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.