Membrane Protein Stoichiometry Determined from the Step‐Wise Photobleaching of Dye‐Labelled Subunits

Das, Sumanta; Darshi, Manjula; Cheley, Stephen; Wallace, Mark I.; Bayley, Hagan

doi:10.1002/cbic.200600474

Cited by 113 publications

(124 citation statements)

References 105 publications

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“…Because CALHM1 is genetically fused to GFP, the number of bleaching steps represents the number of GFP-tagged subunits in the complex. This technique has been used previously to determine the stoichiometry of membrane proteins that have up to seven monomers in the complex (58). The low expression level of the protein reduced the probability of aggregation of multiple complexes in the same pixel region.…”

Section: Similarities Of Calhm1 Connexins and Pannexins/innexinsmentioning

confidence: 99%

Structural and Functional Similarities of Calcium Homeostasis Modulator 1 (CALHM1) Ion Channel with Connexins, Pannexins, and Innexins*

Siebert¹,

Ma²,

Grevet³

et al. 2013

Journal of Biological Chemistry

109

122

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Background: CALHM1 is an ion channel for which structural information is lacking. Results: CALHM1 has poor ion selectivity and a wide (ϳ14 Å) pore and is a hexamer, with monomers having four transmembrane domains with cytoplasmic termini. Conclusion: CALHM1 shares structural features with pannexins, connexins, and innexins. Significance: CALHMs, connexins, and pannexins/innexins are three structurally related protein families with shared and distinct functional properties.

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Section: Similarities Of Calhm1 Connexins and Pannexins/innexinsmentioning

confidence: 99%

Structural and Functional Similarities of Calcium Homeostasis Modulator 1 (CALHM1) Ion Channel with Connexins, Pannexins, and Innexins*

Siebert¹,

Ma²,

Grevet³

et al. 2013

Journal of Biological Chemistry

109

122

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“…Electron microscopy and cross-linking experiments of purified γHL pores on human erythrocyte membranes demonstrated the existence of a heptamer with a 3∶4 or 4∶3 molar ratio of F to S components (20,21), whereas biochemical analyses of pores of engineered covalent γHL heterodimers on erythrocytes and leukocytes suggested octameric stoichiometry (22). Several reports using LUK pores formed on rabbit erythrocytes also demonstrated the existence of an octamer consisting of 4-plus-4 subunits (23)(24)(25). However, hexamer was also proposed based on structure modeling using monomeric structures of PVL (17) and electron microscopy of LUK pores on leukocytes and erythrocytes (26).…”

mentioning

confidence: 99%

Crystal structure of the octameric pore of staphylococcal γ-hemolysin reveals the β-barrel pore formation mechanism by two components

Yamashita

Kawai

Tanaka

et al. 2011

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

154

210

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Staphylococcal γ-hemolysin is a bicomponent pore-forming toxin composed of LukF and Hlg2. These proteins are expressed as watersoluble monomers and then assemble into the oligomeric pore form on the target cell. Here, we report the crystal structure of the octameric pore form of γ-hemolysin at 2.5 Å resolution, which is the first high-resolution structure of a β-barrel transmembrane protein composed of two proteins reported to date. The octameric assembly consists of four molecules of LukF and Hlg2 located alternately in a circular pattern, which explains the biochemical data accumulated over the past two decades. The structure, in combination with the monomeric forms, demonstrates the elaborate molecular machinery involved in pore formation by two different molecules, in which interprotomer electrostatic interactions using loops connecting β2 and β3 (loop A: Asp43-Lys48 of LukF and Lys37-Lys43 of Hlg2) play pivotal roles as the structural determinants for assembly through unwinding of the N-terminal β-strands (aminolatch) of the adjacent protomer, releasing the transmembrane stem domain folded into a β-sheet in the monomer (prestem), and interaction with the adjacent protomer.

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“…For instance, for protein 29 interactions, the model could be extended to situations where the number of label distribution follows different statistical laws. 12,42 …”

Section: Resultsmentioning

confidence: 99%

“…10,11 One possible method to measure the number of dyes present in an aggregate is to count the number of steps observed in the time trace of the fluorescence of single molecules before they are completely photobleached. 1,12,13 More sophisticated methods have also been proposed, that rely on a statistical analysis of the fluorescence trajectories during photobleaching or on the deconvolution of the intensity distribution of single molecules. 14,15 However, all of them require the molecules to be immobilized on a substrate and observed using a high detection efficiency and specificity.…”

Section: Introductionmentioning

confidence: 99%

Measuring, in Solution, Multiple-Fluorophore Labeling by Combining Fluorescence Correlation Spectroscopy and Photobleaching

et al. 2010

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Determining the number of fluorescent entities that are coupled to a given molecule (DNA, protein, etc.) is a key point of numerous biological studies, especially those based on a single molecule approach. Reliable methods are important, in this context, not only to characterize the labeling process, but also to quantify interactions, for instance within molecular complexes. We combined Fluorescence Correlation Spectroscopy (FCS) and photobleaching experiments to measure the effective number of molecules and the molecular brightness as a function of the total fluorescence count rate on solutions of cDNA (containing a few percent of C bases labeled with Alexa Fluor 647). Here, photobleaching is used as a control parameter to vary the experimental outputs (brightness and number of molecules).Assuming a Poissonian distribution of the number of fluorescent labels per cDNA, the FCSphotobleaching data could be easily fit to yield the mean number of fluorescent labels per cDNA strand ( 2). This number could not be determined solely on the basis of the cDNA brightness, because of both the statistical distribution of the number of fluorescent labels and their unknown brightness when incorporated in cDNA. The statistical distribution of the number of fluorophores labeling cDNA was confirmed by analyzing the photon count distribution (with the cumulant method), which showed clearly that the brightness of cDNA strands varies from one molecule to the other. We also performed complementary continuous photobleaching experiments and found that the photobleaching decay rate of Alexa Fluor 647 in the excited state decreases by about 30% when incorporated into cDNA, while its non radiative decay rate is increased such that the brightness of individual Alexa labels is decreased by 25% compared to free Alexa dyes.

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Membrane Protein Stoichiometry Determined from the Step‐Wise Photobleaching of Dye‐Labelled Subunits

Cited by 113 publications

References 105 publications

Structural and Functional Similarities of Calcium Homeostasis Modulator 1 (CALHM1) Ion Channel with Connexins, Pannexins, and Innexins*

Structural and Functional Similarities of Calcium Homeostasis Modulator 1 (CALHM1) Ion Channel with Connexins, Pannexins, and Innexins*

Crystal structure of the octameric pore of staphylococcal γ-hemolysin reveals the β-barrel pore formation mechanism by two components

Measuring, in Solution, Multiple-Fluorophore Labeling by Combining Fluorescence Correlation Spectroscopy and Photobleaching

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