Lipid–protein nanodiscs promote in vitro folding of transmembrane domains of multi-helical and multimeric membrane proteins

Abstract

Production of helical integral membrane proteins (IMPs) in a folded state is a necessary prerequisite for their functional and structural studies. In many cases large-scale expression of IMPs in cell-based and cell-free systems results in misfolded proteins, which should be refolded in vitro. Here using examples of the bacteriorhodopsin ESR from Exiguobacterium sibiricum and full-length homotetrameric K(+) channel KcsA from Streptomyces lividans we found that the efficient in vitro folding of the transmembrane domains of the polytopic and multimeric IMPs could be achieved during the protein encapsulation into the reconstructed high-density lipoprotein particles, also known as lipid-protein nanodiscs. In this case the self-assembly of the IMP/nanodisc complexes from a mixture containing apolipoprotein, lipids and the partially denatured protein solubilized in a harsh detergent induces the folding of the transmembrane domains. The obtained folding yields showed significant dependence on the properties of lipids used for nanodisc formation. The largest recovery of the spectroscopically active ESR (~60%) from the sodium dodecyl sulfate (SDS) was achieved in the nanodiscs containing anionic saturated lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPG) and was approximately twice lower in the zwitterionic DMPC lipid. The reassembly of tetrameric KcsA from the acid-dissociated monomer solubilized in SDS was the most efficient (~80%) in the nanodiscs containing zwitterionic unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The charged and saturated lipids provided lower tetramer quantities, and the lowest yield (<20%) was observed in DMPC. The overall yield of the ESR and KcsA folding was mainly restricted by the efficiency of the protein encapsulation into the nanodiscs.

NMR Dynamics of Transmembrane and Intracellular Domains of p75NTR in Lipid-Protein Nanodiscs

“…Bicelles of a certain composition (DMPC/CHAPS, in this study) can be used, but their applicability is limited to a very narrow and uncomfortable range of protein and lipid concentrations. This presents a nice prospect for the use of LPNs as a membrane mimetic for structural studies of multidomain membrane proteins, especially since LPNs have been shown to be a perfect medium for the refolding of membrane proteins (32). Nanodiscs were first proposed as a reference medium for detergent screening (28) or as a possible environment in which to study the topology of small peptides in the membrane (33)(34)(35)(36); however, recent advances, such as the development of new MSPs that form smaller discoid particles (up to~50 kDa) (31), isotope labeling strategies, and NMR experimental techniques have also made them applicable for structural studies of membrane proteins (37).…”

Characterization of Small Isotropic Bicelles with Various Compositions

“…LPNs, formed by a membrane scaffold protein (MSP), and lipids, contain a large patch of lipid bilayer and represent an almost ideal model of cellular membrane 12 . However, the size of LPNs is far too large for the solution NMR spectroscopy (~120-150 kDa), restricting their usage to application as a reference medium in the detergent screening and as an environment for the protein refolding [13][14][15] . Recent advances, such as modified MSPs, which provide smaller LPNs 16 , allow even the structural studies 17,18 .…”

Oxidative in vitro folding of a cysteine deficient variant of the G protein-coupled neuropeptide Y receptor type 2 improves stability at high concentration

“…Although most suitable for solution NMR studies of integral helical membrane proteins (Gautier and Nietlispach , 2012 ), detergent/ phospholipid micelles provide a fairly unstable environment for GPCRs because of the constant exchange of the detergent molecules between their monomeric and micellar form, and the denaturing properties of detergents at high concentrations (Warschawski et al , 2011 ;Serebryany et al , 2012 ;Shenkarev et al , 2012 ). In addition, a fact that has often been neglected is the formation of non-native disulfide bonds between free cysteines.…”