The parallax method is a method by which the depth of fluorescent molecules within a membrane is calculated from the ratio of quenching induced by two spin-labeled phospholipids at different depths. In this report, the method is extended to measurements of depth in the polar headgroup region of the membrane through use of a lipid with a spin-label attached to the polar choline moiety. Quenching data indicate that the choline-attached nitroxide is close to 19.5 A from the bilayer center, in good agreement with the choline location previously determined by diffraction measurements. By using quenching results obtained with this polar headgroup-labeled phospholipid, depths more accurate than those measured previously can be obtained for fluorophores in the polar region of the membrane. It appears that the most reliable results are obtained when depth is calculated from the quenching of the two spin-labels that quench a specific fluorophore most strongly. Applying this approach to a series of anthroyloxy-labeled fatty acids indicates that the depth of the anthroyloxy group is almost linearly related to the number of carbon atoms between it and the carboxyl group. The fatty acid carboxyl group itself is close to 18.6 A from the bilayer center in the ionized form and 16 A from bilayer center in the protonated form. This is close to the depth of the carboxyl groups on phospholipid fatty acyl chains. More accurate depths have also been obtained for 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) labeled phospholipids using the quenching of the choline-attached spin-label.(ABSTRACT TRUNCATED AT 250 WORDS)
Diphtheria toxin membrane penetration is triggered by the low pH within the endosome lumen. Subsequent exposure to the neutral pH of the cytoplasm is believed to aid in translocation of the catalytic A domain of the toxin into the cytoplasm. To understand the effects of low pH and subsequent exposure to neutral pH on translocation, we studied toxin conformation in solution and in toxin inserted in model membranes. Two conformations were found at low pH. One form, L', predominates below 25-30 degrees C, and the other, L", predominates above 25-30 degrees C and is formed from the L' state by an unfolding event. Both forms are hydrophobic and penetrate deeply into membranes. After pH neutralization, the L' and L'' conformations give rise to two new conformations, R' and R'', respectively. The R' and R" conformations differ from each other in that in the R' state the A domain remains folded, whereas in the R" state the A domain is unfolded. This is confirmed by the finding that only the R' state possesses the capacity to bind and hydrolyze NAD+. It is also supported by the finding that the R'' state can also be formed by thermal unfolding of the R' state. The R conformations differ from the low-pH L conformations in that although they remain largely membrane-inserted, it appears that a large portion of the toxin is no longer in contact with the hydrophobic core of the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)
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