From Rigid to Flexible: Impact of Macrocycle Loss on Tolaasin’s Backbone Dynamics and Activity

Durga Prasad

In this project, we investigate the impact of macrocycle loss on tolaasin activity. Tolaasin, a Cyclic Lipopeptide (CLiP) from Pseudomonas tolaasii, plays a critical role in causing brown blotch disease in mushrooms. Its 18-amino acid sequence features an N-terminal lipid tail and a macrocycle formed via an ester bond. Tolaasin exhibits inhibitory action against fungal and Gram-positive bacteria, underscoring its significance. Studies have shown that hydrolysing the ester bond, potentially opening the macrocycle, can detoxify tolaasin, highlighting the macrocycle’s role in tolaasin’s function. Understanding how specific structural changes alter membrane interactions is crucial for developing novel therapeutics and biocontrol agents.
Our approach involves studying hydrolysed tolaasin in parallel with the native molecule in SDS micelles using NMR spectroscopy. To enable advanced multidimensional structural analysis, we first produce 15N isotope-enriched tolaasin by cultivating the producing bacterium on a minimal medium supplemented with suitable labeled isotopically enriched precursors. Subsequently, isotopically enriched hydrolyzed form was synthesized through controlled alkaline hydrolysis.
A comprehensive analysis, including full resonance assignment and 15N R1, R2, and het-nOe experiments, allows us to investigate peptide backbone dynamics. The order parameters (S2) derived from model-free analysis of relaxation data provide insights into molecular motions occurring on a nanosecond to picosecond timescale. By employing reduced spectral density mapping at Jω0, JωN, and Jω0.87H, we distinguish residues with distinct rigidity and flexibility profiles in both forms of tolaasin. Furthermore, NH R1 rates are determined both in the absence and presence of a soluble paramagnetic relaxation agent. This facilitates mapping the PRE wave of tolaasin, extracting tilt and azimuth angles, and enabling the mapping of helix orientations. Our findings indicate that the opening up of the macrocycle results in a partial loss of peptide backbone rigidity, leading to involvement in microsecond dynamics by later exocyclic residues.