Dominik Ruppelt, Elena L M Ackermann, Tom Robinson, Claudia Steinem
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引用次数: 0
摘要
质子在脂膜上的转运是构成生物体的最基本反应之一。然而,在体外,由于质子浓度、隔室大小、非搅拌层以及缓冲交换和缓冲能力的限制,质子转运反应的研究可能非常具有挑战性。在本研究中,我们开发了一种基于微流控捕获的巨型囊泡的质子渗透检测方法,囊泡中含有对 pH 值敏感的染料吡喃,从而解决了其中的一些难题。pH值快速变化时的时间分辨荧光成像使我们能够研究由通道或载体介导的H+渗透。具体来说,我们比较了质子传输速率与通道γ-呲啶 D 和质子载体羰基氰-间氯苯腙(CCCP)的不同质子梯度的函数关系。我们的研究结果证明了该检测方法在监测质子转运反应以及区分通道类和载体类机制方面的功效。这一突破性成果使我们得以阐明最近发现的天然纤维肽 lugdunin 的质子渗透机制的神秘模式。
Assessing the mechanism of facilitated proton transport across GUVs trapped in a microfluidic device.
Proton transport across lipid membranes is one of the most fundamental reactions that make up living organisms. In vitro, however, the study of proton transport reactions can be very challenging due to limitations imposed by proton concentrations, compartment size, and unstirred layers as well as buffer exchange and buffer capacity. In this study, we have developed a proton permeation assay based on the microfluidic trapping of giant vesicles enclosing the pH-sensitive dye pyranine to address some of these challenges. Time-resolved fluorescence imaging upon a rapid pH shift enabled us to investigate the facilitated H+ permeation mediated by either a channel or a carrier. Specifically, we compared the proton transport rates as a function of different proton gradients of the channel gramicidin D and the proton carrier carbonyl cyanide-m-chlorophenyl hydrazone. Our results demonstrate the efficacy of the assay in monitoring proton transport reactions and distinguishing between a channel-like and a carrier-like mechanism. This groundbreaking result enabled us to elucidate the enigmatic mode of the proton permeation mechanism of the recently discovered natural fibupeptide lugdunin.
期刊介绍:
BJ publishes original articles, letters, and perspectives on important problems in modern biophysics. The papers should be written so as to be of interest to a broad community of biophysicists. BJ welcomes experimental studies that employ quantitative physical approaches for the study of biological systems, including or spanning scales from molecule to whole organism. Experimental studies of a purely descriptive or phenomenological nature, with no theoretical or mechanistic underpinning, are not appropriate for publication in BJ. Theoretical studies should offer new insights into the understanding ofexperimental results or suggest new experimentally testable hypotheses. Articles reporting significant methodological or technological advances, which have potential to open new areas of biophysical investigation, are also suitable for publication in BJ. Papers describing improvements in accuracy or speed of existing methods or extra detail within methods described previously are not suitable for BJ.