A Chemical Mutagenesis Approach to Insert Post-translational Modifications in Aggregation-Prone Proteins

Abstract

Neurodegenerative diseases are a class of disorders linked to the formation in the nervous system of fibrillar protein aggregates called amyloids. This aggregation process is affected by a variety of post-translational modifications, whose specific mechanisms are not fully understood yet. Emerging chemical mutagenesis technology is currently striving to address the challenge of introducing protein post-translational modifications, while maintaining the stability and solubility of the proteins during the modification reaction. Several amyloidogenic proteins are highly aggregationprone, and current modification procedures can lead to unexpected precipitation of these proteins, affecting their yield and downstream characterization. Here, we present a method for maintaining amyloidogenic protein solubility during chemical mutagenesis. As proof-of-principle, we applied our method to mimic the phosphorylation of serine-26 and the acetylation of lysine-28 of the 40-residue long variant of amyloid-β peptide, whose aggregation is linked to Alzheimer's disease.

Kinetic Resolution of Epimeric Proteins Enables Stereoselective Chemical Mutagenesis

“…21,22 The same strategy has also been applied to probe the effect of PTMs in modulating amyloid-β peptide aggregation, which plays a role in Alzheimer's disease. 23 Non-natural amino acids that can be installed via Dha are also beneficial for protein engineering efforts; incorporation of nonstandard functional groups into active sites 24 and binding pockets have been exploited to probe the role of hydrogen bonding in catalysis, 25 to improve substrate recognition in enzymes 26 and antibodies, 27 and to generate de novo binding proteins, 16 among others.…”

“…Chemical mutagenesis via Dha has enabled a range of applications. For example, designer chromatin-bearing biochemically relevant PTMs and their analogues have been generated by functionalizing Dha-containing histones; such investigations have furthered the understanding of how these PTMs are interpreted and edited. ,,, Similarly, mechanistic studies on the site-specific incorporation of phosphorylated Ser and Thr analogues into protein kinases have highlighted the chemical and geometrical features of phosphorylation and kinase activation in regulatory loops. , The same strategy has also been applied to probe the effect of PTMs in modulating amyloid-β peptide aggregation, which plays a role in Alzheimer’s disease . Non-natural amino acids that can be installed via Dha are also beneficial for protein engineering efforts; incorporation of nonstandard functional groups into active sites and binding pockets have been exploited to probe the role of hydrogen bonding in catalysis, to improve substrate recognition in enzymes and antibodies, and to generate de novo binding proteins, among others.…”

Kinetic Resolution of Epimeric Proteins enables Stereoselective Chemical Mutagenesis

“…20,21 The same strategy has also been applied to probe the effect of PTMs in modulating amyloid-β peptide aggregation, which plays a role in Alzheimer's disease. 22 Non-natural amino acids that can be installed via Dha are also beneficial for protein engineering efforts; incorporation of non-standard functional groups into https://doi.org/10.26434/chemrxiv-2023-8099f ORCID: https://orcid.org/0000-0001-6701-0893 Content not peer-reviewed by ChemRxiv. License: CC BY-NC-ND 4.0 active sites 23 and binding pockets have been exploited to probe the role of hydrogen bonding in catalysis, 24 to improve substrate recognition in enzymes 25 and antibodies, 26 and to generate de novo binding proteins, 15 among others.…”