Orientation of the carboxyl and NH2 termini of the membrane-binding segment of cytochrome b5 on the same side of phospholipid bilayers.

H.A Dailey,P Strittmatter

doi:10.1016/s0021-9258(19)69551-7

Abstract

The present data show that the carboxyl terminal end of the membrane binding segment (nonpolar peptide) of cytochrome b5 is present on the same side of phospholipid bilayers as the hydrophilic, heme-containing, NH2-terminal segment. This orientation was determined by observing rapid ionization of both tyrosyl residues at positions 5 and 8 from the carboxyl terminus upon addition of sodium hydroxide to the outer aqueous phase of vesicle preparations, and the reaction of one of these residues with a polar, impermeant reagent, diazotized sulfanilic acid. The rate of ionization of both aromatic residues occurred at least 1 order of magnitude faster than ionization of indigo trisulfonate trapped in the inner aqueous compartment of the vesicles. These data and consideration of our earlier characterization of cytochrome b5 structure and binding to membranes support a model for the membrane binding segment that is highly structured, penetrates to the middle of the bilayer, and loops back to the outer surface to place both the NH2 and the carboxyl termini on the same surface of the bilayer.

Highlights

End of the membrane binding segment of cytochrome bs is present on the same side of segment were prepared as described previously [13].Small unilamelphospholipid bilayers as the hydrophilic, heme-containing, NHz-terminalsegment
1 order of magnitude faster than ionization of indigo trisulfonate trapped in the inner aqueous compartment of the vesicles. These data and consideration ofour earlier characterization of cytochrome bs structure and binding to membranes support a model for the membrane binding segment that is highly structured, penetrates to the middle of the bilayer, and loops back to the outer surface to place both the NH2 and the carboxyl termini on the same surface of the bilayer
The concentration of phospholipid was about 200 VM with 2.6 mM indigo trisulfonate trapped inside the vesicles

Summary

MATERIALS AND METHODS

End of the membrane binding segment (nonpolar pep- Steer livermicrosomal cytochrome b, and the nonpolar peptide tide) of cytochrome bs is present on the same side of segment were prepared as described previously [13].Small unilamelphospholipid bilayers as the hydrophilic, heme-containing, NHz-terminalsegment This orientation was determined by observing rapid ionization of both tyrosy residues at positions 5 and 8 from the carboxyl terminus upon addition ofsodium hydroxide to the outer aqueous phase of vesicle preparations, and the reaction of one of these residues with a polar, impermeant reagent, diazotized sulfanilic acid. In solution "d;ot., BoEunLd" to beginning at residue 94 rather than 91 [4], reacted rapidly rnol/rnol when bound tightly to egg phosphatidylcholine vesicles These Iodine data indicate that all of the nonpolar peptide molecules are NaOH. _I to follow the ionization of tyrosyl hydroxyl groups spectrophotometrically and because it was expected that small unilamellar egg phosphatidylcholine vesicles are not "leaky" to protons, vesicle-bound nonpolar peptide was titrated with. These data are consistent with previous reports that dimyristyplhosphatidylcholine vesicles arme ucmh ore permeablethan egg lecithin [19] and shows that binding protein increases this permeability to protons

Both tyrosyl residuesof the nonpolar peptideionize at pH

DISCUSSION

Vesicle preparation