Multivariate analysis of fluorescence spectra for studying the lipid microenvironment of the nicotinic acetylcholine receptor

WENZ, J.; BARRANTES, F.J.

Montevideo, Uruguay

Congreso; 6th Int. Conference of Biological Physics, 5th Southern Cone Biophysical Congress & 34th Annual Meeting of the Argentinean Biophysical Congress; 2007

IUPAP

Analysis of fluorescent spectra from complex biological systems containing various fluorescent probes with overlapping emission bands is a difficult task. Valuable information can be extracted from the full spectra, however, by using multivariate analysis of measurements at different wavelengths. This analysis can assign a “weight” to each relevant wavelength, discarding those that not correlate well with a given dependent variable (i.e. concentration, molar ratio, etc.). In addition, this approach can usually improves precision and facilitates the detection of errors and/or anomalous samples. Purified nicotinic acetylcholine receptor (AChR) was reconstituted into liposomes containing two extrinsic fluorescent probes (DOPC/DOPA/NBD-cholesterol/pyrene-PC). Resonance energy transfer (FRET) can occur between the protein and pyrene-PC, and between pyrene-PC and NBD-cholesterol, leading to overlapping emission bands. Partial least squares (PLS-1) 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 predicted proportion of the lipid with the highest transition temperature (DOPA) was larger than its actual concentration, indicating that the protein exerts a “rigidizing” effect on its lipid microenvironment. A similar conclusion was reached from the formation of pyrene-PC excimers, a collisional-dependent phenomenon. The excimer/monomer ratio (E/M) was studied for different DOPC/DOPA molar ratios. In AChR-containing samples, the restricted diffusion of the probe resulted in lower E/M values as compared to samples without AChR but with identical lipid composition. The efficiency of FRET from the AChR (donor) to pyrene-PC (acceptor) as a function of temperature was found to increase with increasing temperature, indicating a shorter distance between the FRET partners, i.e. a closer AChR-pyrene PC contact. Taken together, the findings suggest that AChR prefers DOPA in comparison to DOPC in its immediate microenvironment, in agreement with previous results from our laboratory (Wenz & Barrantes, Biochemistry 44  (2005) 398-410), and rigidizes its lipid surroundings.