Chemical Modification Methods for Protein Misfolding Studies
Protein misfolding is the basis of various human diseases, including Parkinson’s disease, Alzheimer’s disease and Type 2 diabetes. When a protein misfolds, it adopts the wrong three dimensional structures that are dysfunctional and sometime pathological. Little structural details are known about this misfolding phenomenon due to the lack of characterization tools. Our group previously demonstrated that a thioamide, a single atom substitution of the peptide bond, could serve as a minimalist fluorescence quencher. In the current study, we showed the development of protein semi-synthesis strategies for the incorporation of thioamides into full-length proteins for misfolding studies. We adopted the native chemical ligation (NCL) method between a C-terminal thioester fragment and an N-terminal Cys fragment. We first devised strategies for the synthesis of thioamide-containing peptide thioesters as NCL substrates, and demonstrated their applications in generating a thioamide/Trp-dually labeled α-synuclein (αS), which was subsequently used in a proof-of-concept misfolding study. To remove the constraint of a Cys at the ligation site, we explored traceless ligation methods that desulfurized Cys into Ala, or β- and γ- thiol analogs into native amino acids after ligation in the presence of thioamides. We further demonstrated that selective deselenization could be achieved in the presence of both Cys residues and thioamides, expanding the scope of thioamide incorporation through traceless ligation to proteins with native Cys. Finally, we showed that hemiselenide protected selenocysteines (Sec) can be incorporated onto the protein N-terminus through chemoenzymatic modification by aminoacyl transferase (AaT) as ligation handles. Further developments are underway in our laboratory to expand the AaT substrate scope for β- and γ- thiol amino acid analogs. In summary, we developed a set of methods that allowed the incorporation of thioamide probes into full-length protein, which enabled the application of this minimalist probe in protein misfolding studies.