Incorporation and Assembly of a Light-Emitting Enzymatic Reaction into Model Protein Condensates

Guan, Muyang; Garabedian, Mikael V.; Leutenegger, Marcel; Schuster, Benjamin S.; Good, Matthew C.; Hammer, Daniel A.

doi:10.1021/acs.biochem.1c00373

Title: Incorporation and Assembly of a Light-Emitting Enzymatic Reaction into Model Protein Condensates

Journal Article · Thu Oct 14 00:00:00 EDT 2021 · Biochemistry

DOI:https://doi.org/10.1021/acs.biochem.1c00373· OSTI ID:1899602

Guan, Muyang ^[1]; Garabedian, Mikael V. ^[1]; Leutenegger, Marcel ^[2]; Schuster, Benjamin S. ^[3];

^[1];

^[1]

Univ. of Pennsylvania, Philadelphia, PA (United States)
Max Planck Inst. for Biophysical Chemistry, Göttingen (Germany)
Rutgers Univ., New Brunswick, NJ (United States)

Eukaryotic cells partition enzymes and other cellular components into distinct subcellular compartments to generate specialized biochemical niches. A subclass of these compartments form in the absence of lipid membranes, via liquid-liquid phase separation of proteins to form biomolecular condensates or “membraneless organelles” such as nucleoli, stress granules, and P-bodies. Because of their ability to form compartments from simple starting materials, membraneless organelles are an attractive target for engineering new functionalities in both living cells and protocells. In this work, we demonstrate incorporation of novel enzymatic activity in protein coacervates with a light-generating enzyme, NanoLuc, to produce bioluminescence. Using condensates comprised of the disordered RGG domain of C. elegans LAF-1, we show functionalization of condensates with enzymatic activity in vitro and that localization to protein coacervates enhances the assembly and activity of split enzymes. To build condensates that function as light emitting reactors, we designed a NanoLuc enzyme flanked by RGG domains. Furthermore, the resulting condensates concentrated NanoLuc by 10-fold over bulk solution and display significantly increased net reaction rates. We further show that condensate viscosity impacts light emission due to diffusion-limited behavior. By splitting NanoLuc enzyme into its constituent components, we demonstrate that NanoLuc activity can be reconstituted via co-condensation. Further, we demonstrate control of the spatial localization of enzyme within condensates by targettng NanoLuc to the surface of in vitro condensates. Collectively, this work demonstrates that membraneless organelles can be endowed with localized enzymatic activity, and that this activity can be spatially and temporally controlled via enzyme reconstitution and design of protein surfactants.

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Research Organization:: Univ. of Pennsylvania, Philadelphia, PA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES)

Grant/Contract Number:: SC0007063

OSTI ID:: 1899602

Journal Information:: Biochemistry, Vol. 60, Issue 42; ISSN 0006-2960

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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Title: Incorporation and Assembly of a Light-Emitting Enzymatic Reaction into Model Protein Condensates

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