RESUMO
We present an elegant synthesis and reconstitution approach for functional studies of voltage responsive membrane proteins. For such studies, we propose a planar architecture of an S-layer-supported lipid membrane as a suitable matrix for presenting unmodified membrane protein species, and here we focus on the voltage-dependent anion channel (VDAC) from human mitochondria. The presented cell-free strategy, in which VDAC proteins are synthesized in bacterial cell lysate, into a membrane structure, offers a great advantage in the study of such subtle membrane proteins over the conventional, cell-based synthesis approach in terms of reproducibility. The material-assay combination is superior over cell-culture related synthesis and purification approaches as here we bypass by a one-step synthesis procedure the complex cell culture, and expression and purification endeavours, and, moreover, the protein of interest never has to be detergent solubilized and had been synthesized de novo. We provide here a detailed description from the all over procedure and our first results, describing in detail the cell-free synthesis and robustness of such a material-assay combination: functional VDAC protein species embedded in a planar membrane architecture and ready for electrochemical characterization.
Assuntos
Bicamadas Lipídicas/química , Lipídeos/química , Proteínas de Membrana/química , Mitocôndrias/química , Ribossomos/química , Canais de Ânion Dependentes de Voltagem/síntese química , Técnicas Eletroquímicas , Humanos , Imidas/química , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Fosfatidiletanolaminas/química , Propilaminas/química , Canais de Ânion Dependentes de Voltagem/química , Canais de Ânion Dependentes de Voltagem/metabolismoRESUMO
The most important aspect of synthetic lipid membrane architectures is their ability to study functional membrane-active peptides and membrane proteins in an environment close to nature. Here, we report on the generation and performance of a biomimetic platform, the S-layer supported lipid membrane (SsLM), to investigate the structural and electrical characteristics of the membrane-active peptide gramicidin and the transmembrane protein α-hemolysin in real-time using a quartz crystal microbalance with dissipation monitoring in combination with electrochemical impedance spectroscopy. A shift in membrane resistance is caused by the interaction of α-hemolysin and gramicidin with SsLMs, even if only an attachment onto, or functional channels through the lipid membrane, respectively, are formed. Moreover, the obtained results did not indicate the formation of functional α-hemolysin pores, but evidence for functional incorporation of gramicidin into this biomimetic architecture is provided.
Assuntos
Biomimética , Peptídeos/metabolismo , Lipossomas Unilamelares/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Espectroscopia Dielétrica , Gramicidina/química , Gramicidina/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/metabolismo , Peptídeos/química , Técnicas de Microbalança de Cristal de Quartzo , Lipossomas Unilamelares/químicaRESUMO
We present a rapid and robust technique for the sampling of membrane-associated proteins from the surface of a single, live cell and their subsequent deposition onto a solid-supported lipid bilayer. As a proof of principle, this method has been used to extract green fluorescent protein (EGFP) labelled K-ras proteins located at the inner leaflet of the plasma membrane of colon carcinoma cells and to transfer them to an S-layer supported lipid bilayer system. The technique is non-destructive, meaning that both the cell and proteins are intact after the sampling operation, offering the potential for repeated measurements of the same cell of interest. This system provides the ideal tool for the investigation of cellular heterogeneity, as well as a platform for the investigation of rare cell types such as circulating tumour cells.
Assuntos
Membrana Celular/química , Proteínas de Membrana/isolamento & purificação , Análise de Célula Única/instrumentação , Linhagem Celular Tumoral , Neoplasias do Colo/química , Proteínas de Fluorescência Verde/isolamento & purificação , Humanos , Proteínas ras/isolamento & purificaçãoRESUMO
Methods for organizing functional materials at the nanometer scale are essential for the development of novel fabrication techniques. One of the most relevant areas of research in nanobiotechnology concerns technological utilization of self-assembly systems, wherein molecules spontaneously associate into reproducible supramolecular structures. For this purpose, the laccase of Bacillus halodurans C-125 was immobilized on the S-layer lattice formed by SbpA of Lysinibacillus sphaericus CCM 2177 either by (i) covalent linkage of the enzyme to the natural protein self-assembly system or (ii) by construction of a fusion protein comprising the S-layer protein and the laccase. The laccase and the S-layer fusion protein were produced heterologously in Escherichia coli. After isolation and purification, the properties of the proteins, as well as the specific activity of the enzyme moiety, were investigated. Interestingly, the S-layer part confers a much higher solubility on the laccase as observed for the sole enzyme. Comparative spectrophotometric measurements of the enzyme activity revealed similar but significantly higher values for rLac and rSbpA/Lac in solution compared to the immobilized state. However, rLac covalently linked to the SbpA monolayer yielded a four to five time higher enzymatic activity than rSbpA/Lac immobilized on a solid support. Combined quartz crystal microbalance with dissipation monitoring (QCM-D) and electrochemical measurements (performed in an electrochemical QCM-D cell) revealed that rLac immobilized on the SbpA lattice had an approximately twofold higher enzymatic activity compared to that obtained with the fusion protein.