Research on structurally rigid and predictable protein fusion using alpha helix structure

Abstract

Introducing covalent modifications to proteins have been researched for the objectives

providing new functions via cross-linking functional modules, fixing proteins in a specific state, or giving extra structural rigidity to the proteins. Since structural stabilization was achieved with fixing the relative positions between domains or protein, researches building artificial nanostructures of proteins have been proposed. Nevertheless most methods for designing artificial structure assembly require intensive computational simulations for designing interfaces or links. Here I propose a novel method for structurally predictable protein fusion via connecting pre-existing alpha helices of two proteins into a long extended helix. I introduce EY-CBS as a probe of alpha helix formation by cross-linking two cysteines in the $i^{th}$ and $i+11^{th}$ positions of the alpha helix, as reported by Zhang et al. And I propose SDS-PAGE as a fast and reliable tool for detecting the binding of EY-CBS. The amplitude of upshift in the SDS-PAGE could distinguish structurally correct bound cross-linker, which was supported by the crystal structure of mbp3-16 - protein A fusion protein with bound EY-CBS. Binding of EY-CBS shows an effect of structural idealization of alpha helix, while the native structure of fusion alpha helix was bent in the crystal structure. Additional screenings of fusion constructs for EY-CBS reactivity show that selection of fusing target affects reactivity, with the relative orientation between two fusing counterparts. The crystal structure from the construct, which did not react with EY-CBS, discovered totally disrupted alpha helix structure upon fusion. The formation of alpha helix in the crystal structure was consistent with the reactivity results of EY-CBS. In addition, I tested various cross-linkers with different reacting groups and backbones for ‘distance-selectivity’ of cross-linkers using constructs with various cys-cys distance. In the experiment cross-linker having haloalkane group with rigid, hydrophilic backbone shows the best distance-selectivity over other cross-linkers. In conclusion, I provide a new method for structurally predictable fusion using alpha helix structure and introduce SDS-PAGE as a fast and reliable detecting tool for structurally sound binding of cross-linker. The binding of cross-linker may represent proper folding of alpha helix upon fusion, and it even structurally idealize the fusion alpha helix. And the cross-linkers with haloalkane groups show better distance-selectivity over maleimide cross-linkers, concluding that selecting reaction group in cross-linker is important for detecting alpha helix structure with high precision.