Photoaffinity Labels for the Study of Cytochrome C and Phospholipid Interactions With Cytochrome C Oxidase

Bisson, Roberto; Montecucco, Cesare; Gutweniger, Heidi

doi:10.1016/b978-0-12-225402-4.50009-7

Cited by 6 publications

(6 citation statements)

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“…The picture emerging for the sidedness of the protein components is in agreement with the three-dimensional reconstruction of the enzyme obtained by R. Henderson (Cambridge) [24,2.5] and may be further specified by the method of photoaffinity labelling [26] with synthetic arylazidophospholipids reported by R. Bisson (Padua) [27,28]. Here 'deep' and 'shallow' probes provide tools for tracing the membrane domains of this [29] and other membrane proteins, among them cytochrome bs and the mitochondrial enzymes succinate ubiquinone reducatase, succinate cytochrome c reductase and ATP synthase.…”

supporting

confidence: 68%

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1981

FEBS Letters

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The Sonderforschungsbereich 160 organized this workshop to discuss and evaluate recent activities and trends in the field of membrane protein structure. Some 20 scientists were invited to present their most recent investigations in an attempt to shed some light on various facets of the structural organization of this rather elusive class of proteins. Despite all efforts there is so far not a single example where the structural determination of a membrane protein has reached the degree of comprehensiveness, accuracy and definiteness which we are eventually seeking in order to begin inquiring sensibly into the way these molecules operate. It was the central aim of the workshop to discuss how the various rapidly advancing physical and chemical approaches presently available can be combined to achieve a 'holistic' picture of the structure of a membrane protein.Ultimately it is only the atomic model which can provide us with the necessary structural information. Most advanced in this respect is bacteriorhodopsin. A model of this protein, obtained by piecing together information from various sources [ 11, was presented by R. Henderson (Cambridge). Low-dose electron microscopy combined with digital image reconstruction techniques provided the basic morphological information: The molecule is composed of seven helical rods traversing the membrane almost perpendicularly [2]. Analysis of a new two-dimensional crystal form obtained by recrystallisation[3] corroborated the boundaries of individual bacteriorhodopsin molecules in the lattice. The amino acid sequence [4] was then fitted tentatively into the morphological model. As important preliminaries the 7 helical segments and the link regions in the sequence were localized by applying the criteria of hydrophobicity and inaccessibility to proteolytic cleavage and assuming that the helices should be comparable in length. In the next step these helical segments had to be identified in the density map. To reduce the enormous number of possible assignments the following criteria were applied: (a) connectivity of non-helical link regions; (b) charge neutralization;(c) correlation of calculated electron scattering cross-sections for the individual helices with the densities determined experimentally. The model emerging as the most probable one exhibits a number of features favourable for a membrane-embedded proton pump. The buried charged amino acids are localized in the center of the molecule, possibly forming a hydrophilic channel through which protons could jump, whereas the hydrophobic, uncharged residues are directed outward towards the lipid environment. This 'inside-out' arrangement has meanwhile been confirmed by neutron diffraction experiments [5].

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confidence: 68%

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1981

FEBS Letters

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“…The present work shows that nitrophenyl azidophospholipids can be used advantageously in protein-topology studies. Indeed we have already reported preliminary studies of well-characterized membrane proteins such as cytochrome b5 (Bisson et al, 1979b), cytochrome c oxidase (Bisson et al, 1979a) and the mitochondrial and (Na+ + K+)activated ATPases (Montecucco et al, 1979(Montecucco et al, , 1980. Nevertheless some points need further investigation.…”

Section: Discussionmentioning

confidence: 99%

“…Its activity measured with an NADH-coupled system ranged from 10.4 to 12.5 units and its lipid/protein ratio from 28 to 37. (Nat + K+)-activated ATPase from pig kidney, cytochrome c oxidase and oligomycinsensitive ATPase from bovine mitochondria were prepared and assayed as reported previously (Montecucco et al, 1979(Montecucco et al, , 1980Bisson et al, 1979a). Protein concentration was determined by the method of Lowry et al (1951) and lipids either by the method of Bartlett (1959) or by g.l.c.…”

Section: Methodsmentioning

confidence: 99%

“…The samples were illuminated at 0°C with a 1OOW mineral lamp (Ultra-Violet Products, San Gabriel, CA, U.S.A.) for various periods of time between 10 and 20min, with a glass/water filter to cut down radiation lower than 300nm. After illumination proteins were recovered, electrophoresed and counted for radioactivity as reported previously (Bisson et al, 1979a;Montecucco et al, 1980).…”

Section: %)mentioning

confidence: 99%

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Photolabelling of membrane proteins with photoactive phospholipids

Bisson¹,

Montecucco²

1981

Biochemical Journal

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Photoactive probes have been introduced recently to study the hydrophobic sector of integral membrane proteins. A simple procedure to synthesize a new series of highly radioactive aryl azido-phospholipids is presented. They effectively exchange with the boundary lipids and, on illumination, the cross-link to several membrane proteins with high efficiency. The procedure and analysis of labelling of ATPase from sarcoplasmic reticulum is reported as an example. The advantages in using these photoactive phospholipids are discussed together with some information obtained on their use.

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“…The use of hydrophobic reagents, such as adamantane diazirine [38] and arylazidophospholipids [39,401, has been of invaluable help in mapping subunit 111. These reagents are lipophilic and thus react with bilayer-intercalated parts of the most hydrophobic subunits, including subunit 111.…”

Section: Chemical Modification Ojsuhunit 111mentioning

confidence: 99%