Quenching of tryptophan fluorescence by brominated phospholipid

Bolen, Elizabeth J.; Holloway, Peter W.

doi:10.1021/bi00493a019

Cited by 175 publications

(171 citation statements)

References 21 publications

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“…1). The 82% loss of fluorescence corresponds to the maximal possible quenching, since the quenching efficiency for the system indole-2,3-dibromobutane is 0.82 (Bolen and Holloway, 1990). This result indicates that all the fluorophores are exposed to the quencher.…”

Section: Effect Of Brolegropgro In the Intrinsic Fluorescence Of Comentioning

confidence: 93%

“…Helices 8 and 9 are well embedded within the hydrophobic core of the membrane, in a parallel orientation with respect to the plane of the membrane. The small displacement of helices 8-10 relative to the helix-3 -helix-7 bundle allows for complete exposure of the tryptophans to the quencher, particularly if we consider the possibility of a distance-dependent quenching mechanism (Bolen and Holloway, 1990).…”

Section: Towards a Model For The Membrane-bound Colicin Amentioning

confidence: 99%

See 1 more Smart Citation

Interacion of the colicin‐A pore‐forming domain with negatively charged phospholipds

González-Mañas¹,

Lakey²,

Pattus³

1993

European Journal of Biochemistry

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The interaction of colicin-A thermolytic fragment with negatively charged liposomes was studied by fluorescence spectroscopy. 1,2-Dioleoyl-sn-glycero-3-phospho-l-sn-glycerol (Ole,GroPGro) containing liposomes do not significantly alter the fluorescence properties of the protein, and thus cannot give much information about this interaction. 1,2-Bis(9,10-dibromooleoyl-sn-glycero-3-phospho-1-sn-glycerol (Br,Ole,GroPGro) is easily synthesized by addition of bromine atoms to the double bond located at the mid-point of the fatty-acid acyl chain of Ole,GroPGro. The brominated phospholipid forms vesicles that strongly quench the protein fluorescence emission. The results presented here show that conversion of Ole,GroPGro to Br,Ole,GroPGro does not change either the affinity for the protein or the extent of lipid binding. This observation allows for the estimation of the distribution of the quenching phospholipid molecules around the fluorophores [Yeager, M. D. & Feigenson G. W. (1990) Biochemistry 29, 4380-43921. Binding of the protein to the vesicles is an irreversible process, since inserted molecules do not dissociate from the vesicle. From steadystate measurements, it can be concluded that in the membrane-bound form, the tryptophans are located within quenching distance of the bromine atoms, i.e. close to the lipid head-grouphydrocarbon boundary, completely accessible to the quencher, protected from the polar phase and that the maximum number of phospholipid molecules in contact with the fluorescent domain of the protein is nine.Colicin A is a plasmid-encoded protein produced by some strains of Escherichia coli, which is active against those strains of E. coli endowed with the necessary receptor. The mechanism of action of colicin A in vivo comprises three steps : binding to the receptor, translocation across the outer membrane (with the intervention of the tolQ, tolR, tolA and tolB proteins) and formation of a voltage-gated pore which depolarizes the cell and kills it. Each of these steps is performed by a different domain in the protein. The central domain binds to the receptor, the N-terminal domain is responsible for protein translocation and the C-terminal domain forms the pore (Pattus et al., 1990).This C-terminal domain can be obtained from the whole colicin-A molecule by thermolysin proteolysis, and thus it is called the thermolytic fragment of colicin A (Tucker et al., 1986). It has been crystallized and its structure determined by X-Ray crystallography. Based on the three-dimensional structure of this fragment, a model for the insertion has been proposed , in which the compact soluble structure, upon interaction with the phospholipid bilayer, un- folds in an umbrella-like fashion. This is the so-called 'umbrella model'.The study of the membrane-bound conformation of colicin A by spectroscopic techniques shows that there is no drastic conformational change upon membrane insertion : the secondary structure of the protein and its fluorescence parameters are largely conserved (Lakey et al., 1991a; Goormagtigh ...

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Section: Effect Of Brolegropgro In the Intrinsic Fluorescence Of Comentioning

confidence: 93%

Section: Towards a Model For The Membrane-bound Colicin Amentioning

confidence: 99%

Interacion of the colicin‐A pore‐forming domain with negatively charged phospholipds

González-Mañas¹,

Lakey²,

Pattus³

1993

European Journal of Biochemistry

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“…Occasionally, the statistical error is smaller than the size of the symbol. Stern-Volmer analysis (Bolen and Holloway, (25)) has been applied to the quenching of tryptophan in SPP buffer. The corresponding plot of F c,0 /F c versus bromide concentration is linear in the range examined with a correlation coefficient of 0.99 and a Stern-Volmer quenching constant of 0.80 M −1 .…”

Section: Supplementary Materialsmentioning

confidence: 99%

Interactions of Tryptophan, Tryptophan Peptides, and Tryptophan Alkyl Esters at Curved Membrane Interfaces

Liu

Caffrey

2006

Biochemistry

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Motivated by on-going efforts to understand the mechanism of membrane protein crystallogenesis and transport in the lipidic cubic phase, the nature of the interaction between tryptophan and the bilayer/aqueous interface of the cubic phase has been investigated. The association was quantified by partitioning measurements which enabled the free energy of interaction to be determined. Temperature-dependent partitioning was used to parse the association free energy change into its enthalpic and entropic components. As has been observed with tryptophan derivatives interacting with glycerophospholipid bilayers in vesicles, tryptophan partitioning in the cubic phase is enthalpy driven. This is in contrast to partitioning into apolar solvents which exhibits the classic hydrophobic effect whose hallmark is a favorable entropy change. These results with tryptophan are somewhat surprising given the simplicity, homogeneity and curvature of the interface that prevails in the case of the cubic phase. Nevertheless, the interaction between tryptophan and the mesophase is very slight as revealed by its low partition coefficient. Additional evidence in support of interaction was obtained by electronic absorption and fluorescence spectroscopy and fluorescence quenching. Partitioning proved insensitive to the lipid composition of the membrane, examined by doping with glycerophospholipids. However, the interaction could be manipulated in meaningful ways by the inclusion in the aqueous medium of salt, glycerol or urea. The effects seen with tryptophan were amplified rationally when measurements were repeated using tryptophan alkyl esters and with tryptophan peptides of increasing length. These findings are interpreted in the context of the insertion, folding and function of proteins in membranes.Keywords cubic phase; fluorescence quenching; lipid bilayers; membrane protein crystallization; small-angle X-ray diffraction Diesenhofer and Michel commented on the "unexpected" non-uniform distribution of tryptophan (W) in the L and M transmembranal subunits of the photosynthetic reaction center (1). Since this was the first membrane protein whose high-resolution, 3-dimensional structure †

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“…30,31 gH625-c-prune, containing the tryptophan residue in the peptide sequence, was added (final concentration of 4 µM) to 2 mL of buffer (5 mM HEPES, 100 mM NaCl, pH 7.4) containing 100 µM of Br-PC/Chol SUV and a total lipid concentration of 400 µM, thus establishing a lipid/peptide molar ratio of 100:1. Emission spectrum of the tryptophan was recorded with excitation set at 295 nm.…”

Section: Tryptophan Fluorescence Experimentsmentioning

confidence: 99%

“…16 To inhibit the endocytic pathways, HeLa cells were treated with sodium azide, cytochalasin D, and low temperature. To be exact, 40 µM sodium azide was added to the cell culture medium for 30 AG, Jena, Germany) equipped with a 488 nm Argon laser line and with a 63× water-immersion objective. Confocal images of all samples were analyzed by ImageJ (National Institutes of Health, Bethesda, MD, USA) software to evaluate the protein uptake in terms of intra cellular fluorescence intensity.…”

Section: Flow Cytometry Of Cell Association Of Gh625-c-prunementioning

confidence: 99%

gH625 is a viral derived peptide for effective delivery of intrinsically disordered proteins

Smaldone¹,

Falanga²,

Capasso³

et al. 2013

IJN

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A genetically modified recombinant gH625-c-prune was prepared through conjugation of c-prune with gH625, a peptide encompassing 625-644 residues of the glycoprotein H of herpes simplex virus 1, which has been proved to possess the ability to carry cargo molecules across cell membranes. C-prune is the C-terminal domain of h-prune, overexpressed in breast, colorectal, and gastric cancers, interacting with multiple partners, and representing an ideal target for inhibition of cancer development. Its C-terminal domain results in an intrinsically disordered domain (IDD), and the peculiar properties of gH625 render it an optimal candidate to act as a carrier for this net negatively charged molecule by comparison with the positively charged TAT. A characterization of the recombinant gH625-c-prune fusion protein was conducted by biochemical, cellular biology and confocal microscopy means in comparison with TAT-c-prune. The results showed that the gH625-c-prune exhibited the ability to cross biomembranes, opening a new scenario on the use of gH625 as a novel multifunctional carrier.

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Quenching of tryptophan fluorescence by brominated phospholipid

Cited by 175 publications

References 21 publications

Interacion of the colicin‐A pore‐forming domain with negatively charged phospholipds

Interacion of the colicin‐A pore‐forming domain with negatively charged phospholipds

Interactions of Tryptophan, Tryptophan Peptides, and Tryptophan Alkyl Esters at Curved Membrane Interfaces

gH625 is a viral derived peptide for effective delivery of intrinsically disordered proteins

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