PT  - JOURNAL ARTICLE
AU  - Ying Ge
AU  - Athina Masoura
AU  - Jingzhou Yang
AU  - Francesco A. Aprile
TI  - A Chemical Mutagenesis Approach to Insert Post-Translational Modifications in Aggregation-Prone Proteins
AID  - 10.1101/2022.01.28.478135
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.01.28.478135
4099  - http://biorxiv.org/content/early/2022/01/28/2022.01.28.478135.short
4100  - http://biorxiv.org/content/early/2022/01/28/2022.01.28.478135.full
AB  - 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 proteins stable and soluble during the modification reaction. Several amyloidogenic proteins are highly aggregation-prone, and current modification procedures lead to unexpected precipitation of these proteins, affecting their yield and downstream characterization. Here, we present a method for maintaining amyloidogenic proteins soluble during chemical mutagenesis. As proof-of-principle, we applied our method to mimic the phosphorylation of the serine 26 and the acetylation of the lysine 28 of the 40-residue long variant of amyloid-β peptide, whose aggregation is linked to Alzheimer’s disease.Competing Interest StatementThe authors have declared no competing interest.ABBREVIATIONSAβamyloid-βAcKacetyllysineAc(S)Kthiol-containing mimic of AcKADAlzheimer’s diseaseAPPamyloid precursor proteinBMEβ-mercaptoethanolDBHDA2,5-dibromohexanediamideDTTdithiothreitolEDTAethylenediaminetetraacetic acidIPTGisopropyl β-D-1-thiogalactopyranosideLC-ESliquid chromatography electrosprayMSmass spectrometrypCphosphocysteinepSphosphoserinePTMpost-translational modificationSDsilk domainSDS-PAGEsodium dodecyl sulphate–polyacrylamide gel electrophoresisSECsize-exclusion chromatographyTCEPtris(2-carboxyethyl)phosphineTEMTransmission electron microscopyTEVTobacco Etch VirusThTThioflavin T