Effect of Lipid Composition on the Affinity and Binding of Dimeric Tubulin to Membranes Studied using Surface Plasmon Resonance, Neutron Reflectivity, Electrophysiology, and AC Electrical Methods

Abstract

Dimeric tubulin has emerged as an important regulatory factor of the permeability of voltage-dependent anion channel (VDAC) in the mitochondrial outer membrane, with implications for mitochondrial energetics as well as the Warburg effect observed in cancers. Previously, single-channel studies revealed that the on-rate of the VDAC-tubulin interaction is strongly dependent on the lipid environment. To understand the nature of the binding of this abundant cytosolic water-soluble protein to lipid membranes, we have employed an array of biophysical techniques using unsupported planar lipid membrane and tethered bilayer lipid membrane (tBLM) platforms. Surface plasmon resonance (SPR) of tBLMs shows that tubulin at concentrations less than 100 nM binds irreversibly to DPhPC and 1:1 DOPC:DOPE membranes. The binding rate is significantly larger in high salt concentrations, suggesting a hydrophobic interaction between tubulin and lipid membrane. No binding to DOPC membranes is observed under the same conditions. Electrochemical impedance spectroscopy (EIS) of tBLMs reveals that only DPhPC membranes are strongly perturbed by the binding of tubulin. Neutron reflectivity (NR) measurements on tBLMs give structural information regarding the penetration of the tubulin into the membranes. Electrophysiological measurements of the tubulin/VDAC interaction on the unsupported bilayers confirm the irreversibility of tubulin binding to DPhPC membranes and membranes with significant DOPE content. Second harmonic analysis of planar lipid membranes’ response to ac excitation suggests that tubulin binding causes significant rearrangements of the lipid headgroups. We conclude that tubulin binds to and modifies the structure of lipid membranes even at nanomolar concentrations. Considering that there is up to 10 μM of free dimeric tubulin in cells, our results suggest a new broad regulatory role of dimeric tubulin in vivo.