Chimeric single α-helical domains as rigid fusion protein connections for protein nanotechnology and structural biology

Fusion proteins are essential tools for heterologous protein expression, purification and crystallization. Non-optimized connections between a fusion protein and the protein of interest are usually flexible. Here we show that the ER/K -helical motif (1) can be seamlessly fused to terminal helices of proteins, forming a chimeric, partially free-standing rigid helix. This modular building block allows the rigid connection of two proteins. Through the intrinsic stability of the ER/K helix, the fusion protein and the protein of interest can be separated by a desired distance, assuring natural freedom of conformation in the latter. We solved the crystal structure of an engineered fusion protein at 1.9 [A] resolution. The construct comprises an N-terminal calmodulin-binding peptide (2,3), an ER/K -helical motif (1), yellow fluorescent protein (YFP)(4), a flexible linker and a C-terminal calmodulin (CaM)(2). The ER/K -helical motif forms a free-standing rigid helix that is continuous with the N-terminal -helix of YFP. Calmodulin is bound to the calmodulin-binding peptide, increasing the mass of the rigid fusion. This molecular biomimetic, protein nanotechnology approach enables new possibilites for the engineering of crystal contacts and for the design of rigid protein particles with increased mass, for example to enable structure elucidation of small proteins by cryo-electron microscopy.