Background

Reconstitution of purified proteins into model membranes is an essential approach for investigating membrane protein function under defined conditions outside the complex cellular environment (Rigaud and Lévy, 2003; Murray et al., 2014; Amati et al., 2020). Typically, proteins are reconstituted with phospholipids into large unilamellar vesicles with 100–200 nm diameters. The orientation of the protein in the liposomal membrane after reconstitution depends on many parameters, including the type of protein, the lipid composition, and the reconstitution procedure (Tunuguntla et al., 2013; Amati et al., 2020). Knowledge of the orientation distribution of the protein is of crucial significance for interpreting its function in the proteoliposomal in vitro system and for the design of proteoliposome experiments. A common approach for determining the orientation of a membrane-inserted protein is the protease-mediated cleavage (e.g., Schuette et al., 2004; Serek et al., 2004; Islam et al., 2013; Eisinger et al., 2018). This assay is based on the principle that an externally added protease can cleave only the accessible portions of the membrane-inserted protein, which requires several critical steps such as limited proteolytic digest, stopping the proteolytic reaction, and fragment analysis (e.g., by SDS-PAGE).

Alternative approaches are based on impermeable labeling reagents for specific amino acids or membrane-impermeable quenchers for fluorescent proteins (e.g., Zhang et al., 2003; Marek et al., 2011; Deutschmann et al., 2022). The protocol described here takes advantage of the SNAP-tag technology (Keppler et al., 2004; Cole, 2013), whereby a protein can be labeled site-specific after expression or purification with a diversity of dyes, which are either membrane-permeable or impermeable (Figure 1). This approach can be extended to other self-labeling enzyme tags including SNAP-tag, HALO-tag, or CLIP-tag and, thus, can be easily customized for the protein of interest. In contrast to other labels such as cysteine labeling, side-specific, stoichiometric 1:1 labeling without changing the amino acid sequence is ensured. Compared to protease-based approaches, labeling with dyes of different permeability allows analysis under nondestructive conditions for the vesicle and the protein. The presented strategy will be useful for the optimization of reconstitution conditions and the analysis of many types of membrane proteins. Thus, this new assay expands the toolbox of fluorescence-based methods for the estimation of membrane protein orientation in liposomes.

Figure 1. SNAP-labeling process. The membrane protein of interest is expressed with SNAP-tag enabling site-specific labeling. Based on the human DNA repair enzyme O⁶-alkylguanine-DNA-alkyltransferase, the SNAP enzyme undergoes self-labeling via a covalent bond with O⁶-benzylguanine derivatives. Depending on the characteristics of the derivative, the labeling substrate is either membrane-permeable like SNAP-647-SIR or membrane-impermeable like SNAP-Alexa488 [chemical structures based on Lukinavičius et al. (2013) and Wilhelm et al. (2021), and drawn with https://chem-space.com].

Things to consider before starting

1. Choice of labeling substrate

   Here, we used a SNAP-tagged fused membrane protein and benzylguanine-derived substrates. Labeling of the SNAP-tag is irreversible and quantitative, and thus well suited for the detection and quantitation of labeled proteins via in-gel fluorescence scanning of SDS-PAGE gels. If alternative labeling systems based on, for example, CLIP-tag or ACP-tag, are used, the according substrate has to be chosen as basis for the labeling dye.

2. Choice of self-labeling fluorophore

   The assay relies on the use of a membrane-impermeable dye in combination with a membrane-permeable dye. In addition, both dyes should be selected for low spectral overlap and based on the imaging system available.

3. Choice of lipid environment

   Here, we describe labeling conditions optimized for proteins reconstituted in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) / 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) (9:1 mol/mol) vesicles. In other lipid environments, labeling duration and label concentrations might need adjustment.

4. Additives in buffer

   A reducing agent (e.g., DTT) is added to the labeling buffer to increase labeling efficiency, due to a reactive cysteine in the SNAP-tag. However, if its presence is affecting the protein, the experiment could be tested without DTT, with optimized labeling time and label concentrations. However, under oxidizing conditions, labeling might not be achieved efficiently. The presence of chelating reagents such as EDTA should be avoided as the SNAP-tag protein contains a structural Zn²⁺ ion.
   

The exemplary detergent-solubilized SNAP-tagged membrane protein is a C-terminally truncated version (∆73) of the Arabidopsis thaliana auto-inhibited H⁺-ATPase isoform 2 (AHA2-SNAP) with additional StrepII and hexahistidine N-terminal tags. AHA2-SNAP was heterologously expressed in the Saccharomyces cerevisiae strain RS-72 (MATa, ade1-100 his4-519 leu2-3,112; endogenous proton pump PMA1 gene is under control of GAL1 promoter) (Cid et al., 1987) and purified via the His-tag resulting in 5–10 mg/mL protein in storage buffer (see Recipe 3), stored at -80 °C (Lanfermeijer et al., 1998).

Note: The protocol can also be applied to purified membrane proteins prepared differently in combination with liposomal reconstitution procedures not based on preformed liposomes, as described here. However, these membrane proteins may have different preferences for lipids, with respect to the phospholipid headgroup and the lipid fatty acid composition.