Secondary structure composition of reconstituted subunit b of the Escherichia coli ATP synthase

Subunit b of the Escherichia coli ATP synthase was isolated by preparative gel electrophoresis, acetone precipitated and after ion-pair extraction red issolved in a buffer either containing n-dodecyl-beta-d-maltoside or sodium cholate. The secondary structure of isolated subunit b was shown to be the same as within the F-O complex, but was strongly dependent on the detergen t used for replacement of the phospholipid environment. This was shown by a n identical tryptic digestion pattern, which was strongly influenced by the detergent used for solubilization. An influence of the detergent n-dodecyl -beta-d-maltoside on the secondary structure of the hydrophilic part of sub unit b was also shown for the soluble part of the polypeptide comprising re sidues Val25 to Leu156 (b(sol)) using CD spectroscopy. In order to determin e the secondary structure of subunit b in its native conformation, isolated subunit b was reconstituted into E. coli lipid vesicles and analyzed with CD spectroscopy. The resulting spectrum revealed a secondary structure comp osition of 80% alpha helix together with 14% beta turn conformation. These results suggest that subunit b is not a rigid rod-like alpha helix simply l inking F-1 to F-O, but rather provides an inherent flexibility for the stor age of elastic energy within the second stalk generated by rotational movem ents within the F1FO complex.