Oligomerization is an important regulatory mechanism for many proteins, including oncoproteins and other pathogenic proteins. The oncoprotein Bcr-Abl relies on oligomerization via its coiled coil domain for its kinase activity, suggesting that a designed coiled coil domain with enhanced binding to Bcr-Abl and reduced self-oligomerization would be therapeutically useful. Key mutations in the coiled coil domain of Bcr-Abl were identified that reduce homo-oligomerization through intermolecular charge-charge repulsion yet increase interaction with the Bcr-Abl coiled coil through additional salt bridges, resulting in an enhanced ability to disrupt the oligomeric state of Bcr-Abl. The mutations were modeled computationally to optimize the design. Assays performed in vitro confirmed the validity and functionality of the optimal mutations, which were found to exhibit reduced homo-oligomerization and increased binding to the Bcr-Abl coiled coil domain. Introduction of the mutant coiled coil into K562 cells resulted in decreased phosphorylation of Bcr-Abl, reduced cell proliferation, and increased caspase-3/7 activity and DNA segmentation. Importantly, the mutant coiled coil domain was more efficacious than the wild type in all experiments performed. The improved inhibition of Bcr-Abl through oligomeric disruption resulting from this modified coiled coil domain represents a viable alternative to small molecule inhibitors for therapeutic intervention.

Photosystem I (PSI) is a key component of oxygenic photosynthetic electron transport because of its light-induced electron transfer to the soluble electron acceptor ferredoxin. This work demonstrates the incorporation of surface assembled cyanobacterial trimeric PSI complexes into a biohybrid system for light-driven current generation. Specifically, this work demonstrates the improved assembly of PSI via electrophoretic deposition, with controllable surface assembled PSI density, on different self-assembled alkanethiol monolayers. Using artificial electron donors and acceptors (Os(bpy)(2)Cl(2) and methyl viologen) we demonstrate photocurrent generation from a single PSI layer, which remains photoactive for at least three hours of intermittent illumination. Photoelectrochemical comparison of the biohybrid systems assembled from different alkanethiols (hexanethiol, aminohexanethiol, mercaptohexanol, and mercaptohexanoic acid) reveals that the PSI generated photocurrent is enhanced by almost 5 times on negatively charged SAM surfaces as compared to positively charged surfaces. These results are discussed in light of how PSI is oriented upon electrodeposition on a SAM.

The α-helical coiled-coil is one of the most common oligomerization motifs found in both native and engineered proteins. To better understand the stability and dynamics of coiled-coil motifs, including those modified by fluorination, several fluorinated and non-fluorinated parallel dimeric coiled-coil protein structures were designed and modeled. We also attempt to investigate how changing the length and geometry of the important stabilizing salt bridges influences the coiledcoil protein structure. Molecular dynamics (MD) and free energy simulations with AMBER employed a particle mesh Ewald treatment of the electrostatics in explicit TIP3P solvent with balanced force field treatments. Preliminary studies with legacy force fields (ff94, ff96, ff99) show a profound instability of the coiled-coil structures in short MD simulation. Significantly better behavior is evident with the more balanced ff99SB and ff03 protein force fields. Overall, the results suggest that the coiled-coil structures can readily accommodate the larger acidic arginine or S-2,7-diaminoheptanedoic acid mutants in the salt bridge, whereas substitution of the smaller Lornithine residue leads to rapid disruption of the coiled-coil structure on the MD simulation time scale. This structural distortion of the secondary structure allows both the formation of large hydration pockets proximal to the charged groups and within the hydrophobic core. Moreover, the increased structural fluctuations and movement lead to a decrease in the water occupancy lifetimes in the hydration pockets. In contrast, analysis of the hydration in the stable dimeric coiled coils shows high occupancy water sites along the backbone residues with no water occupancy in the hydrophobic core, although transitory water interactions with the salt bridge residues are evident. The simulations of the fluorinated coiled-coils suggest that in some cases fluorination electrostatically stabilizes the intermolecular coiled-coil salt bridges. Structural analyses also reveal different side chain rotamer preferences for leucine compared to 5,5,5,5′,5′,5′-hexafluoroleucine mutants. These observed differences in the side chain rotamer populations * To whom correspondence should be addressed at the College of Pharmacy, University of Utah. Phone: (801) 587-9652, FAX: (801) 585-9119, e-mail: tec3@utah.edu. Supplementary MaterialSupplementary material is available that includes full sets of RMSd plots, results from force field comparisons, full description of new parameters, plots of water occupancy, more details regarding the free energy calculations, analysis of rotamer populations, and molecular graphics of the structural distortion in the ornithine substituted coiled-coil. suggest differential changes in the side chain conformational entropy upon coiled-coil formation when the protein is fluorinated. The free energy of hydration of the isolated 5,5,5,5′,5′,5′-hexafluoroleucine amino acid is calculated to be 1.1 kcal/mol less stable than leucine; this hydrophobic penalty in the monomer may ...

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