Conformational folding of mycobacterial methoxyand ketomycolic acids facilitated by a-methyl trans-cyclopropane groups rather than cis-cyclopropane units The oxygenated long-chain mycolic acids from many mycobacteria are characterized by the presence of mid-chain cyclopropane groups, which can have either cis-configuration or transconfiguration with an adjacent methyl branch. To determine the effect of these functional groups on mycolic acid conformation, surface pressure (p) versus mean molecular area isotherms of methoxy-(MeO-) mycolic acids (MAs) from Mycobacterium kansasii, Mycobacterium tuberculosis (Mtb) Canetti and Mtb H37Ra, and of keto-MAs from Mycobacterium avium-intracellulare complex (MAC) and Mtb H37Ra were recorded and analysed. The MeO-and keto-MAs from Mtb H37Ra, containing scarcely any trans-cyclopropyl groups, apparently took no fully folded 'Wform' conformations. Keto-MA from MAC, whose trans-cyclopropyl group content is nearly 90 %, showed a very solid W-form conformation. MeO-MAs from M. kansasii and Mtb Canetti gave stable W-form conformations at lower temperatures and surface pressures and extended conformations at higher temperatures and surface pressures; their W-form conformation was not as stable as expected from their cis-cyclopropyl group content, probably because they had a wide range of constituent homologues. Energy level calculations of cis-or a-methyl transcyclopropane-containing model molecules and computer simulation studies confirmed the superior folding properties of the latter functional unit. The present results were compared with those of MeO-and keto-MAs from Mtb and from M. bovis Bacillus Calmette-Gué rin (BCG) reported previously. Among the oxygenated MAs, those having higher trans-cyclopropane content tended to take W-form conformations more firmly, implying that the meromycolate proximal intrachain a-methyl trans-cyclopropane groups facilitated MA folding more than cis-cyclopropane groups. INTRODUCTIONMycobacterial mycolic acids (MAs) are characteristic components of the mycobacterial cell envelope, where major proportions of the acids are covalently bonded to the cell wall penta-arabinosyl units (Minnikin, 1982;Draper, 1998;Dmitriev et al., 2000). As shown in the general structure below, MAs are long chain 2-alkyl branched, 3-hydroxy fatty acids, with two intra-chain groups X and Y in the major chain, termed the meromycolate chain:According to the distal intra-chain functional group X, the MAs in this study are classified into three major groups: a nonoxygenated a-, and two oxygenated groups, methoxy-(MeO-) and keto-MAs. According to the proximal intra-chain group Abbreviations: BCG, Mycobacterium bovis Bacillus Calmette-Gué rin; MAC, Mycobacterium avium-intracellulare complex; MA, mycolic acid; Mtb, Mycobacterium tuberculosis.