Endoproteolysis of Oligopeptide-Based Coacervates for Enzymatic Modeling

Abstract

Better insights into the fate of membraneless organelles could strengthen the understanding of the transition from prebiotic components to multicellular organisms. Compartmentalized enzyme reactions in a synthetic coacervate have been investigated, yet there remains a gap in understanding the enzyme interactions with coacervate as a substrate hub. Here, we study how the molecularly crowded nature of the coacervate affects the interactions of the embedded substrate with a protease. We design oligopeptide-based coacervates that comprise an anionic Asp-peptide (D10) and a cationic Arg-peptide (R5R5) with a proteolytic cleavage site. The coacervates dissolve in the presence of the main protease (Mpro) implicated in the coronavirus lifecycle. We capitalize on the condensed structure, introduce a self-quenching mechanism, and model the enzyme kinetics by using Cy5.5-labeled peptides. The determined specificity constant (k cat/KM) is 5817 M–1 s–1 and is similar to that of the free substrate. We further show that the enzyme kinetics depend on the type and quantity of dye incorporated into the coacervates. Our work presents a simple design for enzyme-responsive coacervates and provides insights into the interactions between the enzyme and coacervates as a whole.

Activatable prodrug for controlled release of an antimicrobial peptide via the proteases overexpressed in Candida albicans and Porphyromonas gingivalis

“…Once cleaved by SAP9, the dyes separate, causing an increased fluorescent signal. The probe was incubated with recombinant SAP9 (200 nM) at 37 °C and fluorescence was monitored longitudinally ( Supplemental Figure S5 ) to calculate the k cat /K M value as ~55,000 M -1 s -1 ( Figure 1 E ) 48 . The cleavage efficiency of RgpB was measured similarly ( Supplemental Figure S6 ) and a slightly higher k cat /K M value was calculated at ~71,000 M -1 s -1 49 , 50 .…”

Activatable prodrug for controlled release of an antimicrobial peptide via the proteases overexpressed inCandida albicansandPorphyromonas gingivalis

“…This probe was incubated with 200 nM of the recombinant enzyme at 37 °C and fluorescence was monitored longitudinally to calculate the k cat /K M value as ∼55,000 M −1 s −1 ( Supplemental Figure S4 ). [43] The cleavage efficiency of RgpB is known from previous work to be 12,000 M −1 s −1 . [44] [45].…”

Looking back, moving forward: protein corona of lipid nanoparticles

“…Numerous studies have highlighted that the presence of the PC modifies the structure and characteristics of the surface to influence the interactions between NPs and biological entities, consequently altering the in vivo fate. 122,123 This scenario is also applied to the LNPs.…”