Using Optical Tweezers to Dissect Allosteric Communication Networks in Protein Kinases

Abstract

Mutations in protein kinases are often associated with the development of cancer, and application of mutant-specific inhibitors as therapeutic measures have shown a remarkable improvement in prolonging patient survival. However, it has also been observed that tumors bearing certain mutation types are more resistant to current approved drugs. Importantly, many resistant mutations are located in regions outside substrate or inhibitor binding sites, indicating allosteric effects. Understanding how mutations trigger effects over a distant site of the protein requires a deeper investigation of the molecular origin of allosteric regulation networks in kinases. In this chapter, we show the application of single-molecule optical tweezers to selectively manipulate specific regions of proteins to trace allosteric signals, thereby allowing the elucidation of allosteric communication networks. We illustrate this approach using as model system the regulatory subunit of protein kinase A. This single-molecule optical tweezers approach, however, can be readily applicable to study other kinases, and can be further expanded to screen potential allosteric drugs for future therapeutics.

Keywords: Allostery; DNA-protein covalent attachment; Force-extension curves; Kinases; Mechanical fingerprints; Selective manipulation; Single molecule optical tweezers.