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The study of cavitand-based de novo helical bundle proteins Seo, Emily Satoko

Abstract

The template assembly approach for the de novo design of proteins provides a useful tool for studying protein structure and folding. The template employed here is a cavitand, which is a rigid macrocycle used to organize helical bundles, resulting in a structure called a cavitein (derived from cavitand and protein). The linker length between the individual helical peptides and the cavitand has been shown to have a dramatic effect on the structure and properties of the caviteins: this previous work studied a set of caviteins using a sequence designed to link from the hydrophobic/hydrophilic interface of the peptides. Here, a new series of four-helix bundle caviteins was synthesized using a sequence designed to link from the hydrophobic face (AEELLKKLEELLKKG). By changing the attachment point, the linker length requirement was slightly reduced, and in turn, the packing between the helices was improved. The optimal linker for this new sequence was found to be two glycine residues, as this linker resulted in a cavitein with the most native-like characteristics. Molecular dynamics simulations were carried out on the same series of caviteins studied experimentally. The computer modelling results generally agreed with the experimental data in terms of helical content and conformational specificity. These simulation results were used to better comprehend the behaviour of the caviteins in solution. A peptide sequence designed for a four-helix structure (CGGGEELLKKLEELLKKG) was linked onto various-sized [n]cavitands. The four-helix bundle was found to be the most stable and native-like compared to the five- and six-helix bundles, as the design intended, which shows that the same sequence exhibits different native-like properties depending on the number of helices in a bundle. De novo proteins simplify the interactions involved in protein folding by allowing subtle changes in the design to be made. It has been demonstrated that slight modifications in the cavitein design have dramatic effects on their stability and structural properties. With improved understanding of how the different forces interact to influence the overall structure, it should be possible to design more complex caviteins with function.

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