Structure-based biophysical analysis of the interaction of rhodopsin with G protein and arrestin.
Methods Enzymol
; 556: 563-608, 2015.
Article
em En
| MEDLINE
| ID: mdl-25857800
ABSTRACT
In this chapter, we describe a set of complementary techniques that we use to study the activation of rhodopsin, a G protein-coupled receptor (GPCR), and its functional interactions with G protein and arrestin. The protein reagents used for these studies come from native disc membranes or heterologous expression, and G protein and arrestin are often replaced with less complex synthetic peptides derived from key interaction sites of these binding partners (BPs). We first report on our approach to protein X-ray crystallography and describe how protein crystals from native membranes are obtained. The crystal structures provide invaluable resolution, but other techniques are required to assess the dynamic equilibria characteristic for active GPCRs. The simplest approach is "Extra Meta II," which uses UV/Vis absorption spectroscopy to monitor the equilibrium of photoactivated states. Site-specific information about the BPs (e.g., arrestin) is added by fluorescence techniques employing mutants labeled with reporter groups. All functional changes in both the receptor and interacting proteins or peptides are seen with highest precision using Fourier transform infrared (FTIR) difference spectroscopy. In our approach, the lack of site-specific information in FTIR is overcome by parallel molecular dynamics simulations, which are employed to interpret the results and to extend the timescale down to the range of conformational substates.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Rodopsina
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Proteínas de Ligação ao GTP
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Arrestina
/
Mapeamento de Interação de Proteínas
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
Methods Enzymol
Ano de publicação:
2015
Tipo de documento:
Article