Combining non-canonical amino acid mutagenesis and native chemical ligation for multiply modifying proteins: A case study of α-synuclein post-translational modifications.
Methods
; 218: 101-109, 2023 10.
Article
en En
| MEDLINE
| ID: mdl-37549799
ABSTRACT
The Parkinson's disease associated protein α-synuclein (αS) has been found to contain numerous post-translational modifications (PTMs), in both physiological and pathological states. One PTM site of particular interest is serine 87, which is subject to both O-linked ß-N-acetylglucosamine (gS) modification and phosphorylation (pS), with αS-pS87 enriched in Parkinson's disease. An often-overlooked aspect of these PTMs is their effect on the membrane-binding properties of αS, which are important to its role in regulating neurotransmitter release. Here, we show how one can study these effects by synthesizing αS constructs containing authentic PTMs and labels for single molecule fluorescence correlation spectroscopy measurements. We synthesize αS-gS87 and αS-pS87 by combining native chemical ligation with genetic code expansion approaches. We introduce the fluorophore by a click reaction with a non-canonical amino acid. Beyond the specific problem of PTM effects on αS, our studies highlight the value of this combination of methods for multiply modifying proteins.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Enfermedad de Parkinson
/
Alfa-Sinucleína
Límite:
Humans
Idioma:
En
Revista:
Methods
Asunto de la revista:
BIOQUIMICA
Año:
2023
Tipo del documento:
Article
País de afiliación:
Estados Unidos