Boulbrima, Alexandre (2019) Structural and Functional characterization of the Arabidopsis thaliana Voltage Dependent Anion Channel 1 and the Kinesin-like mKlp2 proteins. Doctoral thesis, University of Huddersfield.

The Voltage Dependent Anion Channel (VDAC) is a well characterized mitochondrial porin, known to participate in multiple cellular processes, ensuring the transport of ions, ATP, ADP and proteins of the respiratory chain between intermembrane space (IMS) and the cytosol. Hence, their abundance and strategic localization in the Outer Mitochondrial Membrane (OMM) cast them as a gatekeeper for the transit of ions and metabolites between the mitochondria and the cytosol. These channels were shown to exhibit a strong response to a variation of current, with frequent opening and closure of the channel, and the conservation in their three-dimensional structure attest to their essential role. The study of plant VDACs could offer interesting insights into the cellular and sub-cellular functions of the channel, as the importance of VDACs in plant development, fertility and physiology, was only recently been uncovered. Filling this knowledge gap on plant VDAC could greatly enrich our understanding of VDAC physiology in all eukaryotes.

Here, we report the successful purification of native and mutant Arabidopsis thaliana VDAC1 proteins. We highlight micelle-embedded AtVDAC structures by Small Angle X-Ray Scattering in 0.1 % (v/v) LDAO and show that in vitro refolded AtVDAC1 is mostly composed of beta sheets with a small helicity in circular dichroism experiments, in harmony with previous observations on eukaryotic VDACs. We demonstrate that the AtVDAC1 channel can exhibit voltage mediated gating activity when incorporated into a lipid bilayer, with an open state S0 conductance of 0.54 nS and at least two closed states, S1 and S2, with conductances of 0.43 nS and 0.31 nS, respectively, close to those reported for other VDACs in similar salt concentrations. We also attempt to understand its sensing ability and denote a current-mediated preference for S1 state at -20 mV. Finally, we introduce a new parameter for the electrophysiological characterisation of the channel, the “power of charge” and report similar transfer of charges at -20 mV and -40 mV despite the lower gating activity associated with the S2 state.

In parallel, a potential AtVDAC1-binding partner, mKlp2C-Ter (a truncated version of a mitochondria-restricted kinesin-like protein from A. thaliana), was also heterologously expressed, purified and characterised. We report its behaviour in solution using SAXS, and preliminary functional characterization by Isothermal-Titration Calorimetry (ITC), highlighting an ability to bind zinc ions. These first steps constitute the groundwork for future binding studies focusing on AtVDAC1 interaction with cytosolic partners, and the molecular and electro-physiological consequences of these interactions.

FINAL THESIS - BOULBRIMA.pdf - Accepted Version
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