Interaction of the pore-forming domain of colicin A with phospholipid vesicles

Massotte, Dominique; Dasseux, Jean-Louis; Sauve, Paul; Cyrklaff, Marek; Leonard, Kevin; Pattus, Franc

doi:10.1021/bi00445a029

Cited by 28 publications

(24 citation statements)

References 26 publications

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“…This constant, k, shows pH and negative charge density dependence identical to that observed using completely different experimental approaches (Pattus et al, 1983;Collarini et al, 1987;Massotte et al, 1989). The results obtained confirm the umbrella model for the spontaneous insertion of colicin A thermolytic fragment into vesicles, and the occurrence during this process of a "molten globule" intermediate.…”

supporting

confidence: 86%

“…Bromine atoms are known to quench the intrinsic tryptophan fluorescence of proteins. The mechanism of this quenching is probably collisional (East & Lee, 1982;McIntosh & Holloway, 1987;Yeager & Feigenson, 1990), although a distance-dependent quenching mechanism has recently been proposed (Bolen & Holloway, 1990). The introduction of bromine atoms at different positions along the fatty acid acyl chain of the phospholipid allows for the determination of the depth at which the fluorophore group of the protein is located within the bilayer (McIntosh & Holloway, 1987).…”

mentioning

confidence: 97%

“…The mechanism of this quenching is probably collisional (East & Lee, 1982;McIntosh & Holloway, 1987;Yeager & Feigenson, 1990), although a distance-dependent quenching mechanism has recently been proposed (Bolen & Holloway, 1990). The introduction of bromine atoms at different positions along the fatty acid acyl chain of the phospholipid allows for the determination of the depth at which the fluorophore group of the protein is located within the bilayer (McIntosh & Holloway, 1987). All the reported measurements up to now have been done under steady-state conditions, and the extent of quenching enabled the study of quenching constants (De Kroon et al, 1990), the depth of the fluorophore within the membrane (Markello et al, 1985;De Kroon et al, 1990), lipid affinity (East & Lee, 1982),orsolubilization (DeForestaet al, 1989).…”

mentioning

confidence: 97%

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Brominated phospholipids as a tool for monitoring the membrane insertion of colicin A

González-Mañas

Lakey²,

Pattus³

1992

Biochemistry

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The intrinsic fluorescence of the colicin A thermolytic fragment does not change after insertion into normal phospholipid vesicles and is thus an unsuitable probe for monitoring the membrane insertion process. In this paper, we report the results of studies on the quenching of this fluorescence by brominated dioleoylphosphatidylglycerol (Br-DOPG) vesicles. Bromine atoms located at the midpoint of the phospholipid acyl chain quench the tryptophan fluorescence, indicating contact between fluorophores of the protein and the bilayer's hydrophobic core. Addition of Br-DOPG vesicles to a protein solution quenches the tryptophan fluorescence in a time-dependent manner. This quenching can be fitted to a single-exponential function, and thus interpreted as a one-step process. This allows calculation of an apparent rate constant of protein insertion into the membrane. Parameters known to affect the insertion of the thermolytic fragment into phospholipid monolayers or vesicles (pH and negative charge density) also affect the rate constant in comparable ways. In addition to the information gained concerning membrane exposure in the steady state, this approach provides the first real-time method for measuring the insertion of colicin into membranes. It is highly quantitative and can be used on all versions of the protein, e.g., full size, proteolytic fragments, and mutants. Brominated lipids provide experimental conditions identical to normal lipids and allow for great flexibility in protein/lipid ratios and concentrations. The kinetic analysis shows clearly the existence of a two-step process involving a rapid adsorption of the protein to the lipid surface followed by a slow insertion.

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confidence: 86%

mentioning

confidence: 97%

mentioning

confidence: 97%

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Brominated phospholipids as a tool for monitoring the membrane insertion of colicin A

González-Mañas

Lakey²,

Pattus³

1992

Biochemistry

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“…Therefore, such experiments do not allow one to compare the binding of Br,Ole,GroPGro and Ole,GroPGro to the colicin-A pore-forming domain. However, the value for Br,Ole,GroPGro is very close to that reported by Massotte et al, (1989), using gel chromatography and different lipids. They found that at a lipidprotein ratio of 33 and at acidic pH, all the protein was bound to 1,Z-dimiristoyl-sn-glycero-3-phospho-sn-glycerol (Myr,GroPGro), forming a complex.…”

Section: Irreversibility Of the Binding Of Colicin A To Brolegropgrmentioning

confidence: 57%

“…The lyophilized peptide was dissolved in the desired buffer and the solution was spun in an Eppendorf centrifuge for 15 min to remove suspended particles and nonsolubilized material. The thermolytic fragment concentration was determined from the absorbance at 280nm using the molar absorption coefficient (cZaO 2.43 X 104M-'cm-') determined previously (Massotte et al, 1989).…”

Section: Cell Growth and Purificationmentioning

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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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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Solid‐state NMR studies of the membrane‐bound closed state of the colicin E1 channel domain in lipid bilayers

et al. 1998

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The colicin E1 channel polypeptide was shown to be organized anisotropically in membranes by solid-state NMR analysis of samples of uniformly 15N-labeled protein in oriented planar phospholipid bilayers. The 190 residue C-terminal colicin E1 channel domain is the largest polypeptide to have been characterized by 15N solid-state NMR spectroscopy in oriented membrane bilayers. The 15N-NMR spectra of the colicin E1 show that: (1) the structure and dynamics are independent of anionic lipid content in both oriented and unoriented samples; (2) assuming the secondary structure of the polypeptide is helical, there are both trans-membrane and in-plane helical segments; (3) trans-membrane helices account for approximately 20-25% of the channel polypeptide, which is equivalent to 38-48 residues of the 190-residue polypeptide. The results of the two-dimensional PISEMA spectrum are interpreted in terms of a single trans-membrane helical hairpin inserted into the bilayer from each channel molecule. These data are also consistent with this helical hairpin being derived from the 38-residue hydrophobic segment near the C-terminus of the colicin E1 channel polypeptide.

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Interaction of the pore-forming domain of colicin A with phospholipid vesicles

Cited by 28 publications

References 26 publications

Brominated phospholipids as a tool for monitoring the membrane insertion of colicin A

Brominated phospholipids as a tool for monitoring the membrane insertion of colicin A

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

Solid‐state NMR studies of the membrane‐bound closed state of the colicin E1 channel domain in lipid bilayers

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