2024 Conference Hub

Kiran Kumar – @KiranKumar86269

Lipids are not typically thought to catalyze biological reactions, but evidence shows that certain lipid aggregates can speed up chemical reactions in synthetic organic chemistry. We demonstrate the potential for the hydrophobic region of a lipid bilayer to provide an environment suitable for catalysis as lipozyme. We demonstrate this concept by the ester hydrolysis of calcein-AM to produce calcein as a fluorescent product, Which is a widely used assay for esterase activity in cells. The reaction product was characterized by microscopy and 1H-NMR measurements. Overall, we explore the implications of considering lipid aggregates as catalytic entities

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.

Gajendra Singh

E2-25K (Ube2K) is a ubiquitin-conjugating enzyme that can only synthesize the K48(Lys-48) ubiquitin chain. All ubiquitin E2s have a conserved catalytic ubiquitin-conjugating domain (UBC), whereas E2-25K is the only enzyme that contains additional C-terminal ubiquitin association domain (UBA). We investigated the function of UBA domain in K48 chain elongation. NMR based results suggest that UBA domain provides the binding surface to ubiquitin during the chain elongation. It also holds the di ubiquitin (Ub 2) with different affinity to proximal and distal Ub units. We showed that the UBA domain expedites the polyubiquitin (K48) chain formation. Fluorescent polarization and mutational studies indicated that the UBA domain increases the K48 chain processivity. However, the activity is dependent upon the Ub chain length. Gel-based kinetics results also manifest the function UBA domain in polyubiquitin chain and the results evident that the rate reduces, drastically with truncated UBA domain.

Alexandra Gloria – @_AGloria

Feedlot cattle are crucial to Australia’s livestock industry, supplying beef for domestic and export markets. High summer temperatures and humidity increase heat stress vulnerability, impacting growth, productivity, and welfare. We use NMR-based metabolomics and clinical biochemistry to understand the metabolic effects of heat stress to help mitigate its impact.

Chung-Ta Han

We interrogate the key residue(s) that initiate the pinning of soluble Tau onto existing fibril active ends by tracking the time evolution of 1H-15N correlation spectra along the fibril seeding process of a tau construct composed of 19 amino acids.