Oncogenic mutated Ras is a key player in cancer, but despite intense and expensive approaches its catalytic center seems undruggable. The Ras dimer interface is a possible alternative drug target. Dimerization at the membrane affects cell growth signal transduction. In vivo studies indicate that preventing dimerization of oncogenic mutated Ras inhibits uncontrolled cell growth. Conventional computational drug-screening approaches require a precise atomic dimer model as input to successfully access drug candidates. However, the proposed dimer structural models are controversial. Here, we provide a clear-cut experimentally validated N-Ras dimer structural model. We incorporated unnatural amino acids into Ras to enable the binding of labels at multiple positions via click chemistry. This labeling allowed the determination of multiple distances of the membrane-bound Ras-dimer measured by fluorescence and electron paramagnetic resonance spectroscopy. In combination with protein-protein docking and biomolecular simulations, we identified key residues for dimerization. Site-directed mutations of these residues prevent dimer formation in our experiments, proving our dimer model to be correct. The presented dimer structure enables computational drug-screening studies exploiting the Ras dimer interface as an alternative drug target.
Peptide-functionalized nanoparticles (NPs) often rely on a well-defined peptide structure to function. Here, we report the attachment of model peptides to the ligand shell of AuNPs passivated with oligoethylene glycol (OEG). Specifically, peptides containing the repeating (LLKK)n motif plus either one or two reactive functional groups were covalently linked to OEG-capped, ~5 nm AuNPs via the Cu+-catalyzed azide-alkyne cycloaddition reaction. This work builds on a previous study from our group in which an (LLKK)n peptide having two reactive functional groups was considered. Peptide attachment was confirmed by FTIR spectroscopy. Amino acid analysis was used to determine that 3-4 peptides were immobilized per AuNP. Circular dichroism spectroscopy revealed a structural change from random coil in solution to a-helical upon attachment to OEG-capped AuNPs. The key result of this study is that the nature of the capping layer on the AuNP surface influences peptide structure to a significant degree. Other important findings resulting from this work are that the AuNP-peptide conjugates reported here are water soluble and that the long axis of the helical peptides is oriented tangent to the AuNP surface. The latter point is important for applications involving biorecognition.
