Inside a Shell-Organometallic Catalysis Inside Encapsulin Nanoreactors

Abstract

Compartmentalization of chemical reactions inside cells are a fundamental requirement for life. Encapsulins are self-assembling protein-based nanocompartments from the prokaryotic repertoire that present a highly attractive platform for intracellular compartmentalization of chemical reactions by design. Using single-molecule Förster resonance energy transfer and 3D-MINFLUX analysis, we analyze fluorescently labeled encapsulins on a single-molecule basis. Furthermore, by equipping these capsules with a synthetic ruthenium catalyst via covalent attachment to a non-native host protein, we are able to perform in vitro catalysis and go on to show that engineered encapsulins can be used as hosts for transition metal catalysis inside living cells in confined space.

Keywords: MINFLUX; encapsulins; homogeneous catalysis; self-assembly; single-molecule FRET.

Figure 1

Purification and characterization of encapsulin from M. smegmatis. A) Native and denaturing PAGE analysis of purified ^StrepEnc_SM. The expected mass of a 60‐mer assembly is 1.8 MDa. B) TEM image (negative staining) of empty ^StrepEnc_SM. Number of capsids assessed for diameter calculation (n)=106.

Figure 2

Characterization of ^StrepEnc_SM loaded with eGFP. Four cargo protein constructs, tagged with one short peptide of the putative ELS (6 aa, 9 aa, 12 aa and 19aa long; ctrl. 0 aa and no eGFP). A) Native PAGE analysis of the co‐purified constructs and analysis by Coomassie staining and in‐gel fluorescence. B) Quantitative assessment of the fluorescence intensities detected by in‐gel fluorescence using ImageJ. The analysis was done in triplicate. Error bars represent ± SEM. Key: amino acid (aa), encapsulin localization sequence (ELS).

Figure 3

Quantification of HaloTag labels inside ^StrepEnc_SM by single‐molecule fluorescence. A) smFRET measurements of ^StrepEnc_SM{HaloTag*JF549*JF646}. All photon burst search (APBS), time window 500 μs, threshold 50 photons, FRET‐2CDE filter: 0–10, DA rate: 15–20 kHz, DD rate: 2–20 kHz, burst duration 3–20 ms. B) Dual color burst search (DCBS), time window 500 μs, DD rate: 2–200 kHz, AA rate 5–50 kHz, DA rate: 20–150 kHz, FRET‐2CDE filter: 5–15, burst duration 2–7 ms, 2 Gaussian fits.

Figure 4

MINFLUX measurements of ^StrepEnc_SM{HaloTag‐AF647}. A) Off‐switching count trace with visible off‐switching steps for a single ^StrepEnc_SM{HaloTag‐AF647} construct. B) Estimated number of fluorophores per ^StrepEnc_SM{HaloTag‐AF647} construct for all measurements (histogram, n=125) and median of the distribution (dashed line). C) 3D MINFLUX localizations of the single ^StrepEnc_SM{HaloTag‐AF647} construct. Each scatter dot represents an individual localization estimate for the position of a fluorescent AF647 molecule. The color‐coding describes when the localization was registered after starting the acquisition. Individual fluorescent molecules can deliver tens of localizations. We centered the localization with the origin by subtracting the mean position from all localizations. A sphere of 14 nm diameter (gray) encloses 94 % of localizations.

Figure 5

Uncaging reaction of 1 (125 μM, 1 equiv) to 2. Anaerobic labeling and washing steps with standard aerobic reaction conditions. Yields were determined by fluorescence intensity in three independent experiments (λ _ex=339 nm, λ _em=439 nm). Error bars represent ± SEM. Key: ^StrepEnc_SM{HaloTag‐PEG₃‐Ru} (black square), HaloTag‐PEG₃‐Ru (blue triangle), ^StrepEnc_SM{GFP} (green triangle), CA‐PEG₃‐Ru (5) (turquoise diamond).

Figure 6

Fluorescence microscopy and bright‐field images of J774A.1 monocytes treated with ^StrepEnc_SM{HaloTag‐PEG₃‐Ru} + 1, CA‐PEG₃‐Ru (5) + 1 and 1 only (negative controls). Cells were seeded and treated with 1 μM ^StrepEnc_SM{HaloTag‐PEG₃‐Ru} in DMEM overnight. Subsequently, 1 (20 μM) was applied to the cell supernatant. Following incubation and washing, images were acquired with a digital inverted microscope. Fluorescence microscopy images of monocytes treated with encapsulin show fluorescence from uncaging product 2 that is localized within distinct vesicles. Fluorescence microscopy images show no blue fluorescence in cells treated with 1 only. Key: Dulbecco's Modified Eagle Medium (DMEM).