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

Abstract

SummaryChimeric fusion proteins are essential tools for protein nanotechnology. Non-optimized protein-protein connections are usually flexible, which makes them unsuitable as structural building blocks. Here we show that the ER/K motif, a single α-helical domain (SAH)1, can be seamlessly fused2to terminal helices of proteins, forming an extended and partially free-standing rigid helix. Through the intrinsic stability of the SAH, two domains can be connected with a defined distance and orientation. We designed three constructs termed YFPnano, T4Lnano, and MoStoNano, and we show that a single SAH allows the connection of two separate structural domains with sufficient rigidity to form ordered crystals. The analysis of experimentally determined structures and molecular dynamics simulations reveals a certain degree of plasticity in the connections that allows the adaptation to crystal contact opportunities. Our data show that SAHs can be stably integrated into designed structural elements, enabling new possibilities for protein nanotechnology, for example to improve the exposure of epitopes on nanoparticles (structural vaccinology), to engineer crystal contacts with minimal impact in construct flexibility (for the study of protein dynamics), and to design novel biomaterials.