Real-time monitoring of membrane-protein reconstitution by isothermal titration calorimetry

Analytical Chemistry
Nadin JahnkeSandro Keller

Abstract

Phase diagrams offer a wealth of thermodynamic information on aqueous mixtures of bilayer-forming lipids and micelle-forming detergents, providing a straightforward means of monitoring and adjusting the supramolecular state of such systems. However, equilibrium phase diagrams are of very limited use for the reconstitution of membrane proteins because of the occurrence of irreversible, unproductive processes such as aggregation and precipitation that compete with productive reconstitution. Here, we exemplify this by dissecting the effects of the K(+) channel KcsA on the process of bilayer self-assembly in a mixture of Escherichia coli polar lipid extract and the nonionic detergent octyl-β-d-glucopyranoside. Even at starting concentrations in the low micromolar range, KcsA has a tremendous impact on the supramolecular organization of the system, shifting the critical lipid/detergent ratios at the onset and completion of vesicle formation by more than 2-fold. Thus, equilibrium phase diagrams obtained for protein-free lipid/detergent mixtures would be misleading when used to guide the reconstitution process. To address this issue, we demonstrate that, even under such nonequilibrium conditions, high-sensitivity isothermal titration ...Continue Reading

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Citations

Oct 16, 2015·Biochimica Et Biophysica Acta·Watson LohKam Chiu Tam
Dec 20, 2014·Methods : a Companion to Methods in Enzymology·Thomas H Scheuermann, Chad A Brautigam
Jul 18, 2018·Faraday Discussions·Andreas Horner, Peter Pohl
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Mar 11, 2015·Angewandte Chemie·Erik FrotscherSandro Keller
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Oct 17, 2017·The Journal of Physical Chemistry. B·Aaron J WolfeLiviu Movileanu
Mar 30, 2018·The Journal of Physical Chemistry Letters·Aaron J WolfeLiviu Movileanu
Sep 2, 2014·Journal of the American Chemical Society·Jana BroeckerSandro Keller
Oct 24, 2018·Langmuir : the ACS Journal of Surfaces and Colloids·Na ZhangYilin Wang

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Methods Mentioned

BETA
isothermal
light scattering
fluorescence correlation spectroscopy
FCS

Software Mentioned

NITPIC
WinEDR

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