Synthesis of functional proteins by mixing peptide motifs.

Here, we describe a synthetic approach for generating artificial proteins by the assemblage of naturally occurring peptide motifs. Two motifs respectively related to apoptosis induction and protein transduction were encrypted into different reading frames of an artificial gene (microgene), which was then polymerized; random frame shifts at the junctions between the microgene units yielded combinatorial polymers of three reading frames. Among the proteins created, #284 was found to penetrate through cell membranes and exert a strong apoptotic effect on several cancer cell lines. Because a simple linkage of these motifs was not sufficient to construct a bifunctional peptide, and the successful reconstitution was dependent on how they were joined together, the combinatorial strategy is important for reconstituting functions from mixtures of motifs. This microgene-based approach represents a novel system for creating proteins with desired functions.

Translated products of tandem microgene repeats exhibit diverse properties also seen in natural proteins.

Repetitiousness is often observed in the primary and tertiary structures of proteins. We are intrigued by the potential role played by periodicity in the evolution of proteins and have created artificial repetitious proteins from repeats of short DNA sequences (microgenes). In this paper we characterize the physicochemical properties of six such artificially created proteins, which are the translated products of repeats of three microgenes. Three of the six proteins contain beta-sheet-like structures and are rather hydrophobic in nature. These proteins form macroscopic membranous structures in the presence of monovalent cationic ions, suggesting they have the capacity to promote strong intermolecular interactions. Of the other three proteins, one is comprised of alpha-helices and two have disordered structures. Small angle X-ray scattering analysis indicates that the artificial proteins do not fold as tightly as natural proteins, but are more compact than if completely denatured. One alpha-helical protein whose microgene unit was designed from coiled coil proteins was crystallized, demonstrating that repetitious artificial proteins can undergo transition to a more ordered state under appropriate conditions. Application of this approach to the development of a novel protein engineering system is discussed.

Distinct macroscopic structures developed from solutions of chemical compounds and periodic proteins.

By controlling the growth of inorganic crystals, macro-biomolecules, including proteins, play pivotal roles in modulating biomineralization. Natural proteins that promote biomineralization are often composed of simple repeats of peptide sequences; however, the relationship between these repetitive structures and their functions remains largely unknown. Here we show that an artificial protein containing a repeated peptide sequence allows NaCl, KCl, CuSO(4) and sucrose to form a variety of macroscopic structures, as represented by their dendritic configurations. Mutational analyses revealed that the physicochemical characteristics of the protein, not the peptide sequence per se, were responsible for formation of the dendritic structures. This suggests that proteins that modulate crystal growth may have evolved as repeat-containing forms at a relatively high rate. These observations could serve as the basis for developing new genetic programming systems for creation of artificial proteins able to modulate crystal growth from inorganic compounds, and may thus provide a new tool for nano-biotechnology.