Novel Method for the Design of a New Alpha-Helical Fold

Abstract

Our group developed a novel description of packing in protein structure that provides an amino acid code to protein design. To test the knob-socket model's application in protein design, a protein fold with five α-helices arranged in a five-pointed star topology has been designed: the helical star fold. For α-helical protein design, the knob-socket model is described as a four residue tetrahedral motif, where a single amino acid knob from one α-helix packs into a three amino acid socket within another α-helix. The three residue sockets can be classified into three categories: (1) free, unpacked favoring helical structures, (2) filled, packed favoring inter-helical interactions, and (3) disfavoring α-helical structure. Each type of socket has previously been characterized and show preferences for certain amino acid compositions. A practical application of the amino acid preferences for each socket type is a rational method for design of α-helical structure. To start the helical star fold, a 37 residue α-helix was constructed with only free sockets that served as the basis sequence to each leg of the star. Using known crossing angles between helices, specific packing patterns were designed between helices by changing the composition of free sockets into filled sockets and corresponding knobs. In the helical star fold, each α-helical leg crosses once with two other α-helices, meaning each helical leg has two patches of filled sockets. Short connecting loops between helices were designed to promote the packing between non-consecutive helical legs and therefore favor the star packing. The theoretical amino acid sequence was used for gene synthesis and cloning for over-expression in E. coli. The novel star protein was purified and analyzed for α-helical content using circular dichroism. The results provide further support of the knob-socket model and code for protein packing.