Abstract
It is well established that α-synuclein (α-syn) binding from solution to the surface of membranes composed of negatively charged and/or non-lamellar lipids can be characterized by equilibrium dissociation constants of tens of micromolar. Previously, we have found that a naturally occurring nanopore of the mitochondrial voltage-dependent anion channel (VDAC), reconstituted into planar bilayers of a plant-derived lipid, responds to α-syn at nanomolar solution concentrations. Here, using lipid mixtures that mimic the composition of mitochondrial outer membranes, we show that functionally important binding does indeed take place in the nanomolar range. We demonstrate that the voltage-dependent rate at which a membrane-embedded VDAC nanopore captures α-syn is a strong function of membrane composition. Comparison of the nanopore results with those obtained by the bilayer overtone analysis of membrane binding demonstrates a pronounced correlation between the two datasets. The stronger the binding, the larger the on-rate, but with some notable exceptions. This leads to a tentative model of α-syn-membrane interactions, which assigns different lipid-dependent roles to the N- and C-terminal domains of α-syn accounting for both electrostatic and hydrophobic effects. As a result, the rate of α-syn capture by the nanopore is not simply proportional to the α-syn concentration on the membrane surface but found to be sensitive to the specific interactions of each domain with the membrane and nanopore.
Highlights
Dynamics, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, 20892, USA
We find that functionally important α-syn binding begins at nanomolar solution concentrations, three orders of magnitude smaller than those determined by the macroscopic measurements
Our results suggest that bound α-syn can adopt different sub-conformations on the membrane surface depending on the lipid charge and headgroup packing density, and that each sub-conformation is characterized by a different membrane binding affinity, and by a different availability of the C-terminus for capture by the voltage-dependent anion channel (VDAC) nanopore
Summary
Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, 20892, USA. Based on the detailed kinetic analysis of the blocking events, a hypothetical model for α-syn interaction with the VDAC nanopore was proposed. According to this model, the capture of the negatively charged C-terminal domain of α-syn by the anionic VDAC nanopore, revealed through the characteristic transient ionic current blockages, is necessarily preceded by α-syn binding to the membrane surface[36]. We focus on the rate at which α-syn molecules block the voltage-biased nanopore ionic current (the “on-rate”) These single-molecule results are compared with macroscopic measurements of α-syn binding to planar bilayer architectures using the bilayer overtone analysis (BOA). The VDAC nanopore proves to be an extremely sensitive single-molecule probe for peripheral membrane binding
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
