 contribute toward tailoring silica morphologies in a species-specific manner. We investigated the specific effect of R5 lysine side-chain trimethylation, which adds permanent positive charges, on silica particle formation. Our studies revealed that a doubly trimethylated R5K3,4me3 peptide has reduced maximum activity yet, surprisingly, generates larger silica particles. Molecular dynamics simulations of R5K3,4me3 binding by the precursor orthosilicate anion revealed that orthosilicate preferentially associates with unmodified lysine side-chain amines and the peptide N terminus. Thus, larger silica particles arise from reduced orthosilicate association with trimethylated lysine side chains and their redirection to the N terminus of the R5 peptide.