Label-Free Fluorescence Quantification of Hydrolytic Enzyme Activity on Native Substrates Reveals How Lipase Function Depends on Membrane Curvature

Abstract

Lipases are important hydrolytic enzymes used in a spectrum of technological applications, such as the pharmaceutical and detergent industry. Due to their versatile nature and ability to accept a broad range of substrates they have been extensively used for biotechnological and industrial applications. Current assays to measure lipase activity primarily rely on low sensitivity measurement of pH variations or visible changes on material properties, like hydration, and often require high amount of proteins. Fluorescent readouts on the other hand offer high contrast and even single molecule sensitivity, albeit they are reliant on fluorogenic substrates that structurally resemble the native ones. Here we present a method that combines the highly sensitive readout of fluorescent techniques while reporting enzymatic lipase function on native substrates. The method relies on embedding the environmentally sensitive fluorescent dye pHrodo and native substrates into the bilayer of liposomes. The charged products of the enzymatic hydrolysis alter the local membrane environment and thus the fluorescence intensity of pHrodo. The fluorescence can be accurately quantified and directly assigned to product formation and thus enzymatic activity. We illustrated the capacity of the assay to report function of diverse lipases and phospholipases both in a microplate setup and at the single particle level on individual nanoscale liposomes using Total Internal Reflection Fluorescence (TIRF). The parallelized sensitive readout of microscopy combined with the inherent polydispersity in sizes of liposomes allowed us to screen the effect of membrane curvature on lipase function and identify how mutations in the lid region control the membrane curvature dependent activity. We anticipate this methodology to be applicable for sensitive activity readouts for a spectrum of enzymes where the product of enzymatic reaction is charged..

The dopamine transporter antiports potassium to increase the uptake of dopamine

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Carbohydrate‐Derived Metal‐Chelator‐Triggered Lipids for Liposomal Drug Delivery

“…Hundreds of liposomes per field of view were tethered on poly( l ‐lysine)‐poly(ethylene glycol) (PLL‐PEG)‐passivated and PLL‐PEG biotin‐passivated surfaces [23, 24] . This methodology maintains the spherical shape and structural integrity of the liposomes [25] .…”

“…The parallel readout and analysis of hundreds (>700) of individual liposomes on six surfaces before and after Na 2 EDTA addition allowed us to quantify the fraction of 1⋅Zn liposomes the responded to Zn 2+ removal [23, 25, 26] . It was found that 65.0±4.4 % of the liposomes were active and responded to Na 2 EDTA (Figure 3 g).…”

Single particle combinatorial multiplexed liposome fusion mediated by DNA

“…Fluorescence microscopy is a powerful and sensitive detection tool [17][18][19] , but its capability for multiplexing is limited by the spectral overlap between chromophores, restricting quantitative imaging to a handful of fluorescent colors 20,21 . Fluorescent barcoding technologies can overcome this, offering some success in multiplexing for both in vitro and in vivo imaging.…”

“…The method combines chromophore ratio labelling creating distinct identity encoding barcodes for each DNA encoded nanocontainer and the use of complementary LiNA mediated nanocontainer fusion. Using TIRF microscopy allowed the parallelized imaging of ~8800 individual target lipidic containers [17][18][19]28,29 undergoing >16,000 fusion events with barcoded cargo nanocontainers and up to seven successive rounds of fusion. The fusion sequence is completely stochastic allowing >550 distinct permutations that are directly recorded and precisely classified by machine learning analysis.…”