RESUMEN
Biological membranes display distinct domains that organize membrane proteins and signaling molecules to facilitate efficient and reliable signaling. The organization of rhodopsin, a G protein-coupled receptor, in native rod outer segment disc membranes was investigated by atomic force microscopy. Atomic force microscopy revealed that rhodopsin is arranged into domains of variable size, which we refer to herein as nanodomains, in native membranes. Quantitative analysis of 150 disc membranes revealed that the physical properties of nanodomains are conserved in humans and mice and that the properties of individual disc membranes can be variable. Examining the variable properties of disc membranes revealed some of the factors contributing to the size of rod outer segment discs and the formation of nanodomains in the membrane. The diameter of rod outer segment discs was dependent on the number of rhodopsin molecules incorporated into the membrane but independent of the spatial density of rhodopsin. The number of nanodomains present in a single disc was also dependent on the number of rhodopsin molecules incorporated into the membrane. The size of the nanodomains was largely independent of the number or spatial density of rhodopsin in the membrane.
Asunto(s)
Membrana Celular/metabolismo , Microdominios de Membrana/metabolismo , Rodopsina/metabolismo , Segmento Externo de la Célula en Bastón/metabolismo , Animales , Membrana Celular/química , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Masculino , Microdominios de Membrana/química , Ratones Endogámicos C57BL , Microscopía de Fuerza Atómica , Modelos Moleculares , Datos de Secuencia Molecular , Nanoestructuras/química , Estructura Terciaria de Proteína , Rodopsina/químicaRESUMEN
Membrane proteins are embedded in lipid bilayers and facilitate the communication between the external environment and the interior of the cell. This communication is often mediated by the binding of ligands to the membrane protein. Understanding the nature of the interaction between a ligand and a membrane protein is required to both understand the mechanism of action of these proteins and for the development of novel pharmacological drugs. The highly hydrophobic nature of membrane proteins and the requirement of a lipid bilayer for native function have hampered the structural and molecular characterizations of these proteins under physiologically relevant conditions. Atomic force microscopy offers a solution to studying membrane proteins and their interactions with ligands under physiologically relevant conditions and can provide novel insights about the nature of these critical molecular interactions that facilitate cellular communication. In this review, we provide an overview of the atomic force microscopy technique and discuss its application in the study of a variety of questions related to the interaction between a membrane protein and a ligand. This article is part of a Special Issue entitled: Structural and biophysical characterization of membrane protein-ligand binding.
Asunto(s)
Proteínas de la Membrana/metabolismo , Microscopía de Fuerza Atómica/métodos , Ligandos , Proteínas de la Membrana/química , Sondas Moleculares , Unión ProteicaRESUMEN
Interdigitated electrode (IDE) arrays with nanometer-scale gaps have been utilized to enhance the sensitivity of affinity-based detection. The geometry of nanogap IDEs was first optimized on the basis of simulations of the electric field and current density. It was determined that the gap (G) between the electrodes was the most important geometric parameter in determining the distribution and strength of the electric field and the current density compared to the width (W) and height (H) of the IDEs. Several devices were materialized and analyzed for their sensitivity to the electrochemical environment using faradic electrochemical impedance spectroscopy (EIS) as the detection technique. Nanogap optimized IDEs were then employed as biosensors for the label-free, affinity-based detection of antitissue transglutaminase antibodies (αtTG-Abs), a biomarker for the detection of autoimmune disorder celiac sprue, triggered by ingesting gluten. The label-free biosensor assay was found to be less sensitive compared to on-chip ELISA. Gold nanoparticles (GNPs) were then employed to improve the sensitivity of the nanogap IDE-based biosensor. With GNPs, the transducer sensitivity increased by 350% over that of label-free detection. The suitability of nanogap IDEs as biosensor transducers for EIS in label-free and GNP-labeled formats was established. The immunobiosensor assay detection sensitivity with the GNPs was found comparable to ELISA.
Asunto(s)
Técnicas Biosensibles , Electrodos , Nanopartículas del Metal , Autoanticuerpos/análisis , Ensayo de Inmunoadsorción Enzimática , Oro , Transglutaminasas/inmunologíaRESUMEN
Three-dimensional interdigitated electrodes (IDEs) have been investigated as sensing elements for biosensors. Electric field and current density were simulated in the vicinity of these electrodes as a function of the electrode width, gap, and height to determine the optimum geometry. Both the height and the gap between the electrodes were found to have significant effect on the magnitude and distribution of the electric field and current density near the electrode surface, while the width of the electrodes was found to have a smaller effect on field strength and current density. IDEs were fabricated based on these simulations and their performance tested by detecting C-reactive protein (CRP), a stress-related protein and an important biomarker for inflammation, cardiovascular disease risk indicator, and postsurgical recuperation. CRP-specific antibodies were immobilized on the electrode surface and the formation of an immunocomplex (IC) with CRP was monitored. Electrochemical impedance spectroscopy (EIS) was employed as the detection technique. EIS data at various concentrations (1 pg/mL to 10 microg/mL) of CRP spiked in buffer or diluted human serum was collected and fitted into an equivalent electrical circuit model. Change in resistance was found to be the parameter most sensitive to change in CRP concentration. The sensor response was linear from 0.1 ng/mL to 1 microg/mL in both buffer and 5% human serum samples. The CRP samples were validated using a commercially available ELISA for CRP detection. Hence, the viability of IDEs and EIS for the detection of serum biomarkers was established without using labeled or probe molecules.