Stoichiometry of the α-Complementation Reaction of <i>Escherichia coli</i> β-Galactosidase As Revealed through Single-Molecule Studies

Mogalisetti, Pratyusha; Walt, David R.

doi:10.1021/bi5015024

Cited by 10 publications

(18 citation statements)

References 21 publications

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“…The measurements detect individual bursts of fluorescence when a single protein or single oligomer diffuses within the detection volume. Although proteins transiting through the focal volume produce signals with different intensities, depending on their individual trajectories, sufficient sampling of many trajectories can be provide information on the number of proteins in each oligomer [ 80 , 81 , 82 , 83 ]. To simplify, the maximal number of photons collected in a burst of a millisecond can be compared with the number of photons emitted by a monomeric fluorophore, and interpreted in terms of the number of proteins in an oligomer [ 84 ].…”

Section: Introductionmentioning

confidence: 99%

Confocal Spectroscopy to Study Dimerization, Oligomerization and Aggregation of Proteins: A Practical Guide

Gambin

Polinkovsky

François

et al. 2016

IJMS

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Protein self-association is a key feature that can modulate the physiological role of proteins or lead to deleterious effects when uncontrolled. Protein oligomerization is a simple way to modify the activity of a protein, as the modulation of binding interfaces allows for self-activation or inhibition, or variation in the selectivity of binding partners. As such, dimerization and higher order oligomerization is a common feature in signaling proteins, for example, and more than 70% of enzymes have the potential to self-associate. On the other hand, protein aggregation can overcome the regulatory mechanisms of the cell and can have disastrous physiological effects. This is the case in a number of neurodegenerative diseases, where proteins, due to mutation or dysregulation later in life, start polymerizing and often fibrillate, leading to the creation of protein inclusion bodies in cells. Dimerization, well-defined oligomerization and random aggregation are often difficult to differentiate and characterize experimentally. Single molecule “counting” methods are particularly well suited to the study of self-oligomerization as they allow observation and quantification of behaviors in heterogeneous conditions. However, the extreme dilution of samples often causes weak complexes to dissociate, and rare events can be overlooked. Here, we discuss a straightforward alternative where the principles of single molecule detection are used at higher protein concentrations to quantify oligomers and aggregates in a background of monomers. We propose a practical guide for the use of confocal spectroscopy to quantify protein oligomerization status and also discuss about its use in monitoring changes in protein aggregation in drug screening assays.

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Section: Introductionmentioning

confidence: 99%

Confocal Spectroscopy to Study Dimerization, Oligomerization and Aggregation of Proteins: A Practical Guide

Gambin

Polinkovsky

François

et al. 2016

IJMS

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“…These observations have led to the assumption that β-gal is either active or inactive. Although there have been several studies suggesting that β-gal has different activities depending on activation of different monomers within the tetramer, it is not clear whether the activity can be tuned by modifying individual monomers within a tetramer (7,8).In this work, we take a bottom-up approach that synthesizes tetrameric β-gal with four different levels of activity, which is determined by the number of active monomers in intact tetramers. We demonstrate that the β-gal enzyme can function even if it contains both active and inactive monomers, as long as the intact tetrameric structure is maintained ( Fig.…”

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confidence: 99%

mentioning

confidence: 99%

Bottom-up single-molecule strategy for understanding subunit function of tetrameric β-galactosidase

Jiang

Chong

et al. 2018

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

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In this paper, we report an example of the engineered expression of tetrameric β-galactosidase (β-gal) containing varying numbers of active monomers. Specifically, by combining wild-type and single-nucleotide polymorphism plasmids at varying ratios, tetrameric β-gal was expressed in vitro with one to four active monomers. The kinetics of individual enzyme molecules revealed four distinct populations, corresponding to the number of active monomers in the enzyme. Using single-molecule-level enzyme kinetics, we were able to measure an accurate in vitro mistranslation frequency (5.8 × 10 per base). In addition, we studied the kinetics of the mistranslated β-gal at the single-molecule level.

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“…Recent advances have explored electrochemical detection of proteins with antibodies and aptamers with great success for temporal quantification of protein from purified samples, several with picomolar detection capabilities 2, 3, 4, 5 . Fluorescence labeling of target proteins has provided the additional dimension of spatial information for protein detection and studying protein-protein interactions, though most protein detection strategies are for intracellular protein detection 6, 7, 8 . However, such methods rely on fluorescent modification of the target protein, and are restricted to use inside the cell.…”

Section: Introductionmentioning

confidence: 99%

Single-molecule detection of protein efflux from microorganisms using fluorescent single-walled carbon nanotube sensor arrays

et al. 2017

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A distinct advantage of nanosensor arrays is their ability to achieve ultralow detection limits in solution by proximity placement to an analyte. Here, we demonstrate label-free detection of individual proteins from Escherichia coli (bacteria) and Pichia pastoris (yeast) immobilized in a microfluidic chamber, measuring protein efflux from single organisms in real time. The array is fabricated using non-covalent conjugation of an aptamer-anchor polynucleotide sequence to near-infrared emissive single-walled carbon nanotubes, using a variable chemical spacer shown to optimize sensor response. Unlabelled RAP1 GTPase and HIV integrase proteins were selectively detected from various cell lines, via large near-infrared fluorescent turn-on responses. We show that the process of E. coli induction, protein synthesis and protein export is highly stochastic, yielding variability in protein secretion, with E. coli cells undergoing division under starved conditions producing 66% fewer secreted protein products than their non-dividing counterparts. We further demonstrate the detection of a unique protein product resulting from T7 bacteriophage infection of E. coli, illustrating that nanosensor arrays can enable real-time, single-cell analysis of a broad range of protein products from various cell types.

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Stoichiometry of the α-Complementation Reaction of Escherichia coli β-Galactosidase As Revealed through Single-Molecule Studies

Cited by 10 publications

References 21 publications

Confocal Spectroscopy to Study Dimerization, Oligomerization and Aggregation of Proteins: A Practical Guide

Confocal Spectroscopy to Study Dimerization, Oligomerization and Aggregation of Proteins: A Practical Guide

Bottom-up single-molecule strategy for understanding subunit function of tetrameric β-galactosidase

Single-molecule detection of protein efflux from microorganisms using fluorescent single-walled carbon nanotube sensor arrays

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