Exceptionally high-affinity Ras binders that remodel its effector domain.

The Ras proteins are aberrantly activated in a wide range of human cancers, often endowing tumors with aggressive properties and resistance to therapy. Decades of effort to develop direct Ras inhibitors for clinical use have thus far failed, largely because of a lack of adequate small-molecule-binding pockets on the Ras surface. Here, we report the discovery of Ras-binding miniproteins from a naïve library and their evolution to afford versions with midpicomolar affinity to Ras. A series of biochemical experiments indicated that these miniproteins bind to the Ras effector domain as dimers, and high-resolution crystal structures revealed that these miniprotein dimers bind Ras in an unprecedented mode in which the Ras effector domain is remodeled to expose an extended pocket that connects two isolated pockets previously found to engage small-molecule ligands. We also report a Ras point mutant that stabilizes the protein in the open conformation trapped by these miniproteins. These findings provide new tools for studying Ras structure and function and present opportunities for the development of both miniprotein and small-molecule inhibitors that directly target the Ras proteins.

A new i, i + 3 peptide stapling system for α-helix stabilization.

We have previously shown that the incorporation of an 8-atom all-hydrocarbon 'staple' at positions i and i + 3 of a synthetic peptide results in substantial stabilization of the α-helical conformation. As part of our ongoing effort to explore the scope and utility of all-hydrocarbon stapling systems, we have investigated and report herein the properties of a new i, i + 3 stapling system that employs a 6-carbon cross-link.

Stepwise silver-staining-based immunosorbent assay for amyloid-beta autoantibody detection.

AIMS: To detect amyloid-beta (Abeta) autoantibodies in a reliable and high-throughput fashion, we developed a stepwise silver-staining-based immunosorbent assay in a 96-well-plate platform. MATERIALS & METHODS: Abeta autoantibodies were incubated in an Abeta-immobilized 96-well microplate. Antihuman IgG-modified gold nanoparticle probes were then used to bind to the autoantibodies and signal enhancement was carried out with stepwise silver-staining on immobilized gold nanoparticle probes. A microplate reader was used to quantify silver-stained gold nanoparticles on a well-plate surface. RESULTS & DISCUSSION: Stepwise silver-staining at low temperature enables long-term silver-staining with minimal increase of background signal. This stepwise staining method helps solve the problems of one-step staining, such as nonspecific binding or nonuniformity in silver precipitation after prolonged silver-staining for signal enhancement. CONCLUSION: A stepwise silver-staining strategy could be useful in minimizing nonspecific background signals. This 96-well-plate-based Abeta antibody detection assay could be useful in studying and diagnosing Alzheimer's disease.

Ultrasensitive optical biodiagnostic methods using metallic nanoparticles.

Dramatic progress has been made over the recent decade in the applications of metallic nanoparticles in the field of biomolecule detection. The useful physical and chemical properties (e.g., availability of various synthetic methods of size- and shape-controlled nanoparticles, size- and shape-dependent optical properties, availability of various surface chemistries and biocompatibility) of metallic nanoparticles have brought development to the ultrasensitive detection of biomolecules at the attomolar level and this sensitivity enables the diagnosis of otherwise undetectable biomarkers of many fatal diseases, including Alzheimer's disease. Furthermore, coupled with the strong physical properties and biocompatible nature of gold nanoparticles in in vivo conditions, the scope of applications for these particles have been broadened into the field of in vivo imaging, such as X-ray contrasting agents, and also cellular tracking. Here, we review synthetic methods and optical properties of metallic nanoparticles and their use in ultrasensitive, in vitro and in vivo biodiagnostic methods.