Identifying regions of membrane proteins in contact with phospholipid head groups: covalent attachment of a new class of aldehyde lipid labels to cytochrome c oxidase

Abstract

A series of amine-specific reagents based on the benzaldehyde reactive group have been synthesized, characterized, and used to study beef heart cytochrome c oxidase reconstituted in phospholipid bilayers. The series contained three classes of reagents: lipid-soluble phosphodiesters having a single hydrocarbon chain, phospholipid analogues, and a water-soluble benzaldehyde. All reagents were either radiolabeled or spin-labeled or both. The Schiff bases formed by these benzaldehydes with amines were found to be reversible until the addition of the reducing agent sodium cyanoborohydride, whereas attachment of lipid-derived aliphatic aldehydes was not readily reversible in the absence of the reducing agent. The benzaldehyde group provides a convenient method of controlling and delaying permanent attachment to integral membrane proteins until after the reconstitution steps. This ensures that the lipid analogues are located properly to identify amine groups at the lipid-protein interface rather than reacting indiscriminately with amines of the hydrophilic domains of the protein. The benzaldehyde lipid labels attach to cytochrome c oxidase with high efficiency. Typically, 20% of the amount of lipid label present was covalently attached to the protein, and the number of moles of label incorporated per mole of protein ranged from 1 to 6, depending on the molar ratios of label, lipid, and protein. The efficiency of labeling by the water-soluble benzaldehyde was much less than that observed for any of the lipid labels because of dilution effects, but equivalent levels of incorporation were achieved by increasing the label concentration. Electron spin resonance spectra of a nitroxide-containing phospholipid analogue covalently attached to reconstituted cytochrome c oxidase exhibited a large motion-restricted component, which is characteristic of spin-labeled lipids in contact with the hydrophobic surfaces of membrane proteins. The line shape and splittings were similar for covalently attached label and label free to diffuse and contact the protein molecules in the bilayer, providing independent evidence that the coupling occurs at the protein-lipid interface. The distribution of the benzaldehyde reagents attached to the polypeptide components of cytochrome c oxidase was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The labeling pattern observed for the lipid analogues was not affected by the presence of the nitroxide moiety on the acyl chains but was dependent on the molar ratio of labeling reagent to protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Preparation of schiff base adducts of phosphatidylcholine core aldehydes and aminophospholipids, amino acids, and myoglobin

“…In these instances, the reaction products are stabilized by secondary reactions, although the structures have not been completely established in all instances. The simple aliphatic aldehydes yield Schiff bases that are more easily dissociated than those of the bifunctional aldehydes and require chemical reduction for stabilization (12,25).…”

Lipid‐protein interactions in membranes

“…The latter may be of functional significance in the inner mitochondrial membrane, since cardiolipin is found to be an efficient activator of cytochrome oxidase ( [7,8] indicate that the source of the selectivity is not in al1 cases solely electrostatic. Covalent modification has indicated that lysine residues are involved in the lipid selectivity of cytochrome oxidase [ 131, and the subunits bearing some of the lysine groups involved have been identified [15]. The polar region (residues 116-150) that makes a major contribu-tion to the lipid selectivity of the myelin proteolipid protein has been located with the help of the DM-20 proteolipid mutant which lacks this portion of the sequence [la.…”

Topology of subunits of the mammalian cytochrome c oxidase: relationship to the assembly of the enzyme complex

“…When detergent-dissolved cytochrome c oxidase was reacted with [35S]-NAP-taurine, there was heavy labeling of Va but no labeling of Vb (results not shown). A set of experiments were also conducted with a second membrane-impermeant reagent methyl 4-[3H] formylphenyl phosphate (MFPP), which reacts to form a Schiff base with lysine residues in the presence of borohydride (McMillen et al, 1986). Analysis of enzyme labeled with MFPP in reconstituted vesicles and in detergent-dispersed form showed similar results to those with NAP-taurine: subunit Va was labeled in intact vesicles ( Figure 3A), detergent disruption of these vesicles gave only a small amount of additional labeling of this subunit, while Vb was not labeled even in detergent-solubilized cytochrome c oxidase (result not shown).…”

“…Sodium methyl 4-[3H]formylphenyl phosphate (MFF'P) (5.5 mCi/mmol) was synthesized according to the procedure of McMillen et al (1986). MFPP was placed in a glass vial and solvent removed under nitrogen for 15 min.…”