Site-specific resolution of anionic residues in proteins using solid-state NMR spectroscopy.
J Biomol NMR
; 74(6-7): 355-363, 2020 Jul.
Article
en En
| MEDLINE
| ID: mdl-32514875
ABSTRACT
NMR spectroscopy is commonly used to infer site-specific acid dissociation constants (pKa) since the chemical shift is sensitive to the protonation state. Methods that probe atoms nearest to the functional groups involved in acid/base chemistry are the most sensitive for determining the protonation state. In this work, we describe a magic-angle-spinning (MAS) solid-state NMR approach to measure chemical shifts on the side chain of the anionic residues aspartate and glutamate. This method involves a combination of double quantum spectroscopy in the indirect dimension and REDOR dephasing to provide a sensitive and resolved view of these amino acid residues that are commonly involved in enzyme catalysis and membrane protein transport. To demonstrate the applicability of the approach, we carried out measurements using a microcrystalline soluble protein (ubiquitin) and a membrane protein embedded in lipid bilayers (EmrE). Overall, the resolution available from the double quantum dimension and confidence in identification of aspartate and glutamate residues from the REDOR filter make this method the most convenient for characterizing protonation states and deriving pKa values using MAS solid-state NMR.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas
/
Resonancia Magnética Nuclear Biomolecular
Tipo de estudio:
Prognostic_studies
Idioma:
En
Revista:
J Biomol NMR
Asunto de la revista:
BIOLOGIA MOLECULAR
/
DIAGNOSTICO POR IMAGEM
/
MEDICINA NUCLEAR
Año:
2020
Tipo del documento:
Article
País de afiliación:
Estados Unidos