期刊
BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
卷 1828, 期 2, 页码 776-784出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbamem.2012.11.005
关键词
Bacteriorhodopsin ESR; Potassium channel KcsA; Reconstructed high-density lipoprotein; Membrane protein folding; Membrane mimetic; Cell-free expression
资金
- Russian Foundation for Basic Research [10-04-01752, 12-04-01712, 12-04-31485]
- Russian Federal Target Program Scientific and Science-Educational Personnel of Innovative Russia [8789, 8268]
- Russian Academy of Sciences
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 (similar to 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 (similar to 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. (C), 2012 Elsevier B.V. All rights reserved.
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