RESUMEN
The insulin-responsive 12-transmembrane transporter GLUT4 changes conformation between an inward-open state and an outward-open state to actively facilitate cellular glucose uptake. Because of the difficulties of generating conformational mAbs against complex and highly conserved membrane proteins, no reliable tools exist to measure GLUT4 at the cell surface, follow its trafficking, or detect the conformational state of the protein. Here we report the isolation and characterization of conformational mAbs that recognize the extracellular and intracellular domains of GLUT4, including mAbs that are specific for the inward-open and outward-open states of GLUT4. mAbs against GLUT4 were generated using virus-like particles to present this complex membrane protein in its native conformation and using a divergent host species (chicken) for immunization to overcome immune tolerance. As a result, the isolated mAbs recognize conformational epitopes on native GLUT4 in cells, with apparent affinities as high as 1 pM and with specificity for GLUT4 across the human membrane proteome. Epitope mapping using shotgun mutagenesis alanine scanning across the 509 amino acids of GLUT4 identified the binding epitopes for mAbs specific for the states of GLUT4 and allowed the comprehensive identification of the residues that functionally control the GLUT4 inward-open and outward-open states. The mAbs identified here will be valuable molecular tools for monitoring GLUT4 structure, function, and trafficking, for differentiating GLUT4 conformational states, and for the development of novel therapeutics for the treatment of diabetes.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Transportador de Glucosa de Tipo 4/inmunología , Transportador de Glucosa de Tipo 4/metabolismo , Vacunas de Partículas Similares a Virus/inmunología , Animales , Pollos , Mapeo Epitopo , Transportador de Glucosa de Tipo 4/química , Transportador de Glucosa de Tipo 4/genética , Células HEK293 , Humanos , Virus de la Leucemia Murina/genética , Modelos Moleculares , Dominios Proteicos , Vacunas de Partículas Similares a Virus/químicaRESUMEN
In this article, we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization.
Asunto(s)
Proteínas Fluorescentes Verdes/química , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/química , Secuencia de Aminoácidos , Amiloide/química , Bioensayo , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Calor , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Datos de Secuencia Molecular , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Electricidad EstáticaRESUMEN
To better understand how detergents disrupt enveloped viruses, we monitored the biophysical stability of murine leukemia virus (MLV) virus-like particles (VLPs) against a panel of commonly used detergents using real-time biosensor measurements. Although exposure to many detergents, such as Triton X-100 and Empigen, results in lysis of VLP membranes, VLPs appeared resistant to complete membrane lysis by a significant number of detergents, including Tween 20, Tween 80, Lubrol, and Saponin. VLPs maintained their structural integrity after exposure to Tween 20 at concentrations up to 500-fold above its CMC. Remarkably, VLPs containing immature cores composed of unprocessed (uncleaved) Gag polyprotein were significantly more resistant to detergent lysis than VLPs with mature cores. Although the maturity of retroviral Gag is known to influence the stability of the protein core structure itself, our studies suggest that the maturity of the Gag core also influences the stability of the lipid bilayer surrounding the core.