RESUMEN
Generating stable conditions for membrane proteins after extraction from their lipid bilayer environment is essential for subsequent characterization. Detergents are the most widely used means to obtain this stable environment; however, different types of membrane proteins have been found to require detergents with varying properties for optimal extraction efficiency and stability after extraction. The extraction profiles of several detergent types have been examined for membranes isolated from bacteria and yeast, and for a set of recombinant target proteins. The extraction efficiencies of these detergents increase at higher concentrations, and were shown to correlate with their respective CMC values. Two alkyl sugar detergents, octyl-ß-d-glucoside (OG) and 5-cyclohexyl-1-pentyl-ß-d-maltoside (Cymal-5), and a zwitterionic surfactant, N-decylphosphocholine (Fos-choline-10), were generally effective in the extraction of a broad range of membrane proteins. However, certain detergents were more effective than others in the extraction of specific classes of integral membrane proteins, offering guidelines for initial detergent selection. The differences in extraction efficiencies among this small set of detergents supports the value of detergent screening and optimization to increase the yields of targeted membrane proteins.
Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Detergentes/química , Proteínas Fúngicas/aislamiento & purificación , Glucósidos/química , Proteínas de la Membrana/aislamiento & purificación , Proteínas Bacterianas/química , Membrana Celular/química , Electroforesis en Gel de Poliacrilamida , Escherichia coli/química , Proteínas Fúngicas/química , Proteínas de la Membrana/química , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Pichia/química , Desnaturalización Proteica , Estabilidad Proteica , Solubilidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Tensoactivos/químicaRESUMEN
The aspartate biosynthetic pathway provides essential metabolites for many important biological functions, including the production of four essential amino acids. As this critical pathway is only present in plants and microbes, any disruptions will be fatal to these organisms. An early pathway enzyme, l-aspartate-ß-semialdehyde dehydrogenase, produces a key intermediate at the first branch point of this pathway. Developing potent and selective inhibitors against several orthologs in the l-aspartate-ß-semialdehyde dehydrogenase family can serve as lead compounds for antibiotic development. Kinetic studies of two small molecule fragment libraries have identified inhibitors that show good selectivity against l-aspartate-ß-semialdehyde dehydrogenases from two different bacterial species, Streptococcus pneumoniae and Vibrio cholerae, despite the presence of an identical constellation of active site amino acids in this homologous enzyme family. Structural characterization of enzyme-inhibitor complexes have elucidated different modes of binding between these structurally related enzymes. This information provides the basis for a structure-guided approach to the development of more potent and more selective inhibitors.