Research on the construction of complex protein using an alpha helix fusion method and its application

Abstract

To accurately design and generate artificial proteins and protein complexes, the method which can stably and predictably connect protein components is required. In this research, we have shown that ‘alpha helix fusion method’ can rigidly and predictably connect protein components. The successful design of these novel fusion proteins was confirmed by X-ray crystallography. Repeat proteins such as ankyrin repeats or leucine-rich repeat (LRR) proteins have multiple copies of repeating units. They have applications in protein engineering and drug design, and we could generate several repeat proteins which are highly similar to the shape of ankyrin or LRR proteins. Therefore, ‘alpha helix fusion method’ can be used to develop novel proteins mimicking the structure or function of biologically and therapeutically important natural proteins. For single particle analysis using cryo-EM, molecular weight of protein needs to be higher than 100 kDa, and existence of symmetry at the target protein is helpful. By using the alpha helix fusion method, we could generate tetrameric protein complex. Since its high molecular weight (>200 kDa) and twofold symmetry, this tetrameric complex generated by the alpha helix fusion method may be a convenient tool for the cryo-EM analysis of small proteins.