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Functional delivery of a membrane protein into oocyte membranes using bicelles.

ABSTRACT: Voltage-gated potassium channel modulatory membrane protein KCNE3 was overexpressed and purified into both micelles and bicelles. Remarkably, microinjection of KCNE3 in bicelles into Xenopus oocytes resulted in functional co-assembly with the human KCNQ1 channel expressed therein. Microinjection of LMPC micelles containing KCNE3 did not result in channel modulation, indicating that bicelles sometimes succeed at delivering a membrane protein into a cellular membrane when classical micelles fail. Backbone NMR resonance assignments were completed for KCNE3 in both bicelles and LMPC, indicating that the secondary structure distribution in KCNE3's N-terminus and transmembrane domains exhibits only modest differences from that of KCNE1, even though these KCNE family members have very different effects on KCNQ1 channel function.

SUBMITTER: Kang C

PROVIDER: S-EPMC2811756 | biostudies-other | 2010 Feb

REPOSITORIES: biostudies-other

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Functional delivery of a membrane protein into oocyte membranes using bicelles.

Kang Congbao C Vanoye Carlos G CG Welch Richard C RC Van Horn Wade D WD Sanders Charles R CR

Biochemistry 20100201 4

Voltage-gated potassium channel modulatory membrane protein KCNE3 was overexpressed and purified into both micelles and bicelles. Remarkably, microinjection of KCNE3 in bicelles into Xenopus oocytes resulted in functional co-assembly with the human KCNQ1 channel expressed therein. Microinjection of LMPC micelles containing KCNE3 did not result in channel modulation, indicating that bicelles sometimes succeed at delivering a membrane protein into a cellular membrane when classical micelles fail. ...[more]

PMID: 20044833

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Project description:Membrane proteins are essential in the exchange processes of cells. In spite of great breakthrough in soluble proteins studies, membrane proteins structures, functions and interactions are still a challenge because of the difficulties related to their hydrophobic properties. Most of the experiments are performed with detergent-solubilized membrane proteins. However widely used micellar systems are far from the biological two-dimensions membrane. The development of new biomimetic membrane systems is fundamental to tackle this issue.We present an original approach that combines the Fluorescence Recovery After fringe Pattern Photobleaching technique and the use of a versatile sponge phase that makes it possible to extract crucial informations about interactions between membrane proteins embedded in the bilayers of a sponge phase. The clear advantage lies in the ability to adjust at will the spacing between two adjacent bilayers. When the membranes are far apart, the only possible interactions occur laterally between proteins embedded within the same bilayer, whereas when membranes get closer to each other, interactions between proteins embedded in facing membranes may occur as well.After validating our approach on the streptavidin-biotinylated peptide complex, we study the interactions between two membrane proteins, MexA and OprM, from a Pseudomonas aeruginosa efflux pump. The mode of interaction, the size of the protein complex and its potential stoichiometry are determined. In particular, we demonstrate that: MexA is effectively embedded in the bilayer; MexA and OprM do not interact laterally but can form a complex if they are embedded in opposite bilayers; the population of bound proteins is at its maximum for bilayers separated by a distance of about 200 A, which is the periplasmic thickness of Pseudomonas aeruginosa. We also show that the MexA-OprM association is enhanced when the position and orientation of the protein is restricted by the bilayers. We extract a stoichiometry for the complex that exhibits a strong pH dependance: from 2 to 6 MexA per OprM trimer when the pH decreases from 7.5 to 5.5.Our technique allows to study membrane protein associations in a membrane environment. It provides some challenging information about complexes such as geometry and stoichiometry.

Dataset Information

Functional delivery of a membrane protein into oocyte membranes using bicelles.

Publications

Functional delivery of a membrane protein into oocyte membranes using bicelles.

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