Ubiquitin Designer Proteins as a New Additive Generation towards Controlling Crystallization

Abstract

Proteins controlling mineralization in vivo are diverse, suggesting that there are various ways by which mineralization can be directed in bio-inspired approaches. While well-defined three-dimensional (3D) structures occur in biomineralization proteins, the design of synthetic, soluble, bio-inspired macromolecules with specific, reproducible and predictable 3D arrangements of mineral-interacting functions poses an ultimate challenge. Thus, the question how certain arrangements of such functions on protein surfaces influence mineralization, and in which way, subsequently, specific alterations affect this process, remains elusive. Here, we used genetically engineered Ubiquitin (Ub) proteins in order to overcome the limitations of generic bio-inspired additive systems. By advancing existing protocols, we introduced an unnatural amino acid and, subsequently, mineral-interacting functions via selective pressure incorporation and click chemistry, respectively, without affecting the Ub secondary structure. Indeed, as-obtained Ub with three phosphate functions at defined positions shows unique effects, based on a yet unmatched capability towards the stabilization of a film of a dense liquid mineral phase visible even by naked eye, its transformation into amorphous nanoparticles, and afterwards crystals with complex shapes. We thereby demonstrate that Ub designer proteins pose a unique, new generation of crystallization additives where the 3D arrangement of mineral-interacting functions can be designed at will, promising a future use for programmable, target-oriented mineralization control.