RESUMEN
Identifying stabilising variants of membrane protein targets is often required for structure determination. Our new computational pipeline, the Integral Membrane Protein Stability Selector (IMPROvER) provides a rational approach to variant selection by employing three independent approaches: deep-sequence, model-based and data-driven. In silico tests using known stability data, and in vitro tests using three membrane protein targets with 7, 11 and 16 transmembrane helices provided measures of success. In vitro, individual approaches alone all identified stabilising variants at a rate better than expected by random selection. Low numbers of overlapping predictions between approaches meant a greater success rate was achieved (fourfold better than random) when approaches were combined and selections restricted to the highest ranked sites. The mix of information IMPROvER uses can be extracted for any helical membrane protein. We have developed the first general-purpose tool for selecting stabilising variants of [Formula: see text]-helical membrane proteins, increasing efficiency and reducing workload. IMPROvER can be accessed at http://improver.ddns.net/IMPROvER/ .
Asunto(s)
Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ingeniería de Proteínas , Estabilidad Proteica , Programas Informáticos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Clostridium/química , Clostridium/genética , Simulación por Computador , Tranportador Equilibrativo 1 de Nucleósido/química , Tranportador Equilibrativo 1 de Nucleósido/genética , Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Modelos Moleculares , Conformación Proteica en Hélice alfa/genética , Desnaturalización Proteica , Pirofosfatasas/química , Pirofosfatasas/genética , Receptor de Hormona Paratiroídea Tipo 1/química , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Alineación de Secuencia , Análisis de Secuencia de Proteína , Homología Estructural de ProteínaRESUMEN
Integral membrane proteins have a critical role in fundamental biological processes; they are major drug targets and therefore of high research interest. Recombinant protein production is the first step in the protein tool generation for biochemical and biophysical studies. Here, we provide simplified protocols that facilitate the generation of high-quality virus and initial expression analysis for integral membrane protein targets utilizing the baculovirus-mediated expression system in insect cells. The protocol steps include generation of viruses, virus quality control, and initial expression trials utilizing standard commercial baculovirus vector systems and are exemplified for G protein-coupled receptor targets. The viral quality, quantity, and recombinant protein expression are evaluated by microscopy, flow cytometry, fluorimetry, and SDS-PAGE, using either covalently fused fluorescent proteins or co-expressed fluorescence markers. Moreover, integral membrane protein expression levels, approximate molecular mass, and stability can be evaluated from small-scale expression and purification trials.
Asunto(s)
Baculoviridae/genética , Clonación Molecular/métodos , Vectores Genéticos , Proteínas de la Membrana/genética , Spodoptera/citología , Animales , Baculoviridae/crecimiento & desarrollo , Reactores Biológicos , Técnicas de Cultivo de Célula/métodos , Línea Celular , Regulación Viral de la Expresión Génica , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/aislamiento & purificación , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Spodoptera/genética , Spodoptera/crecimiento & desarrollo , Spodoptera/metabolismo , Transducción Genética/métodos , Transfección/métodosRESUMEN
The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species1. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family2 and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation3-5. Because SUCNR1 senses succinate as an immunological danger signal6-which has relevance for diseases including ulcerative colitis, liver fibrosis7, diabetes and rheumatoid arthritis3,8-it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo.