Single molecule kinetics of horseradish peroxidase exposed in large arrays of femtoliter-sized fused silica chambers

Ehrl, Benno N.; Liebherr, Raphaela B.; Gorris, Hans H.

doi:10.1039/c3an00809f

Cited by 24 publications

(21 citation statements)

References 29 publications

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“…However, the complexity is narrowed or even eliminated when the enzyme is deformed or unfolded under magnetic pulling force. microseconds it provides a direct readout of enzymatic reactions with single turnover resolution (5,8,9,(22)(23)(24). Additionally, fluorescent product molecules are produced continuously in turnover cycles and this eliminates the negative impact of photobleaching, and thereby long time trajectories from a single enzyme can be recorded (8,9).…”

Section: Significancementioning

confidence: 99%

Probing conformational dynamics of an enzymatic active site by an in situ single fluorogenic probe under piconewton force manipulation

Pal

2016

Proc. Natl. Acad. Sci. U.S.A.

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Unraveling the conformational details of an enzyme during the essential steps of a catalytic reaction (i.e., enzyme-substrate interaction, enzyme-substrate active complex formation, nascent product formation, and product release) is challenging due to the transient nature of intermediate conformational states, conformational fluctuations, and the associated complex dynamics. Here we report our study on the conformational dynamics of horseradish peroxidase using single-molecule multiparameter photon time-stamping spectroscopy with mechanical force manipulation, a newly developed single-molecule fluorescence imaging magnetic tweezers nanoscopic approach. A nascent-formed fluorogenic product molecule serves as a probe, perfectly fitting in the enzymatic reaction active site for probing the enzymatic conformational dynamics. Interestingly, the product releasing dynamics shows the complex conformational behavior with multiple product releasing pathways. However, under magnetic force manipulation, the complex nature of the multiple product releasing pathways disappears and more simplistic conformations of the active site are populated.enzymatic conformational dynamics | magnetic tweezers | mechanical force manipulation | enzymatic product releasing | fluorogenic substrate E nzymes are capable of enhancing biological reaction activity by millions or even billions of times (1). A typical enzymatic turnover cycle involves multiple steps: substrate (S) binding to the active site of the enzyme (E) in forming an enzyme-substrate complex E+S→[E · S]; enzymatic reaction converting substrate to nascent product (P), [E · S]→[E · P]; and product releasing from the enzyme active site, [E · P]→E+P, which completes an enzymatic reaction turnover (2-4). In recent years, it has been widely identified that conformational dynamics plays a crucial role in regulating enzymatic reactions (2, 4-7). The advent of sitespecific mutagenesis, single-molecule (SM) spectroscopic technique, and molecular dynamics simulations have demonstrated complex dynamics involving multiple conformational states during a catalytic cycle (4, 5, 7-13). Fundamentally, not only the enzymatic active site conformational dynamics but also the overall conformational fluctuation dynamics of the protein matrix, providing the required entropy, enthalpy, and corresponding energy landscape, has a profound impact on an enzyme to ultimately possess the power of enhancing reaction rates. Nevertheless, capturing an individual snapshot of each intermediate conformational step remains challenging, mainly due to the transient nature of those intermediate structures, inadequacy of the conventional structure analysis methods to seize those fluctuating structures, and lack of molecular probes that can specifically report the active site environment at each step of an enzymatic reaction. More often than not, the rate-limiting step is the product release from the active site (14). However, a thorough and comprehensive picture of the product release mechanism is still insufficient, mos...

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Section: Significancementioning

confidence: 99%

Probing conformational dynamics of an enzymatic active site by an in situ single fluorogenic probe under piconewton force manipulation

Pal

2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Because of the small dimensions of the fl-containers, they can be arranged in very high density arrays [91]. For example, fl-arrays etched into the surface of a glass coverslip have a density of 10,000 mm −2 [92]. When these arrays are filled with an enzyme solution, thousands of individual molecules can be observed in parallel, enabling excellent statistical analysis and high quality data evaluation.…”

Section: Single Molecule Enzymology In Femtoliter Arraysmentioning

confidence: 99%

“…The fluorescent product of the enzyme reaction was then monitored over time by wide-field fluorescence microscopy. In the fused silica array system, we investigated the oxidation of the fluorogenic substrate Amplex Red to fluorescent resorufin by hundreds of individual horseradish peroxidase (HRP) molecules [92]. The fluorescence excitation and the detection scheme were optimized to minimize the photooxidation of Amplex Red and photobleaching of resorufin.…”

Section: Femtoliter Arrays Fabricated In Glass Slides By Photolithmentioning

confidence: 99%

“…For example, non-specific binding of the enzyme can be avoided by adding an excess of blocking reagents that are not involved in the reaction [11]. Furthermore, we have found that the signal generation of horseradish peroxidase enclosed in a femtoliter well was ten times lower than in bulk reaction [92,98]. This can be explained by a two-step reaction mechanism which leads to the formation of a radical intermediate that can also react with the well surface instead of forming the fluorescent product.…”

Section: Challenges and Opportunities For Analyzing Single Enzyme mentioning

confidence: 99%

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Enzyme Molecules in Solitary Confinement

Liebherr

Gorris

2014

Molecules

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Large arrays of homogeneous microwells each defining a femtoliter volume are a versatile platform for monitoring the substrate turnover of many individual enzyme molecules in parallel. The high degree of parallelization enables the analysis of a statistically representative enzyme population. Enclosing individual enzyme molecules in microwells does not require any surface immobilization step and enables the kinetic investigation of enzymes free in solution. This review describes various microwell array formats and explores their applications for the detection and investigation of single enzyme molecules. The development of new fabrication techniques and sensitive detection methods drives the field of single molecule enzymology. Here, we introduce recent progress in single enzyme molecule analysis in microwell arrays and discuss the challenges and opportunities.

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“…1–4 In such assays, a sample is divided into a large number of equal volume subunits to isolate single entities, namely, “digitization”. This “digitization” endows digital assays with a number of outstanding abilities, including: i) absolutely quantifying biomolecules without reference standards; 5–7 ii) measuring single-molecule kinetics; 8–10 iii) probing single-cell phenotype and genotype; 11 iv) studying temporal processes of chemical or biochemical reactions on microscales. 12–14 These abilities prompt the widespread application of digital assays, especially in the detection of very minute quantities of diagnostic markers, quantitative assays for DNA and protein markers.…”

Section: Figurementioning

confidence: 99%

A Facile and Rapid Route to Self-Digitization of Samples into a High Density Microwell Array for Digital Bioassays

Cui

Chen

et al. 2021

Preprint

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Digital bioassays are powerful methods to detect rare analytes from complex mixtures and study the temporal processes of individual entities within biological systems. In digital bioassays, a crucial first step is the discretization of samples into a large number of identical independent partitions. Here, we developed a rapid and facile sample partitioning method for versatile digital bioassays. This method is based on a detachable self-digitization (DSD) chip which couples a reversible assembly configuration and a predegassing-based self-pumping mechanism to achieve an easy, fast and large-scale sample partitioning. The DSD chip consists of a channel layer used for loading sample and a microwell layer used for holding the sample partitions. Benefitting from its detachability, the chip avoids a lengthy oil flushing process used to remove the excess sample in loading channels and can compartmentalize a sample into more than 100,000 wells of picoliter volume with densities up to 14,000 wells/cm2 in less than 30 s. We also demonstrated the utility of the proposed method by applying it to digital PCR and digital microbial assays.

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Single molecule kinetics of horseradish peroxidase exposed in large arrays of femtoliter-sized fused silica chambers

Abstract: We have further confirmed that the product formation of HRP in femtoliter chambers is 10-fold lower than that in the bulk solution due to the particular two-step redox reaction mechanism of HRP.

Cited by 24 publications

References 29 publications

Probing conformational dynamics of an enzymatic active site by an in situ single fluorogenic probe under piconewton force manipulation

Probing conformational dynamics of an enzymatic active site by an in situ single fluorogenic probe under piconewton force manipulation

Enzyme Molecules in Solitary Confinement

A Facile and Rapid Route to Self-Digitization of Samples into a High Density Microwell Array for Digital Bioassays

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