Biophysical characterization and stabilization of detergent-solubilized lipoprotein N-acyl transferase from P. aeruginosa and E. coli.

Lipoproteins are important for bacterial growth and virulence and interest in them as targets for antibiotic development is growing. Lipoprotein N-acyl transferase (Lnt) catalyzes the final step in the lipoprotein posttranslational processing pathway. The mature lipoprotein can remain in the inner membrane or be trafficked to the outer membrane in the case of diderm prokaryotes. With a view to obtaining high-resolution crystal structures of membrane integral Lnt for use in drug discovery a program was undertaken to generate milligram quantities of stable, homogenous and functional protein. This involved screening across bacterial species for suitable orthologues and optimization at the level of protein expression, solubilization and stability. Combining biophysical and functional characterization, orthologous Lnt from Escherichia coli and the opportunistic human pathogen Pseudomonas aeruginosa was identified as suitable for the proposed structure determination campaign that ultimately yielded crystal structures. The rational approaches taken that eventually provided structure-quality protein are presented in this report.

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