The phase separation of biomolecules
has become the focus of intense
research in the past decade, with a growing body of research implicating
this phenomenon in essentially all biological functions, including
but not limited to homeostasis, stress responses, gene regulation,
cell differentiation, and disease. Excellent reviews have been published
previously on the underlying physical basis of liquid–liquid
phase separation (LLPS) of biological molecules (Nat. Phys.
2015, 11, 899–904) and LLPS
as it occurs natively in physiology and disease (Science
2017, 357, eaaf4382; Biochemistry
2018, 57, 2479–2487; Chem. Rev.
2014, 114, 6844–6879).
Here, we review how the theoretical physical basis of LLPS has been
used to better understand the behavior of biomolecules that undergo
LLPS in natural systems and how this understanding has also led to
the development of novel synthetic systems that exhibit biomolecular
phase separation, and technologies that exploit these phenomena. In
part 1 of this Review, we explore the theory behind the phase separation
of biomolecules and synthetic macromolecules and introduce a few notable
phase-separating biomolecules. In part 2, we cover experimental and
computational methods used to study phase-separating proteins and
how these techniques have uncovered the mechanisms underlying phase
separation in physiology and disease. Finally, in part 3, we cover
the development and applications of engineered phase-separating polypeptides,
ranging from control of their self-assembly to create defined supramolecular
architectures to reprogramming biological processes using engineered
IDPs that exhibit LLPS.
Advances in synthetic biology permit the genetic encoding of synthetic chemistries at monomeric precision, enabling the synthesis of programmable proteins with tunable properties. Bacterial pili serve as an attractive biomaterial for the development of engineered protein materials due to their ability to self-assemble into mechanically robust filaments. However, most biomaterials lack electronic functionality and atomic structures of putative conductive proteins are not known. Here, we engineer high electronic conductivity in pili produced by a genomically-recoded E. coli strain. Incorporation of tryptophan into pili increased conductivity of individual filaments >80-fold. Computationally-guided ordering of the pili into nanostructures increased conductivity 5-fold compared to unordered pili networks. Site-specific conjugation of pili with gold nanoparticles, facilitated by incorporating the nonstandard amino acid propargyloxy-phenylalanine, increased filament conductivity ~170-fold. This work demonstrates the sequence-defined production of highly-conductive protein nanowires and hybrid organic-inorganic biomaterials with genetically-programmable electronic functionalities not accessible in nature or through chemical-based synthesis.
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