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1.
Biophys J ; 112(1): 99-108, 2017 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-28076820

RESUMO

Ion conduction across the cellular membrane requires the simultaneous opening of activation and inactivation gates of the K+ channel pore. The bacterial KcsA channel has served as a powerful system for dissecting the structural changes that are related to four major functional states associated with K+ gating. Yet, the direct observation of the full gating cycle of KcsA has remained structurally elusive, and crystal structures mimicking these gating events require mutations in or stabilization of functionally relevant channel segments. Here, we found that changes in lipid composition strongly increased the KcsA open probability. This enabled us to probe all four major gating states in native-like membranes by combining electrophysiological and solid-state NMR experiments. In contrast to previous crystallographic views, we found that the selectivity filter and turret region, coupled to the surrounding bilayer, were actively involved in channel gating. The increase in overall steady-state open probability was accompanied by a reduction in activation-gate opening, underscoring the important role of the surrounding lipid bilayer in the delicate conformational coupling of the inactivation and activation gates.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ativação do Canal Iônico , Bicamadas Lipídicas/metabolismo , Canais de Potássio/química , Canais de Potássio/metabolismo , Cardiolipinas/metabolismo , Membrana Celular/metabolismo , Modelos Moleculares , Conformação Proteica
2.
Proc Natl Acad Sci U S A ; 111(52): 18607-12, 2014 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-25512535

RESUMO

A major obstacle in the study of membrane proteins is their solubilization in a stable and active conformation when using detergents. Here, we explored a detergent-free approach to isolating the tetrameric potassium channel KcsA directly from the membrane of Escherichia coli, using a styrene-maleic acid copolymer. This polymer self-inserts into membranes and is capable of extracting membrane patches in the form of nanosize discoidal proteolipid particles or "native nanodiscs." Using circular dichroism and tryptophan fluorescence spectroscopy, we show that the conformation of KcsA in native nanodiscs is very similar to that in detergent micelles, but that the thermal stability of the protein is higher in the nanodiscs. Furthermore, as a promising new application, we show that quantitative analysis of the co-isolated lipids in purified KcsA-containing nanodiscs allows determination of preferential lipid-protein interactions. Thin-layer chromatography experiments revealed an enrichment of the anionic lipids cardiolipin and phosphatidylglycerol, indicating their close proximity to the channel in biological membranes and supporting their functional relevance. Finally, we demonstrate that KcsA can be reconstituted into planar lipid bilayers directly from native nanodiscs, which enables functional characterization of the channel by electrophysiology without first depriving the protein of its native environment. Together, these findings highlight the potential of the use of native nanodiscs as a tool in the study of ion channels, and of membrane proteins in general.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Bicamadas Lipídicas/química , Nanoestruturas/química , Canais de Potássio/química , Canais de Potássio/isolamento & purificação , Streptomyces lividans/química , Proteínas de Bactérias/genética , Cardiolipinas/química , Escherichia coli/genética , Fosfatidilgliceróis/química , Canais de Potássio/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Streptomyces lividans/genética
3.
Lab Chip ; 14(23): 4461-4, 2014 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-25284632

RESUMO

We report a wafer-scale fabrication process for the production of glass-FEP-glass microdevices using UV-curable adhesive (NOA81) as gluing material, which is applied using a novel "spin & roll" approach. Devices are characterized for the uniformity of the gluing layer, presence of glue in the microchannels, and alignment precision. Experiments on lipid bilayers with electrophysiological recordings using a model pore-forming polypeptide are demonstrated.


Assuntos
Vidro/química , Bicamadas Lipídicas/química , Técnicas Analíticas Microfluídicas/instrumentação , Politetrafluoretileno/análogos & derivados , Adesivos , Eletrofisiologia , Desenho de Equipamento , Politetrafluoretileno/química
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