Resolution of complex fluorescence spectra of lipids and nicotinic acetylcholine receptor by multivariate analysis reveals protein-mediated effects on the receptor's immediate lipid microenvironment

Abstract

Analysis of fluorescent spectra from comp lex biological systems containing various fluorescent probes with overlapping emissi on bands is a challenging task. Valuable information can be extracted from the full spectra, however, by using multivariate analysis (MA) of measurements at differen t wavelengths. We a pplied MA to spectral data of purified Torpedo nicotinic acetylcholine receptor (AChR) protein reconstituted into liposomes made up of diol eoylphosphatidic acid (DOPA) and dioleoylphosphatidylcholine (DOPC) doped with two ex trinsic fluorescent probes (NBD-cholesterol/pyrene-PC). Förster re sonance energy transfer (FRET) was observed between the protein and pyre ne-PC and between pyrene-PC and NBD- cholesterol, leading to overlapping emission bands. Partial least squares analysis was applied to fluorescence spectra of pyrene -PC in liposomes with different DOPC/DOPA ratios, generating a model that was tested by an internal validation (leave-one-out cross-validation) and was further used to predict the apparent lipid molar ratio in AChR-containing samples. The values predi cted for DOPA, the lipid with the highest Tm, indicate that the protein exerts a rigidi fying effect on its lipid microenvironment. A similar conclusion was reached from excime r formation of pyrene -PC, a collisional- dependent phenomenon. The excimer/monome r ratio (E/M) at different DOPC/DOPA molar ratios revealed the restricted diffusio n of the probe in AChR-containing samples in comparison to pure lipid samples devoid of protein. FRET from the AChR (donor) to pyrene-PC (acceptor) as a function of temperature was found to increase with increasing temperature, suggesting a shorte r distance between AChR and pyrene PC. Taken together, the results obtained by MA on complex spectra indicate that the AChR rigidifies its surrounding lipid and prefers DOPA rather than DOPC in its immediate microenvironment.