TY - JOUR
T1 - Reconstitution of respiratory oxidases in membrane nanodiscs for investigation of proton-coupled electron transfer
AU - Näsvik Öjemyr, Linda
AU - Von Ballmoos, Christoph
AU - Gennis, Robert B.
AU - Sligar, Stephen G.
AU - Brzezinski, Peter
N1 - Funding Information:
These studies were supported by a grant from the Swedish Research Council (to P.B.) and, by grants HL 16101 (to R.B.G) and GM 33775 (to S.G.S.) from the National Institutes of Health. C.v.B. is supported by a fellowship from the Swiss National Science Foundation (SNF).
PY - 2012/3/9
Y1 - 2012/3/9
N2 - The function of membrane-bound transporters is commonly affected by the milieu of the hydrophobic, membrane-spanning part of the transmembrane protein. Consequently, functional studies of these proteins often involve incorporation into a native-like bilayer where the lipid components of the membrane can be controlled. The classical approach is to reconstitute the purified protein into liposomes. Even though the use of such liposomes is essential for studies of transmembrane transport processes in general, functional studies of the transporters themselves in liposomes suffer from several disadvantages. For example, transmembrane proteins can adopt two different orientations when reconstituted into liposomes, and one of these populations may be inaccessible to ligands, to changes in pH or ion concentration in the external solution. Furthermore, optical studies of proteins reconstituted in liposomes suffer from significant light scattering, which diminishes the signal-to-noise value of the measurements. One attractive approach to circumvent these problems is to use nanodiscs, which are phospholipid bilayers encircled by a stabilizing amphipathic helical membrane scaffold protein. These membrane nanodiscs are stable, soluble in aqueous solution without detergent and do not scatter light significantly. In the present study, we have developed a protocol for reconstitution of the aa 3- and ba 3-type cytochrome c oxidases into nanodiscs. Furthermore, we studied proton-coupled electron-transfer reactions in these enzymes with microsecond time resolution. The data show that the nanodisc membrane environment accelerates proton uptake in both oxidases.
AB - The function of membrane-bound transporters is commonly affected by the milieu of the hydrophobic, membrane-spanning part of the transmembrane protein. Consequently, functional studies of these proteins often involve incorporation into a native-like bilayer where the lipid components of the membrane can be controlled. The classical approach is to reconstitute the purified protein into liposomes. Even though the use of such liposomes is essential for studies of transmembrane transport processes in general, functional studies of the transporters themselves in liposomes suffer from several disadvantages. For example, transmembrane proteins can adopt two different orientations when reconstituted into liposomes, and one of these populations may be inaccessible to ligands, to changes in pH or ion concentration in the external solution. Furthermore, optical studies of proteins reconstituted in liposomes suffer from significant light scattering, which diminishes the signal-to-noise value of the measurements. One attractive approach to circumvent these problems is to use nanodiscs, which are phospholipid bilayers encircled by a stabilizing amphipathic helical membrane scaffold protein. These membrane nanodiscs are stable, soluble in aqueous solution without detergent and do not scatter light significantly. In the present study, we have developed a protocol for reconstitution of the aa 3- and ba 3-type cytochrome c oxidases into nanodiscs. Furthermore, we studied proton-coupled electron-transfer reactions in these enzymes with microsecond time resolution. The data show that the nanodisc membrane environment accelerates proton uptake in both oxidases.
KW - Cytochrome aa
KW - Cytochrome ba
KW - Cytochrome c oxidase
KW - Energy transduction
KW - Lipid
KW - Membrane protein
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U2 - 10.1016/j.febslet.2011.12.023
DO - 10.1016/j.febslet.2011.12.023
M3 - Review article
C2 - 22209982
AN - SCOPUS:84857923404
SN - 0014-5793
VL - 586
SP - 640
EP - 645
JO - FEBS Letters
JF - FEBS Letters
IS - 5
ER -