Electrostatic interactions are fundamental forces governing the behavior of charged molecules at the lipid membrane interface. These interactions, among other factors, influence ion distribution, membrane protein orientation, and ion channel activity. One method for assessing surface electrostatics involves determining the p K a shift of protonatable molecular probes positioned at the lipid bilayer interface. Previously, we successfully employed EPR of phospholipids labeled at the headgroup with a pH-sensitive nitroxide to map bilayer interfacial electrostatics. Here, we report on a comparative study of the surface electrostatics of multilamellar (MLVs) and unilamellar vesicles (ULVs) composed of either POPG or POPC lipids. For vesicles composed of negatively charged POPG, the effective p K a measured for MLVs was significantly, up to 0.9 pH unit, higher than that for ULVs across all three probes. The larger local electrostatic potential experienced by probes within the inner lamellae is likely due to an influence of charges on the adjacent lamellae. For zwitterionic POPC, the effective p K a for MLVs was only slightly lower than that for ULVs, suggesting that the electrostatic environment in the inner lamellae closely resembles that of the outermost and innermost layers, with minor differences potentially arising from variations in hydration and ion distribution. We then measured the protonation state of probes at the surfaces of outer or inner leaflets of DMPG ULVs and observed a significant shift in pK a , indicating a difference in electrostatic or local dielectric environments of the probes. The results further highlight the utility of the spin-labeled phospholipids in biophysical EPR spectroscopy and reveal unexpectedly large changes in layer-dependent electrostatic environments in MLVs and ULVs. This material is based upon work supported by the National Science Foundation under grant no. 2305172.
Nguyen et al. (Sun,) studied this question.