Molecular dynamics simulations of wild-type and mutant forms of the Mycobacterium tuberculosis MscL channel.

The crystal structure of the Mycobacterium tuberculosis homolog of the bacterial mechanosensitive channel of large conductance (Tb-MscL) provides a unique opportunity to consider mechanosensitive signal transduction at the atomic level. Molecular dynamics simulations of the Tb-MscL channel embedded in an explicit lipid bilayer and of its C-terminal helical bundle alone in aqueous solvent were performed. C-terminal calculations imply that although the helix bundle structure is relatively unstable at physiological pH, it may have been stabilized under low pH conditions such as those used in the crystallization of the channel. Specific mutations to the C-terminal region, which cause a similar conservation of the crystal structure conformation, have also been identified. Full channel simulations were performed for the wild-type channel and two experimentally characterized gain-of-function mutants, V21A and Q51E. The wild-type Tb-MscL trajectory gives insight into regions of relative structural stability and instability in the channel structure. Channel mutations led to observable changes in the trajectories, such as an alteration of intersubunit interactions in the Q51E mutant. In addition, interesting patterns of protein-lipid interactions, such as hydrogen bonding, arose in the simulations. These and other observations from the simulations are relevant to previous and ongoing experimental studies focusing on characterization of the channel.

A computational study of nicotine conformations in the gas phase and in water.

The conformational preferences of nicotine in three protonation states and in the gas phase as well as aqueous solution are investigated using several computational procedures. Conformational aspects emphasized are N-methyl stereochemistry, relative rotation of the pyridine and pyrrolidine rings, and pyrrolidine ring conformation. All methods consistently predicted that the N-methyl trans species are most stable for all protonation states in both gas phase and in water. However, the cis/trans energy gap is significantly reduced in water. Additionally, the two pyridine ring rotamers, which are energetically equivalent in the gas phase, experience different solvation energies in water.

Comparing and contrasting Escherichia coli and Mycobacterium tuberculosis mechanosensitive channels (MscL). New gain of function mutations in the loop region.

Sequence analysis of 35 putative MscL homologues was used to develop an optimal alignment for Escherichia coli and Mycobacterium tuberculosis MscL and to place these homologues into sequence subfamilies. By using this alignment, previously identified E. coli MscL mutants that displayed severe and very severe gain of function phenotypes were mapped onto the M. tuberculosis MscL sequence. Not all of the resulting M. tuberculosis mutants displayed a gain of function phenotype; for instance, normal phenotypes were noted for mutations at Ala(20), the analogue of the highly sensitive Gly(22) site in E. coli. A previously unnoticed intersubunit hydrogen bond in the extracellular loop region of the M. tuberculosis MscL crystal structure has been analyzed. Cross-linkable residues were substituted for the residues involved in the hydrogen bond, and cross-linking studies indicated that these sites are spatially close under physiological conditions. In general, mutation at these positions results in a gain of function phenotype, which provides strong evidence for the importance of the loop region in MscL channel function. No analogue to this interesting interaction could be found in E. coli MscL by sequence alignment. Taken together, these results indicate that caution should be exercised in using the M. tuberculosis MscL crystal structure to analyze previous functional studies of E. coli MscL.