Use of a heterodimeric coiled-coil system for biosensor application and affinity purification.

The two-stranded alpha-helical coiled-coil is now recognized as one of nature's favorite ways of creating a dimerization motif. Based on the knowledge of protein folding studies and de novo design model systems, a novel heterodimeric coiled-coil protein was synthesized. The heterodimeric E/K coiled-coil was constructed with two distinct peptides (E and K) that will spontaneously associate into a full helical coiled-coil structure in solution. Equilibrium CD, NMR and real time biosensor kinetics experiments showed that the E/K coiled-coil is both structurally (deltaG(unfold)=11.3 kcal/mol) and kinetically (Kd approximately 1 nM) stable in solution at neutral pH. The engineered coiled-coil had been applied as a dimerization and capture domain for biosensor based applications and used in an expression/detection/affinity chromatography system. Specific test examples demonstrated the usefulness of the E/K heterodimeric system in these applications. The universality of coiled-coil as a dimerization motif in nature and our ability to design and synthesize these proteins suggest a wide variety of applications.