Inside a Shell—Organometallic Catalysis Inside Encapsulin Nanoreactors

Abstract

Compartmentalization of chemical reactions inside cells are af undamental requirement for life.E ncapsulins are self-assembling protein-based nanocompartments from the prokaryotic repertoire that present ahighly attractive platform for intracellular compartmentalization of chemical reactions by design. Using single-molecule Fçrster resonance energy transfer and 3D-MINFLUX analysis,weanalyzefluorescently labeled encapsulins on as ingle-molecule basis.F urthermore, by equipping these capsules with asynthetic ruthenium catalyst via covalent attachment to an on-native host protein, we are able to perform in vitro catalysis and go on to showt hat engineered encapsulins can be used as hosts for transition metal catalysis inside living cells in confined space.

Sequential, all-bioorthogonal reaction cascade catalyzed by a dual functional artificial metalloenzyme inside encapsulin

“…A crucial step in ArM synthesis is targeted and exhaustive metal to protein binding as well as subsequent removal of unbound components. 5 For establishing a labeling strategy of the dual functional guest protein inside encapsulin, we developed a pulse chase experiment. It includes the TM-complexes RuH and AuB besides the fluorescent Halo ligand AlexaFluor660 (AF660L, λexc=668 nm, λem=698 nm) and the avidin ligand, fluorescein-4-biotin (f4b, λexc=498 nm, λem=517 nm).…”

“…4 Previously, we described the application of a nanoreactor for bioorthogonal pro-fluorophore activation inside living cells using encapsulin from Mycobacterium smegmatis (M. smegmatis). 5 The smallest representatives of Encs known to date consist of 60 protein monomers, which self-assemble in a pentagon dodecahedron-like structure with a sphere diameter of 24 nm. Small molecule reactants can pass the proteinaceous shell via pores lying in the symmetry axes of the dodecahedral structure.…”

“…Moreover, future perspectives in ArM and nanoreactor research are shown. 5,[13][14][15] Here, we describe the first proteinaceous nanoreactor catalyzing a two-step, bimetallic, sequential, allbioorthogonal reaction cascade. In this context, Figure 1 shows previous research, the present work and potential developing goals being addressed.…”

“…In this context, Figure 1 shows previous research, the present work and potential developing goals being addressed. For stable immobilization of two orthogonal transition metal (TM) complexes inside Enc, we designed a dual functional fusion protein based on HaloTag (Halo) 5,16 and monomeric Biotin-(strept)avidin. 17 Several monomeric analogues of avidin-type proteins have been described, but only one report of a monomeric avidin based ArM exists.…”

“…The first step of the presented reaction cascade involves the alloc-deprotection of a secondary amine catalyzed by a ruthenium catalyst (RuH, see Figure 2) covalently bound to HaloTag. 5 The deprotected amine serves as nucleophile in a subsequent gold-catalyzed hydroamination reaction at the monomeric avidin analogue. 23 This reaction is catalyzed by a Buchwald-type Au(I)-phosphine complex (AuB, see Figure 2), that we introduce here as a novel bioorthogonal catalyst.…”