Fig. 3: Substrate and product localisation inside HH_min/peptide coacervate droplets.

Ai HH_min structure (in black) shows substrate (in blue) binding and its cleavage location (red arrow). 3′ Alexa 532 (red) and 5′ FAM (green) dyes indicate the position of the FRET pair on the substrate. Aii Merged excitation and emission spectra of FAM and Alexa 532 dyes used in the FRET substrate. Emission band filters used to detect the two dyes are λ_Em1 = 614–660 nm for the substrate and λ_Em2 = 490–509 nm for the product, marked as the red- and green-checked regions with fixed excitation at λ = 488 nm. Bi Confocal microscopy images show the localisation of the FRET-substrate (top: λ_Em1) and cleaved product (bottom: λ_Em2) inside a HH_min/P-3 coacervate droplet 5 min, 20 min and 60 min after the addition of substrate. Scale bar: 5 µm. Bii Line profiles showing the fluorescence distribution along the droplet diameter (yellow dotted line of Bi). Ci Confocal microscopy images showing localisation of FRET-substrate (top: λ_Em1) and cleaved product (bottom: λ_Em2) inside a Mut-HH/P-3 coacervate droplet 5 min, 20 min and 60 min after the addition of substrate. Fluorescence intensity in the product channel was much weaker with Mut-HH relative to HH_min. Scale bar: 5 µm. Cii Line profiles showing the fluorescence distribution along the droplet diameter (yellow dotted line of Ci). Di Comparative fluorescence intensities of the substrate (λ_Em1) as a function of time obtained from analysis of whole droplet microscopy images of HH_min/P-3 and Mut-HH/P-3 taken under identical conditions. Curves show the average mean intensity of two droplets for each coacervate system and the shaded regions show the standard deviation. Dii Same comparison as above for the product (λ_Em2). The experiments were repeated at least three times. Source data is provided in the repository.