Design and laboratory evolution of artificial proteins

点击翻译按钮获取中文摘要

To shed light on the principles of protein architecture in a new aspect that is unattainable by orthodox biochemistry of natural proteins, we have developed a methodology to design artificial proteins with native-like properties as follows.1.We analyzed the distribution of the side-chain rotational isomers (rotamers) of hydrophobic amino acids in protein tertiary structures and derived ΔS^<contact>, the conformational-entropy changes of side chains by residue-residue contacts in each secondary structure to find the origin of the structural uniqueness in native amino acid sequences, which can be used as a specificity parameter for designing artificial proteins with a unique structure.2.We derived a new potential function for protein design from a protein 3D structural database. This function can assess fitness of each amino-acid rotamer to a site environment in protein 3D structures. By using this function, an artificial sequence of the sixty amino acids to stabilize the backbone tertiary structure was designed and synthesized. The synthesized artificial protein folds into the targeted dimer with the secondary structure contents similar to those of native λCro.3.We prepared biotinylated heme and investigated its usage for detection and purification of hemoproteins. The purification of native and artificial heme-binding proteins from recombinant cell extracts using biotinyl heme was demonstrated. Thus the molecule is useful for laboratory evolution of designed hemoproteins.