Abstract
Many proteins and peptides fold upon binding another protein. Mutagenesis has proved an essential tool in the study of these multi-step molecular recognition processes. By comparing the biophysical behavior of carefully selected mutants, the concert of interactions and conformational changes that occur during folding and binding can be separated and assessed. Recently, this mutagenesis approach has been radically expanded by deep mutational scanning methods, which allow for many thousands of mutations to be examined in parallel. Furthermore, these high-throughput mutagenesis methods have been expanded to include mutations to non-canonical amino acids, returning peptide structure-activity relationships with unprecedented depth and detail. These developments are timely, as the insights they provide can guide the optimization of de novo cyclic peptides, a promising new modality for chemical probes and therapeutic agents.
Highlights
Interactions between proteins are essential for the working of the cell (Rual et al, 2005)
These peptide folding and binding reactions are widespread in biology (Tompa et al, 2014; Yan et al, 2016) and are especially enriched in eukaryotes and in proteins associated with disease (Uversky et al, 2008, 2014)
Mutant peptides have been tested by one-at-a-time synthesis and biophysical analysis
Summary
Interactions between proteins are essential for the working of the cell (Rual et al, 2005). Backbone interactions are an important component of folding and binding, as these govern chain dynamics, secondary structure formation, and, occasionally, direct backbone Hbonding with the partner protein These interactions are challenging to study using canonical mutations. With access to non-canonical amino acids, many more conservative side-chain mutations are possible This is useful when mutation to alanine would be too destabilizing for the method in question or the role of a particular functional group or aliphatic carbon is to be studied. Mutating amides involved in inter-chain H-bonding produced a similar destabilization to those involved in intra-chain helix formation (Bachmann et al, 2011) These studies, much like one-at-a-time canonical mutagenesis, suffer from time-intensive peptide synthesis and purification, followed by low-throughput biophysical data collection. Mutational scans with greater depth and coverage require a wholly different experimental approach
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