Quantitative Analysis of Protein-Lipid Interactions Using Tryptophan Fluorescence

Kraft, Catherine A.; Garrido, José; Leiva‐Vega, Luis; Romero, Guillermo

doi:10.1126/scisignal.299pl4

Cited by 36 publications

(31 citation statements)

References 26 publications

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“…The Trp fluorescence quenching assay, a popular method for investigating lipid-protein interactions (Dua et al, 1995; Kraft et al, 2009), was chosen because BFT2 contains 4 Trp residues in its catalytic site. Proteinase K was used as a negative control, and BSA, which is known to bind lipids in the medium nanomolar range (Charbonneau and Tajmir-Riahi, 2010), was used as a positive control.…”

Section: Resultsmentioning

confidence: 99%

Interaction of Bacteroides fragilis Toxin with Outer Membrane Vesicles Reveals New Mechanism of Its Secretion and Delivery

Zakharzhevskaya

Цветков

Vanyushkina

et al. 2017

Front. Cell. Infect. Microbiol.

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The only recognized virulence factor of enterotoxigenic Bacteroides fragilis (ETBF) that accompanies bloodstream infections is the zinc-dependent non-lethal metalloprotease B. fragilis toxin (BFT). The isolated toxin stimulates intestinal secretion, resulting in epithelial damage and necrosis. Numerous publications have focused on the interrelation of BFT with intestinal inflammation and colorectal neoplasia, but nothing is known about the mechanism of its secretion and delivery to host cells. However, recent studies of gram-negative bacteria have shown that outer membrane vesicles (OMVs) could be an essential mechanism for the spread of a large number of virulence factors. Here, we show for the first time that BFT is not a freely secreted protease but is associated with OMVs. Our findings indicate that only outer surface-exposed BFT causes epithelial cell contact disruption. According to our in silico models confirmed by Trp quenching assay and NMR, BFT has special interactions with outer membrane components such as phospholipids and is secreted during vesicle formation. Moreover, the strong cooperation of BFT with polysaccharides is similar to the behavior of lectins. Understanding the molecular mechanisms of BFT secretion provides new perspectives for investigating intestinal inflammation pathogenesis and its prevention.

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Section: Resultsmentioning

confidence: 99%

Interaction of Bacteroides fragilis Toxin with Outer Membrane Vesicles Reveals New Mechanism of Its Secretion and Delivery

Zakharzhevskaya

Цветков

Vanyushkina

et al. 2017

Front. Cell. Infect. Microbiol.

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“…Membrane-bound peptides often possess intrinsic fluorescence caused by aromatic amino acids, with tryptophan residues giving by far the highest quantum yield [92, 93]. Tryptophan fluorescence is strongly influenced by other amino acids or lipids in the vicinity.…”

Section: Fluorescence Methodsmentioning

confidence: 99%

Determining the Orientation and Localization of Membrane-Bound Peptides

Hohlweg

Kosol²,

Zangger³

2012

CPPS

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Many naturally occurring bioactive peptides bind to biological membranes. Studying and elucidating the mode of interaction is often an essential step to understand their molecular and biological functions. To obtain the complete orientation and immersion depth of such compounds in the membrane or a membrane-mimetic system, a number of methods are available, which are separated in this review into four main classes: solution NMR, solid-state NMR, EPR and other methods. Solution NMR methods include the Nuclear Overhauser Effect (NOE) between peptide and membrane signals, residual dipolar couplings and the use of paramagnetic probes, either within the membrane-mimetic or in the solvent. The vast array of solid state NMR methods to study membrane-bound peptide orientation and localization includes the anisotropic chemical shift, PISA wheels, dipolar waves, the GALA, MAOS and REDOR methods and again the use of paramagnetic additives on relaxation rates. Paramagnetic additives, with their effect on spectral linewidths, have also been used in EPR spectroscopy. Additionally, the orientation of a peptide within a membrane can be obtained by the anisotropic hyperfine tensor of a rigidly attached nitroxide label. Besides these magnetic resonance techniques a series of other methods to probe the orientation of peptides in membranes has been developed, consisting of fluorescence-, infrared- and oriented circular dichroism spectroscopy, colorimetry, interface-sensitive X-ray and neutron scattering and Quartz crystal microbalance.

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“…Various fl uorescence techniques have been employed to monitor membrane-protein interaction. The change in Trp fl uorescence emission intensity in terms of an increase ( 15 ), quenching ( 16 ), and fl uorescence resonance energy transfer ( 17,18 ) can be monitored as a protein binds the membrane if it contains a Trp residue(s) on the membrane binding surface. Although rapid, convenient, and potentially sensitive, this method is not generally applicable because there are many membrane binding proteins without Trp on their membrane binding surfaces and because genetic introduction of Trp to the membrane binding surface can dramatically change the membrane binding property of the protein ( 19 ).…”

Section: Protein Expression and Purifi Cationmentioning

confidence: 99%

High-throughput fluorescence assay for membrane-protein interaction

Kim

Afsari

Cho

2013

Journal of Lipid Research

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Membrane‐protein interactions play key roles in a wide variety of biological processes, including cell signaling. Although various methods have been employed to measure membrane binding of soluble proteins, a robust high‐throughput assay that is universally applicable to all proteins is lacking at present. Here we report a new fluorescence quenching assay utilizing enhanced green fluorescence protein (EGFP)‐fusion proteins and a lipid containing a dark quencher, N‐dimethylaminoazobenzenesulfonyl‐phosphatidylethanolamine (dabsyl‐PE). The EGFP fluorescence emission intensity showed a large decrease when EGFP‐fusion proteins bound the vesicles containing 5–10 mole% dabsyl‐PE. This simple assay, which can be performed using either a cuvette‐based spectrofluorometer or a fluorescence plate reader, allowed rapid, sensitive, and accurate determination of lipid specificity and affinity for various lipid‐binding domains, including two pleckstrin homology (PH) domains, an epsin N‐terminal homology (ENTH) domain, and a phox (PX) domain. The assay was also successfully applied to high‐throughput screening of small molecules that modulate membrane binding of proteins.

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Quantitative Analysis of Protein-Lipid Interactions Using Tryptophan Fluorescence

Cited by 36 publications

References 26 publications

Interaction of Bacteroides fragilis Toxin with Outer Membrane Vesicles Reveals New Mechanism of Its Secretion and Delivery

Interaction of Bacteroides fragilis Toxin with Outer Membrane Vesicles Reveals New Mechanism of Its Secretion and Delivery

Determining the Orientation and Localization of Membrane-Bound Peptides

High-throughput fluorescence assay for membrane-protein interaction

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