Carbon nanomembranes as scaffold for studying nanoscale heterogeneity and vectorial functionality of protein assemblies
Statistical Physics, Nonlinear Dynamics, Complex Systems, Soft and Fluid Matter, Biological Physics
Final Report Abstract
Carbon nanomembranes (CNMs) synthesized by irradiation of self-assembled 4’-nitro- 1,1’-biphenyl-4-thiol (NBPT) display a thickness of around 1 nm. The hypothesis of our work was, that such membranes may serve as scaffold for arranging functional proteins and thereby to develop a novel hybrid material. First, the project aimed to develop functionalization regimes to bind proteins to the exposed amino group or/and after release from the gold surface, to the thiol group in order to obtain single sided or double sided functionalized membranes. The reliability of the published method employing 3-(maleimido)propionic acid N-hydroxysuccinimide ester (MI) as well as N-[N ,N- Bis(carboxymethyl)-L-lysine]-12-mercapto-dodecanamide (L-lysine-NTA) was insufficient in our hands. Therefore, we allocated significant time to establish alternative functionalization methods by, for example, binding biotin to immobilize streptavidin-tagged proteins, or by introducing maleimide moieties to amino-terminated CNMs using a copperfree click reaction employing 2-azidoacetyl chloride (2-AAC) and dibenzocyclooctynemaleimide (DBCOM). CNM functionalized with maleimide moieties and locally treated with drops of modified Cysx3 bound yellow fluorescent mVenus proteins. Due to the intensive search of a reliable method for the protein immobilization using the CNM’s functional amino groups, the exploration of an appropriate binding strategy for the thiol-terminated site of the carbon nanomembrane could not be performed any more. However, considering the facts gained during this investigation, the usage of modified proteins containing a readily clonable cysteine-tag is assumed to be an effective way also on this side. A second set of experiments studied the permeance of CNMs for water, CO2 and H2PO4- using a two compartment device with an orifice covered by CNM and [3H]H2O, [14C]NaHCO3, and [32P]H3PO4 as radioactive labeled molecules. Our data prove that the TPT-CNM is a highly selective and semi-permeable membrane in aqueous environment and expand previously published knowledge on permeance in gas phase and on impermeability for ions. In principle, the presented diffusion experiments are sensitive to neutral and ionic tracer species. However, a selective detection of the neutral form occurs for water as the concentration of its ions is negligible in respect to neutral water molecules at pH = 7. For carbon dioxide, we reason sole transport by neutral CO2 molecules across CNMs, based on previous measurements which indicate negligible transport of ions through CNMs in aqueous solution. The impermeability of the CNM for [32P]H3PO4 enables the use of this compound for assessing the quality and intactness of the CNM at the time of the measurement. Furthermore, we demonstrated in this project that alkanethiols can serve as molecular building blocks to prepare self-supporting CNMs with unique perforation properties. We cloned, validated, expressed and characterized the dominant chloroplast carbonic anhydrase from Arabidopsis thaliana β-CA1 and a second one from Sulfurihydrogenibium azorense. We generated site directed mutated variants which are inactive. This will be important as control in future immobilization experiments. The inactivity of β-CA1 after recombinant expression and purification prompted us to study the reason for inactivation. This study proved reversible thiol-redox state-dependent switching of β-CA1 activity and dependence on specific chloroplast thioredoxins. We also established that β-CA1 thiol state changes in planta (published in “Biomolecules” and “Antioxidants”. We also established the assay to quantify the activity of ribulose-1,5-bisphosphate carboxylase. Thus, all protein tools needed to start the immobilization and analyze the vectorial CO2 transport in hybrid CNMs are at hand. Due to the difficulties in establishing the functionalization regimes, these work packages will have to be addressed in future work.
Publications
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(2020) Redox conformationspecific protein-protein interactions of the 2-cysteine peroxiredoxin in Arabidopsis. Antioxidants 9 (6), 515
Liebthal M, Schuetze J, Dreyer A, Mock HP, Dietz KJ
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(2020) Selective diffusion of CO2 and H2O through carbon nanomembranes in aqueous solution as studied with radioactive tracers. The Journal of Physical Chemistry Letters, 2020
Dalpke R, Dreyer A, Korzetz R, Dietz KJ, Beyer A
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(2020) Thiol redox regulation of plant β-carbonic anhydrase. Biomolecules, 2020
Dreyer A, Schackmann A, Kriznik A, Chibani K, Wesemann C, Vogelsang L, Beyer A, Dietz KJ
