Site-directed fluorescence labeling of a membrane protein with BADAN: probing protein topology and local environment

We present a new and simple method based on site-directed fluorescence labeling using the BADAN label that allows to examine protein-lipid interactions in great detail. We apply this approach to a membrane-embedded mainly -helical reference protein, the M13 major coat protein, of which in a high-throughput approach 40 site-specific cysteine mutants were prepared of the 50-residues long protein. The steady-state fluorescence spectra are analyzed using a three-component spectral model that enables to separate Stokes shift contributions from water and internal label dynamics, and protein topology. It is found that most of the fluorescence originates from BADAN labels that are hydrogen bonded to water molecules even within the hydrophobic core of the membrane. Our spectral decomposition method reveals the embedment and topology of the labeled protein in the membrane bilayer under various conditions of headgroup charge and lipid chain length, as well as key characteristics of the membrane, such a hydration level and local polarity, given by the local dielectric constant