Using Fluorescence Quenching Titration to Determine the Orientation of a Model Transmembrane Protein in Mimic Membranes

Abstract

After synthesis of transmembrane proteins (TMPs), they are transferred and inserted into plasma membranes to play biological functions. Crucially, orientation of TMPs in membranes determines whether they have biological activities. In cellular environments, a number of cofactors, such as translocon, can assist TMPs to be inserted into membranes in defined orientations. During in vitro reconstitution of TMPs with mimic membranes, both insertion and orientation of TMPs are primarily determined by interactions with the membrane. Yet the knowledge is limited, hindering the in vitro applications of TMPs. Here, we take Bacteriorhodopsin (bR) as a model TMP, using fluorescence quenching titration experiment to identify orientation of bR in mimic membranes, examining effects of a number of factors, including lipid composition, pH value, ionic strength and membrane curvature. The most effective determinant is the lipid type, which modulates insertion and orientation of bR in membranes by changing the membrane surface charge and the membrane fluidity. Both the pH value and the ionic strength play secondary roles by tuning the nature of the electrostatic interaction. The membrane curvature was found to have a minor effect on orientation of bR in membranes. By comparing orientations of bR in folded and unfolded states, no obvious change was observed, informing that nascent proteins could be inserted into membranes in defined orientations before folding into the native state inside the membrane.

Keywords: fluorescence quenching titration; insertion; mimic membrane; orientation; transmembrane protein.

Figure 1

Molecular structures of both TAMRA and different types of lipids used in our experiments.

Figure 2

Fluorescence spectra (a) and UV-Vis absorption spectra (b) of bR proving its successful labeling with TAMRA.

Figure 3

DLS data and Negative-Stain TEM images of lipid vesicles prepared by extrusion (a) 100 nm POPC; (b) 100 nm POPG; (c) 100 nm DOPC.

Figure 4

The fluorescence anisotropy of TAMRA-bR incorporated in an increasing concentration of lipid vesicles. Three types of lipids were used, including POPC (a); POPG (b) and DOPC (c), the 50 nm and 100 nm indicate the size of mimic membrane. We have conducted a systematic study of the concentration of phospholipids impact on the scattered light of mimic membranes in our early studies and obtained the optimal concentration of bR to be 4 µM and the mimic membranes to be 3 mg/mL.

Figure 5

The fluorescence quenching titration data for TAMRA-bR incorporated into three types of lipid vesicles.

Figure 6

Structural and electrostatic properties of bR (PDB code: 1AP9). (a–d) Ribbon representations of the crystal structure from different views. (e–h) Electrostatic surface potential calculated with the aid of the Protein Continuum Electrostatics tool. b and f are the top views with the C terminus pointing upwards, whereas d and h are those viewing form the N terminus.