Final Report Summary - SINGLE-BIOET (Single-molecule junction capabilities to map the electron pathways in redox bio-molecular architectures) The project objectives lean on three central blocks:(i) Setting up the single-molecule junction system. Realization of control experiments with a list of simple chemical blocks. e.g. variable length aliphatic chains and poly-phenyl wires, whose single-molecule conductance signatures are well-known.(ii) Elaboration of a simple site-directed mutagenesis plan to perform point-site mutations on the wild-type Azurin structure that will allow single-molecule bridging between the two electrodes. In a second stage of this block, inner mutations at the first coordination sphere of the Cu center will be proposed (target positions for hopping electron transfer (ET)).(iii) Realization of single-molecule junction experiments on the synthesized Azurin mutants. This step involves the exploitation of several scanning tunneling microscopy (STM)-based approaches to reliably identify single-Azurin bridges formation and evaluate its life time, mechanical stability, current level flow, etc. 2-dimensional maps or Conductograms containing all the spectroscopic and spatial information will be generated for the wild-type and for each mutant in order to deliver the final charge transport landscape of a single-biomolecule device.The working plan of this project was structured around the three main blocks configuring the main goals (see Table I of the original proposal). To summarize all the work done in this project, Block 1 and 2 tasks has been completed in full (100%) and the corresponding goals accomplished, and the last Block 3 has been almost accomplished with minor deviations from the original plan. The results from the latter Block are in the process of publication (one full paper has been already submitted and a second is in the writing stage). Overall, we have achieved the most relevant objectives for this period of the project with some minor modifications from the original plan in relation to Blocks 2 and 3. We can also add that Block 1 has been accomplished beyond the original expectations. Translating into work done, so far we have setup and successfully tested three fully equipped setups with single-molecule junction capabilities (one more equipment over the initial plan). Moreover, the three setups have been gifted with brand new code that allows a more robust automated use as well as more sophisticated tools for the data analysis. A site-directed mutagenesis scheme has been fully implemented in our lab and the final 9 selected residues of the outer Cu-Azurin structure (second coordination sphere of the metal center: K41C, V43C, Q12C, A65C, L68C, D76C, S89C, P115C, L120C) has been produced and conveniently purified by standard procedures. Note that the mutated positions do not significantly differ from those initially proposed for the outer protein sphere (see original proposal Figure 2), and that the inner target positions to study hopping ET have been ruled out for the reasons explained in the Block 3 results below. All 9 purified Cu-Azurin mutants have been characterized to assess their activity as well as structural folding. Electrochemical assays for each mutant revealed that 6 out of the 9 protein variants presented redox activity (naming, V43C, L68C, D76C, S89C, L120C and K41C). In order to approach the Block 3 objectives in the safest way, we have characterize the charge transport properties single-protein devices for those mutant presenting minor structural modifications in the blue Cu site, i.e. L120C, S89C, D76C, K41C and V43C, as obtained from the detailed spectroscopic electronic characterization by UV-visible and fluorescence. By the end of this period, we have performed single-protein transport measurements for L120C, S89C, D76C and K41C, which constitutes 80% of the proposed tasks for this Block.The comparison of the wild type single-protein transport to that of the protein variants has brought first hand information on the transport pathways through the complex metalloprotein structure, as well as important technical details on the single-protein bridge formation for the mutant cases. Briefly, the single-molecule conductance measurement on the modified protein shows much less dispersion as compare to the wild type. This is a clear indication of the more restricted protein orientation in the former case thanks to its stronger thiol attachment to the two junction electrodes. For the same reason, the single-protein lifetime has increased substantially in the mutant cases, as measured by our newly implemented blinking tool. Second, the electrochemical gating response of the single-mutant wires deviates completely from its wild type counterpart, which demonstrated for the first time the feasibility of bioengineering charge transport in a nanoscale biomolecular wire. This result also suggested that outer modifications of the protein backbone (at the secondary Cu coordination sphere) are enough to produce pronounced changes in the protein charge transport schemes. Therefore, the inner point-site mutations initially proposed were ruled out in this project period, turning out not to be necessary for a mechanistic study of the charge transport as a function of structural point modifications of the protein. We have achieved a complete mechanistic interpretation of the observed single-protein transport results for the K41C mutant (publication under review) by developing ab initio computational calculations within the relevant protein fragment for charge transport. We have also achieved important advances for the rest of the measured mutant systems. As the main conclusions, the final results of the single-protein transport demonstrate the feasibility to bioengineer the charge transport in a single-biomolecule device. By rationally point-modify the outer sphere of the protein, we can have a fine control of the transport channel dominating the charge transport through the protein backbones, which results in a very particular transport regime of the single-biomolecule electrical device. Our experimental results in combination with computational data brought us the mechanistic description of the transport for each particular mutation. Such information will end in the exact location of the electrical plugs in a complex biomolecular structure and the capability to tailor them to generate the desired electrical behavior in the nanoscale bioelectronic device.These results foresee a huge impact in the emerging field of Bioelectronics that exploits the use of biomolecular structures as active components in optoelectronic devices. The final picture that will be achieved here will serve to clarify key aspects concerning the electrical communication between an electrically active biomolecule and the solid electrode platform for signal transduction. The observed variations of the transport regime as a function of the mutated position will also impact the field of biological ET where specific structural variations of the ET molecular machinery in basic biological processes such us the respiratory chain have been ascribed to certain mitochondria dysfunction that leads to well-described health condition.
