Electron spin resonance in membrane research: protein–lipid interactions from challenging beginnings to state of the art

Conventional electron paramagnetic resonance (EPR) spectra of lipids that are spin-labelled close to the terminal methyl end of the acyl chains are able to resolve the lipids directly contacting the protein from those in the fluid bilayer regions of the membrane. This allows determination of both the stoichiometry of lipid–protein interaction (i.e., number of lipid sites at the protein perimeter) and the selectivity of the protein for different lipid species (i.e., association constants relative to the background lipid). Spin-label EPR data are summarised for 20 or more different transmembrane peptides and proteins, and 7 distinct species of lipids. Lineshape simulations of the two-component conventional spin-label EPR spectra allow estimation of the rate at which protein-associated lipids exchange with those in the bulk fluid regions of the membrane. For lipids that do not display a selectivity for the protein, the intrinsic off-rates for exchange are in the region of 10 MHz: less than 10× slower than the rates of diffusive exchange in fluid lipid membranes. Lipids with an affinity for the protein, relative to the background lipid, have off-rates for leaving the protein that are correspondingly slower. Non-linear EPR, which depends on saturation of the spectrum at high radiation intensities, is optimally sensitive to dynamics on the timescale of spin-lattice relaxation, i.e., the microsecond regime. Both progressive saturation and saturation transfer EPR experiments provide definitive evidence that lipids at the protein interface are exchanging on this timescale. The sensitivity of non-linear EPR to low frequencies of spin exchange also allows the location of spin-labelled membrane protein residues relative to those of spin-labelled lipids, in double-labelling experiments

Influencia del Estado de Fase del Lipido Sobre la Interaccion de la Proteina Periferica L-BABP con Menbranas Anionicas

Influencia del Estado de Fase del Lipido Sobre la Interaccion de la Proteina Periferica L-BABP con Menbranas Anionica

Interactions of chicken liver basic fatty acid-binding protein with lipid membranes

The interactions of chicken liver basic fatty acid-binding protein (Lb-FABP) with large unilamellar vesicles (LUVs) of palmitoyloleoyl phosphatidylcholine (POPC) and palmitoyloleoyl phosphatidylglycerol (POPG) were studied by binding assays, Fourier transform infrared (FT-IR) spectroscopy, monolayers at air-water interface, and low-angle X-ray diffraction. Lb-FABP binds to POPG LUVs at low ionic strength but not at 0.1 M NaCl. The infrared (IR) spectra of the POPG membrane-bound protein showed a decrease of the band corresponding to beta-structures as compared to the protein in solution. In addition, a cooperative decrease of the beta-edge band above 70 degrees C in solution was also evident, while the transition was less cooperative and took place at lower temperature for the POPG membrane-bound protein. Low- and wide-angle X-ray diffraction experiments with lipid multilayers indicate that binding of the protein produces a rearrangement of the membrane structure, increasing the interlamellar spacing and decreasing the compactness of the lipids

Thermodynamic characterization of the association of small basic peptides with membranes containing acidic lipids

We measured the binding of the peptide acetyl-Trp-Lys7-amide to membranes formed from mixtures of the zwitterionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC) and the acidic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (PG). Direct filtration and equilibrium dialysis measurements demonstrate that binding increases sigmoidally with the mole fraction of PG as predicted from a simple Gouy-Chapman/mass action theoretical model. We used these measurements to calibrate two binding assays, one based on the increase in Trp fluorescence that occurs when the peptide binds to the membrane, the other on the quenching of Trp fluorescence that occurs when the peptide binds to membranes containing fluorescent lipids. Both fluorescence assays demonstrate that binding does not depend strongly on temperature, which suggests the enthalpy change, delta H, is small. Calorimetric measurements demonstrate this directly for the analogous basic peptide Lys5: delta H congruent to +1 kcal/mol for the binding of Lys5 to sonicated phospholipid vesicles and delta H congruent to 0 kcal/mol for its binding to large unilamellar vesicles. Thus, the decrease in the free energy that occurs when these peptides bind to the membrane is due to a positive change in the entropy of the system. Fluorescence measurements demonstrate the binding of the Trp-containing peptide to 4:1 PC/PG membranes is independent of pressure up to 2 kbar, which suggests that binding occurs without a significant change in volume

Component analysis of the FTIR spectra.

<p>ReP1-NCXSQ in solution (panel A) and in the presence of anionic membranes of DMPG (panel B) and cationic membranes of DMTAP (panel C) and EDMPC (panel D). Spectra were collected a 33 ^oC. Measured spectra (lower continuous black trace), Fourier self-deconvolutions (upper continuous gray trace), using bandwidth of 18 cm^-1 and factor k = 2. Band components are in dashed lines.</p

Trajectories of the molecular dynamics simulations of adsorption.

<p>The <i>z</i>_<i>L-P</i> (panel A and B), the <i>θ</i>_{<i>μ-plane x-y</i>} (panel C and D) and the root mean square deviation (RMSD) (panel E and F) for the four simulations of ReP1-NCXSQ with cationic membranes of EDMPC (R-EDMPC1-4) and the two of L-BABP with EDMPC (B-EDMPC1-2) in panels A, C and E. In order to have a reference, data from simulations of ReP1-NCXSQ with anionic membranes of POPG (R-POPG1-3) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194154#pone.0194154.ref008" target="_blank">8</a>] are displayed in panels B, D and F.</p