The lipid bilayer determines helical tilt angle and function in lactose permease of Escherichia coli

The structure of lactose permease from Escherichia coli in its lipid environment was studied by attenuated total reflection Fourier transform infrared spectroscopy. The protein exhibits an α-helical content of about 65% and about 25% β-sheet. Unusually fast hydrogen/deuterium (H/D) exchange to 90–95% completion suggests a structure that is highly accessible to the aqueous phase. An average tilt angle of 33° for the helices was found with respect to the bilayer normal at a lipid-to-protein ratio of ≈800:1 (mol/mol), and the permease exhibits optimal activity under these conditions. However, upon decreasing the lipid-to-protein ratio, activity decreases continuously in a manner that correlates with the decrease in the lipid order parameter and the increase in the average helical tilt angle. Taken together, the data indicate that the structure and function of the permease are strongly dependent on the order and integrity of the lipid bilayer.

Determination of the angle between the anticodon and aminoacyl acceptor stems of yeast phenylalanyl tRNA in solution.

A principal feature of the crystal structures of tRNAs is an L-shaped tertiary conformation in which the aminoacyl acceptor stem and the anticodon stem are approximately perpendicular. However, the anticodon-acceptor interstem angle has not been precisely quantified in solution for any tRNA. Such a determination would represent an important test of the predicted global conformation of tRNAs in solution. To this end, we have constructed a yeast tRNA(Phe) heteroduplex RNA molecule in which the anticodon and acceptor stems of the tRNA have each been extended by approximately 70 base pairs. A comparison of the rotational decay times of the heteroduplex molecule and a linear control yields an interstem angle of 89 +/- 4 degrees in 4 mM magnesium chloride/100 microM spermine hydrochloride, essentially identical to the corresponding angle observed in the crystal under similar buffer and temperature conditions. The current approach is applicable to the study of a wide variety of RNA molecules that possess elements of nonhelical structure.