Rotational mobility and orientational stability of a transport protein in lipid membranes

A single-cysteine mutant of the lactose transport protein LacS(C320A/W399C) from Streptococcus thermophilus was selectively labeled with a nitroxide s pin label, and its mobility in lipid membranes was studied as a function of its concentration in the membrane by saturation-transfer electron spin res onance. Bovine rhodopsin was also selectively spin-labeled and studied to a id the interpretation of the measurements. Observations of spin-labeled pro teins in macroscopically aligned bilayers indicated that the spin label ten ds to orient so as to reflect the transmembrane orientation of the protein. Rotational correlation times of 1-2 mu s for purified spin-labeled bovine rhodopsin in lipid membranes led to viscosities of 2.2 poise for bilayers o f dimyristoylphosphatidylcholine (28 degrees C) and 3.0 poise for the speci fic mixture of lipids used to reconstitute LacS (30 degrees C). The rotatio nal correlation time for LacS did not vary significantly over the range of low concentrations in lipid bilayers, where optimal activity was seen to de crease sharply and was determined to be 9 +/- 1 mu s (mean +/- SD) for thes e samples. This mobility was interpreted as being too low for a monomer but could correspond to a dimer if the protein self-associates into an elongat ed configuration within the membrane. Rather than changing its oligomeric s tate, LacS appeared to become less ordered at the concentrations in aligned membranes exceeding 1:100 (w/w) with respect to the lipid.