Isolation of yeast complex IV in native lipid nanodiscs

Abstract

We used the amphipathic styrene maleic acid (SMA) co-polymer to extract cytochrome c oxidase (CytcO) in its native lipid environment from S. cerevisiae mitochondria. Native nanodiscs containing one CytcO per disc were purified using affinity chromatography. The longest cross-sections of the native nanodiscs were 11 nm x 14 nm. Based on this size we estimated that each CytcO was surrounded by ~100 phospholipids. The native nanodiscs contained the same major phospholipids as those found in the mitochondrial inner membrane.Even though CytcO forms a supercomplex with cytochrome bc 1 in the mitochondrial membrane, cyt. bc 1 was not found in the native nanodiscs. Yet, the loosely-bound Respiratory

Examining the stability of membrane proteins within SMALPs

“…The sensitivity to Ni 2+ was particularly interesting given that we routinely use Ni-NTA affinity chromatography to purify SMALPencapsulated proteins. Binding of SMALP-encapsulated proteins to Ni-NTA resin has previously been reported to be problematic at times [5,14,18,29], but this has generally been considered to result from interactions between the resin and excess free SMA [18,29], and despite these issues there have been many reported success stories using Ni-NTA [6-11, 14, 16, 17, 29]. This suggests that the effective concentration of Ni 2+ in the bead slurry must be below 2 mM or perhaps the spatial arrangement of Ni 2+ attached to the beads doesn't allow multiple nickel ions to bind to the same SMALP in the manner that leads to precipitation.…”

Membrane biology visualized in nanometer-sized discs formed by styrene maleic acid polymers

“…The recombinant constituent proteins can also be purified using C-terminal His 7 tags [38]. The latter particles do not resemble round discs, instead displaying oblong dimensions of 11 nm and 14 nm by negative stain EM.…”

The effectiveness of styrene-maleic acid (SMA) copolymers for solubilisation of integral membrane proteins from SMA-accessible and SMA-resistant membranes

“…These typically range in size from 10 to 15 nm, and estimates of the number of lipids they contain have ranged from 11 to 150 (see [2] for a review). A number of recent studies have shown that SMA can be used to produce highly pure preparations of integral membrane proteins from a variety of bacterial and eukaryotic sources [5] , [6] , [7] , [8] , [9] , [10] , [11] , [12] , [13] , [14] . Modification with a His-tag greatly assists this process by providing a means to separate nanodiscs containing the target protein from the heterogeneous population produced from a solubilised membrane.…”