Single Enzyme Studies Reveal the Existence of Discrete Functional States for Monomeric Enzymes and How They Are “Selected” upon Allosteric Regulation

Hatzakis, Nikos S.; Li, Wei; Jørgensen, Sune K.; Kunding, Andreas Hjarne; Bolinger, Pierre-Yves; Ehrlich, Nicky; Makarov, Ivan; Skjøt, Michael; Svendsen, Allan; Hedegård, Per; Stamou, Dimitrios

doi:10.1021/ja3011429

Cited by 34 publications

(67 citation statements)

References 51 publications

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“…Single-molecule studies on the other hand offer the exquisite ability to directly observe the temporal fluctuations of individual enzymes and their role in regulation of their activities. 29,30 Single-enzyme studies were pioneered for ion channel current recordings and, more recently, single-enzyme studies on transporters have visualized the structural dynamics of transmembrane helices. 31 However, neither method can directly report on proton pumping during continuous turnover.…”

Section: Introductionmentioning

confidence: 99%

Single Enzyme Experiments Reveal a Long-Lifetime Proton Leak State in a Heme-Copper Oxidase

Jørgensen

McMillan

et al. 2015

J. Am. Chem. Soc.

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Heme-copper oxidases (HCOs) are key enzymes in prokaryotes and eukaryotes for energy production during aerobic respiration. They catalyze the reduction of the terminal electron acceptor, oxygen, and utilize the Gibbs free energy to transport protons across a membrane to generate a proton (ΔpH) and electrochemical gradient termed proton motive force (PMF), which provides the driving force for the adenosine triphosphate (ATP) synthesis. Excessive PMF is known to limit the turnover of HCOs, but the molecular mechanism of this regulatory feedback remains relatively unexplored. Here we present a single-enzyme study that reveals that cytochrome bo3 from Escherichia coli, an HCO closely homologous to Complex IV in human mitochondria, can enter a rare, long-lifetime leak state during which proton flow is reversed. The probability of entering the leak state is increased at higher ΔpH. By rapidly dissipating the PMF, we propose that this leak state may enable cytochrome bo3, and possibly other HCOs, to maintain a suitable ΔpH under extreme redox conditions.

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Section: Introductionmentioning

confidence: 99%

Single Enzyme Experiments Reveal a Long-Lifetime Proton Leak State in a Heme-Copper Oxidase

Jørgensen

McMillan

et al. 2015

J. Am. Chem. Soc.

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“…15). Recent experiments of this category are focused at interaction of peptides and proteins with vesicles, function of single membrane-active enzymes, [19][20][21] encapsulation efficiency, 22 and vesicle docking and fusion. 23 Alternatively, suspended vesicles may interact with various species attached to a supported lipid bilayer.…”

Section: Introductionmentioning

confidence: 99%

“…24 In all these experiments, the kinetics were simultaneously tracked on a large number of individual fluorescent dye-labelled vesicles by using total internal reflection fluorescence microscopy (TIRFM) 17,20,21,24 or confocal fluorescence microscopy (CFM). 14,15,19,22,23 (Historically, TIRFM was introduced to study various surface bioprocesses in the early 1980s; 25,26 for CFM and combination of TIRFM and CFM, see Refs. 27 and 28, respectively; the application of TIRFM and CFM to immobilized vesicles started in the early 2000s.…”

Section: Introductionmentioning

confidence: 99%

Total internal reflection fluorescence microscopy for determination of size of individual immobilized vesicles: Theory and experiment

Olsson

Zhdanov

Höök

2015

Journal of Applied Physics

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Lipid vesicles immobilized via molecular linkers at a solid support represent a convenient platform for basic and applied studies of biological processes occurring at lipid membranes. Using total internal reflection fluorescence microscopy (TIRFM), one can track such processes at the level of individual vesicles provided that they contain dyes. In such experiments, it is desirable to determine the size of each vesicle, which may be in the range from 50 to 1000 nm. Fortunately, TIRFM in combination with nanoparticle tracking analysis makes it possible to solve this problem as well. Herein, we present the formalism allowing one to interpret the TIRFM measurements of the latter category. The analysis is focused primarily on the case of unpolarized light. The specifics of the use of polarized light are also discussed. In addition, we show the expected difference in size distribution of suspended and immobilized vesicles under the assumption that the latter ones are deposited under diffusion-controlled conditions. In the experimental part of our work, we provide representative results, showing explicit advantages and some shortcomings of the use of TIRFM in the context under consideration, as well as how our refined formalism improves previously suggested approaches. V C 2015 AIP Publishing LLC. [http://dx

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“…Single particle tracking studies on the other hand report the spatiotemporal localization of enzymes, providing unprecedented insights on the behavior of diverse mutants but to date rely on correlations of diffusion to function [27][28][29][30] . Assays based on fluorogenic substrates offers low consumption and high sensitivity, even at the fundamental limit of single turnover cycles [31][32][33] , albeit at the cost of using substrate analogues that resemble the native enzyme substrates.…”

Section: Introductionmentioning

confidence: 99%

Label free fluorescence quantification of hydrolytic enzyme activity on native substrates reveal how lipase function depends on membrane curvature

Bohr

Thorlaksen

Kühnel

et al. 2020

Preprint

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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.

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Single Enzyme Studies Reveal the Existence of Discrete Functional States for Monomeric Enzymes and How They Are “Selected” upon Allosteric Regulation

Cited by 34 publications

References 51 publications

Single Enzyme Experiments Reveal a Long-Lifetime Proton Leak State in a Heme-Copper Oxidase

Single Enzyme Experiments Reveal a Long-Lifetime Proton Leak State in a Heme-Copper Oxidase

Total internal reflection fluorescence microscopy for determination of size of individual immobilized vesicles: Theory and experiment

Label free fluorescence quantification of hydrolytic enzyme activity on native substrates reveal how lipase function depends on membrane curvature

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