Conformational folding of mycobacterial methoxy- and ketomycolic acids facilitated by α-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 trans-configuration with an adjacent methyl branch. To determine the effect of these functional groups on mycolic acid conformation, surface pressure (π) 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 'W-form' 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 α-methyl trans-cyclopropane-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-Guérin (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 intra-chain α-methyl trans-cyclopropane groups facilitated MA folding more than cis-cyclopropane groups.

Differential conformational behaviors of alpha-mycolic acids in Langmuir monolayers and computer simulations.

Phase diagrams of Langmuir monolayers of alpha-mycolic acids (alpha-MAs) from representative slow growing mycobacteria were intensively analyzed over a range of temperatures, by using so-called type-1 alpha-MAs having two cis-cyclopropyl groups from Mycobacterium tuberculosis, Mycobacterium kansasii and Mycobacterium avium-intracellulare complex (MAC) and type-3 alpha-MA, having one cis-cyclopropyl and one cis-double bond from Mycobacterium bovis BCG and MAC. Their intrachain groups are either cis-cyclopropyl or cis-double bond but the methylene chain segment lengths vary greatly. However, their monolayer features were alike: at lower temperatures and surface pressures, the molecules seemed to be in folded conformations, requiring larger mean molecular areas, whereas at higher temperatures and surface pressures, to be in extended conformations, requiring smaller average molecular areas approximately equivalent to cross sectional area of two hydrocarbon chains in a condensed film. In phase diagrams, the region for the folded forms of alpha-MAs was very limited, in contrast to the previously established behavior of ketomycolates, where complete folding predominated. Easy conversion of folded to extended conformations was indicated by thermodynamic studies of the Langmuir monolayer results and computer simulations.

Conformational behavior of oxygenated mycobacterial mycolic acids from Mycobacterium bovis BCG.

Phase diagrams of Langmuir monolayers of oxygenated mycolic acids, i.e. methoxy mycolic acid (MeO-MA), ketomycolic acid (Keto-MA), and artificially obtained deoxo-mycolic acid (deoxo-MA) from Mycobacterium bovis BCG were obtained by thermodynamic analysis of the surface pressure (pi) vs. average molecular area (A) isotherms. At lower temperatures and lower surface pressures, both Keto- and MeO-MAs formed rigid condensed monolayers where each MA molecule was considered to be in a 4-chain form, in which the three carbon chain segments due to bending of the 3-hydroxy aliphatic carboxylate chain and the 2-side chain were in compact parallel arrangement. At higher temperatures and surface pressures, MeO-MA and deoxo-MA tended to take stretched-out conformations in which the 3-hydroxy aliphatic carboxylate chain was more or less in an extended form, but Keto-MA retained the original 4-chain structure. The thickness measurement of the monolayers in situ by ellipsometry at different pi values and temperatures supported the above conclusions derived from the phase diagrams. The enthalpy changes associated with the phase transitions of MeO-MA and deoxo-MA implied that the MeO-MA needed larger energy to change from a compact conformation to an extended one, possibly and partly due to the dehydration of the methoxy group from water surface involved. Molecular dynamics studies of MA models derived from Monte Carlo calculations were also performed, which confirmed the conformational behavior of MAs suggested by the thermodynamic studies on the Langmuir monolayers.

Temperature dependence of the Langmuir monolayer packing of mycolic acids from Mycobacterium tuberculosis.

Phase diagrams of the Langmuir monolayer of dicyclopropyl alpha mycolic acid (alpha-MA), cyclopropyl methoxy mycolic acid (MeO-MA), and cyclopropyl ketomycolic acids (Keto-MA) from Mycobacterium tuberculosis were obtained by thermodynamic analysis of the surface pressure (pi) vs. average molecular area (A) isotherms at temperatures in the range of 10-46 degrees C. The Langmuir monolayers of MAs were shown to exhibit various phases depending on the temperature (T) and the pi values. In the Langmuir monolayer of Keto-MA, the carbonyl group in the meromycolate chain apparently touches the water surface to give the molecule a W-shape in all the temperatures and surface pressures studied. Keto-MA formed a rigid solid condensed film, with four hydrocarbon chains packing together, not observed in the others. In contrast, the monolayer films of alpha-and MeO-MAs having no such highly hydrophilic intra-chain groups in the meromycolate chain were mostly in liquid condensed phase. This novel insight into the packing of mycolic acids opens up new avenues for the study of the role of mycolic acids in the mycobacterial cell envelopes and pathogenic processes.