Galiakhmetov, A. | bioRxiv Science

bioRxiv2018

Extraction of a recombinant full-length NADPH-cytochrome P450 oxidoreductase from bacterial membranes: effect of detergents and additives

NADPH-cytochrome P450 oxidoreductase (POR) is a membrane protein in the endoplasmic reticulum of eukaryotic cells. POR is as a key reducing partner for a number of cytochrome P450 proteins involved in different metabolic degradation and signaling pathways. Preparation of the full-length recombinant POR expressed in bacteria has been reported and, typically, involved the use of Triton X-100 detergent for extraction of the overexpressed POR from bacterial membranes. However, extraction efficiency is always relatively low hindering structural studies, particularly--the NMR spectroscopy requiring isotopic enrichment. In this paper, we assessed the effect of a variety of detergents and additives on the efficiency of the membrane-extraction step in POR preparation protocol. We evaluated non-ionic detergents with the variable hydrophobicity (Triton X-100, X-114, and X-405) and structure (Triton X-100, TWEEN-20, Brij-35), a zwitterionic/non-ionic detergent combination (Triton X-100 and CHAPS), as well as a range of alkylamines and polyamines as additives to the conventional extraction buffer containing Triton X-100. None of the detergents or detergent-additive combinations yielded better extraction efficiency than the conventional protocol with the Triton X-100. Lack of variation of the extraction yield allows to hypothesize that the conventional protocol extracts all of the available natively-folded monomeric POR while the remaining fraction is possibly an unfolded aggregated POR, which did not insert in the membranes during expression. We propose that the yield of soluble POR may be increased by a careful optimization of expression conditions while monitoring the distribution of POR between soluble and insoluble fractions in the detergent extraction step.

biochemistry↗

bioRxiv2017

Partitioning of semisynthetic lipidated N-Ras in lipid raft nanodomains determined by FRET to lipid domain markers

Cellular membranes are heterogeneous planar lipid bilayers displaying lateral phase separation with the nanometer-scale liquid-ordered phase (aka \"lipid rafts\" or Lo) surrounded by the liquid-disordered phase (Ld). Many membrane-associated proteins were found to stably integrate in the rafts, which is critical for their biological function. Isoforms H and N of Ras GTPase possess a unique ability to switch their lipid domain preference depending on the type of bound guanine nucleotide (GDP or GTP). This behavior, however, has never been reproduced in vitro in model bilayers with recombinant proteins, and therefore has been attributed to action of other proteins binding Ras at the membrane surface. In this paper, we report the observation of the nucleotide-dependent switch of lipid domain preferences of the semisynthetic lipidated N-Ras in raft lipid vesicles in the absence of other proteins. To detect segregation of Ras molecules in raft and disordered lipid domains, we measured Forster Resonance Energy Transfer (FRET) between the donor fluorophore, mant, attached to the protein-bound guanine nucleotides, and the acceptor, rhodamine-conjugated lipid, localized to the liquid-disordered domains. We demonstrated that N-Ras preferentially populated raft domains when bound to mant-GDP, while losing preference for rafts when it was associated with a GTP mimic, mant-GppNHp. At the same time, the isolated lipidated C-terminal peptide of N-Ras was found localized outside of the liquid-ordered rafts, most likely--in the bulk disordered lipid.

biochemistry↗