Semisynthesis and Enzymatic Preparation of Post-translationally Modified α-Synuclein
Posttranslational modifications (PTMs) serve as molecular switches for regulating protein folding, function, and interactome and have been implicated in the misfolding and amyloid formation by several proteins linked to neurodegenerative diseases, including Alzheimer's and Parkinson's disease. Understanding the role of individual PTMs in protein misfolding and aggregation requires the preparation of site-specifically modified proteins, as well as the identification of the enzymes involved in regulating these PTMs. Recently, our group has pioneered the development of enzymatic, synthetic, and semisynthetic strategies that allow site-specific introduction of PTMs at single or multiple sites and generation of modified proteins in milligram quantities. In this chapter, we provide detailed description of enzymatic and semisynthetic strategies for the generation of the phosphorylated α-Synuclein (α-Syn) at S129, (pS129), which has been identified as a pathological hallmark of Parkinson's disease. The semisynthetic method described for generation of α-Syn-pS129 requires expertise with protein chemical ligation, but can be used to incorporate other PTMs (single or multiple) within the α-Syn C-terminus if desired. On the other hand, the in vitro kinase-mediated phosphorylation strategy does not require any special setup and is rather easy to apply, but its application is restricted to the generation of α-Syn_pS129. These methods have the potential to increase the availability of pure and homogenous modified α-Syn reagents, which may be used as standards in numerous applications, including the search for potential biomarkers of synucleinopathies.
Record created on 2016-03-04, modified on 2016-08-09