Heavy Water Reduces the Electronic Conductance of Protein Wires via Deuteron Interactions with Aromatic Residues.

Proteins are versatile, self-assembling nanoelectronic components, but their hopping conductivity is expected to be influenced by solvent fluctuations. The role of the solvent was investigated by measuring the single molecule conductance of several proteins in both H2O and D2O. The conductance of a homologous series of protein wires decreases more rapidly with length in D2O, indicating a 6-fold decrease in carrier diffusion constant relative to the same protein in H2O. The effect was found to depend on the specific aromatic amino acid composition. A tryptophan zipper protein showed a decrease in conductance similar to that of the protein wires, whereas a phenylalanine zipper protein was insensitive to solvent changes. Tryptophan contains an indole amine, whereas the phenylalanine aromatic ring has no exchangeable protons, so the effect of heavy water on conductance is a consequence of specific D- or H-interactions with the aromatic residues.

Engineering gain-of-function mutants of a WW domain by dynamics and structural analysis.

Proteins gain optimal fitness such as foldability and function through evolutionary selection. However, classical studies have found that evolutionarily designed protein sequences alone cannot guarantee foldability, or at least not without considering local contacts associated with the initial folding steps. We previously showed that foldability and function can be restored by removing frustration in the folding energy landscape of a model WW domain protein, CC16, which was designed based on Statistical Coupling Analysis (SCA). Substitutions ensuring the formation of five local contacts identified as "on-path" were selected using the closest homolog native folded sequence, N21. Surprisingly, the resulting sequence, CC16-N21, bound to Group I peptides, while N21 did not. Here, we identified single-point mutations that enable N21 to bind a Group I peptide ligand through structure and dynamic-based computational design. Comparison of the docked position of the CC16-N21/ligand complex with the N21 structure showed that residues at positions 9 and 19 are important for peptide binding, whereas the dynamic profiles identified position 10 as allosterically coupled to the binding site and exhibiting different dynamics between N21 and CC16-N21. We found that swapping these positions in N21 with matched residues from CC16-N21 recovers nature-like binding affinity to N21. This study validates the use of dynamic profiles as guiding principles for affecting the binding affinity of small proteins.

Light-Driven CO2 Reduction by Co-Cytochrome b 562.

The current trend in atmospheric carbon dioxide concentrations is causing increasing concerns for its environmental impacts, and spurring the developments of sustainable methods to reduce CO2 to usable molecules. We report the light-driven CO2 reduction in water in mild conditions by artificial protein catalysts based on cytochrome b 562 and incorporating cobalt protoporphyrin IX as cofactor. Incorporation into the protein scaffolds enhances the intrinsic reactivity of the cobalt porphyrin toward proton reduction and CO generation. Mutations around the binding site modulate the activity of the enzyme, pointing to the possibility of further improving catalytic activity through rational design or directed evolution.

Local Interactions That Contribute Minimal Frustration Determine Foldability.

Earlier experiments suggest that the evolutionary information (conservation and coevolution) encoded in protein sequences is necessary and sufficient to specify the fold of a protein family. However, there is no computational work to quantify the effect of such evolutionary information on the folding process. Here we explore the role of early folding steps for sequences designed using coevolution and conservation through a combination of computational and experimental methods. We simulated a repertoire of native and designed WW domain sequences to analyze early local contact formation and found that the N-terminal ß-hairpin turn would not form correctly due to strong non-native local contacts in unfoldable sequences. Through a maximum likelihood approach, we identified five local contacts that play a critical role in folding, suggesting that a small subset of amino acid pairs can be used to solve the "needle in the haystack" problem to design foldable sequences. Thus, using the contact probability of those five local contacts that form during the early stage of folding, we built a classification model that predicts the foldability of a WW sequence with 81% accuracy. This classification model was used to redesign WW domain sequences that could not fold due to frustration and make them foldable by introducing a few mutations that led to the stabilization of these critical local contacts. The experimental analysis shows that a redesigned sequence folds and binds to polyproline peptides with a similar affinity as those observed for native WW domains. Overall, our analysis shows that evolutionary-designed sequences should not only satisfy the folding stability but also ensure a minimally frustrated folding landscape.

De Novo Design of Iron-Sulfur Proteins.

Iron-sulfur proteins are one of the most abundant and functionally pliable redox proteins found in all living organisms. Because of their crucial role in mediating electron transfer processes, minimalist model systems have been developed as a proxy to study natural Fe-S redox proteins and to dissect rules to enable tuning of their redox and electron transfer activities. This goal has been pursued through computational design, mutagenesis in the first and second coordination sphere, metal substitution, cofactor replacement, and the use of unnatural amino acids to stabilize a given cluster. In this chapter, we discuss the most recent design strategies to introduce various Fe-S clusters into natural and artificial protein scaffolds. Practical approaches for the cluster reconstitution, hydrogen production, and electrochemical characterization are mentioned.