Probing Cascade complex composition and stability using native mass spectrometry techniques.
Methods Enzymol
; 616: 87-116, 2019.
Article
em En
| MEDLINE
| ID: mdl-30691656
ABSTRACT
Adaptive prokaryotic immune systems rely on clustered regularly interspaced short palindromic repeats and their associated genes to provide the components necessary to clear infection by foreign genetic elements. These immune systems are based on highly specific nucleases that bind DNA or RNA and, upon sequence recognition, degrade the bound nucleic acid. Because of their specificity, CRISPR-Cas systems are being co-opted to edit genes in eukaryotic cells. While the general function of these systems is well understood, an understanding of mechanistic details to facilitate engineering and application to this new arena remains a topic of intense study. Here, we present two methods that have been successfully used to study the structure and mechanism of the Type IE CRISPR system, Cascade, from Escherichia coli. We provide the protocol for a typical native mass spectrometry experiment which, because it allows for analysis of a protein complex without disruption of the noncovalent interactions within the complex, can be used to determine complex composition, architecture, and relative affinity between subunits. We, also, provide the protocol for intact protein hydrogen-deuterium exchange mass spectrometry, which provides insight into the overall conformational stability of the complex and changes in complex stability based on conditions such as substrate binding. Investigating the solution-phase structure, stability, and dynamics of these complexes improves the overall understanding of the mechanism facilitating engineered adjustments to function or utility.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Bactérias
/
Proteínas de Bactérias
/
Proteínas Associadas a CRISPR
Idioma:
En
Revista:
Methods Enzymol
Ano de publicação:
2019
Tipo de documento:
Article