Design, synthesis and characterization of artificial proteins for biomolecular materials

Ting Xu, Department of Chemistry, University of Pennsylvania, and Cold neutrons for biology and technology, NIST

Artificial protein models, based on both alpha-helical bundle and beta-sheet structural motifs, can be designed to incorporate biological cofactors and retain a range of specific functional elements of their natural counterparts, but within much more simple structures. Amphiphilic 4-helix bundle peptides have been previously designed to selectively incorporate heme and other natural cofactors within both the hydrophilic and hydrophobic domains and have proven to possess characteristic electronic and optical properties of natural electron-transfer proteins. However, it is non-trivial to incorporate both the electron donor and acceptor inside the 4-helix bundles in a controlled manner. Extended pi-electron systems have been designed and tailored, with appropriate donors, acceptors and constituents, to exhibit selected light-induced electron transport and/or proton translocation over large distances. These non-biological cofactors have the advantage of including the electron donor and the acceptor within the same prosthetic group and offer an independent means to modulate the properties of electronically excited states, intrinsic cofactor midpoint potentials, cofactor-cofactor electronic coupling, and the nature of the charge distribution.

We studied the binding between a series of non-biological metalloporphyrin cofactors and the designed amphiphilic 4-helix bundle peptides at selected locations. The interior of the artificial protein was used to control the solubility, position, orientation of the cofactors, while the exterior was used to control the macroscopic orientation. Designed amphiphilic peptides are cable of binding the non-biological cofactor, Zn33Zn, at selected locations via bis-histidyl ligation. Incorporation of the Zn33Zn into the 4-helix bundles did not interfere the protein's secondary structure nor the 4-helix bundle formation. The cofactor binding occurs with a broad range of the ionic strength and surfactant concentration. The cofactor/peptide stochiometry can be tuned from 1 to 4 cofactors per 4-helix bundle simply by varying the peptide oligomeric state and surfactant concentration. The Zn33Zn binding affinities are comparable at different binding sites along the helix. This offers great control over the exact location of the cofactor within the peptide scaffold. The protein/cofactor complexes are very robust and are ideal building blocks toward biomaterials. The amphiphilic protein/cofactor complexes are thermally stable up to 85C and maintain more than 85% of the original helicity up to 80C. This development may potentially lead to functional biomaterials with novel electron transfer properties and it is crucial to control the macroscopic ordering of the artificial proteins in one, two or three dimensions. The artificial protein Langmuir monolayers, both the apo- and holo-form, can be oriented vectorially at the air/water interface upon compression as shown by the x-ray reflectivity data. However, Grazing Incidence X-ray Diffraction data from Langmuir monlayers at higher surface pressure show a broad maximum for momentum transfer parallel to the monolayer plane. This diffraction arises from the interference between parallel helices and demonstrates that the di-helices aggregate to form 4-helix bundles with glass-like inter-bundle positional ordering in the monolayer plane. Nanoporous thin films made from diblock copolymers are ideal templates to assemble the artificial proteins with laterally hexagonal order. We will also discuss the efforts on re-designing the artificial proteins and incorporate them into block copolymer based nanoporous templates.

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