Tag: Biological NMR

Tobias Schubeis – @TSchubeis

The CorA channel is the major Mg2+-influx pathway in prokaryotes. The mechanistic details of the channel gating and the transport of the metal ions are still not entirely understood. Here we investigate the dynamics of CorA in DMPC lipid bilayers with 1H-detected solid-state NMR at 100 kHz MAS.

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.

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

Malitha Chathuranga Dickwella Widanage – @MalithaChathur4

Intermediates in fibrillation pathways, toxic and responsible for amyloid diseases, are hard to study due to their heterogeneity and instability. We introduce a novel MAS methodology to produce stable hIAPP oligomers, which are heterogeneous and β-sheet-rich, capable of seeding fibrillation. MAS NMR shows their nature is sample condition-sensitive.

Kamil Aysin – @KamilNAysin

Specialized diet for obese people together with clear markers of metabolic status is a must. We took 48 volunteers’ urine samples before/after two weeks of food supplementation with β-Hydroxybutyrate. Compare to the control group we observed altered concentrations of several metabolites showing a positive effect of BHB food supplementation.

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.

Shreya Pandey – @shreyapandey171

The global prevalence of NASH is 1.5-6.5%. In India, NAFLD affects 9-32%, with unclear NASH prevalence. NAFLD is marked by excess liver triglycerides (TG), while NASH includes steatosis, hepatocyte damage, inflammation, and fibrosis, leading to severe liver issues. Current NASH diagnosis relies on biopsies, but metabolomic and lipidomic profiling offers non-invasive diagnostic potential. This study aims to identify specific metabolites to improve non-invasive diagnosis and treatment.

Sofia Mariasina, Ulyana Bulgakova, Olga Gavrilova, Anna Kuzmina – @SMariasina with @uliana_buv, @GavrilovaO25732 and @AnnaKuz38797

Kidney disorders dramatically change the blood composition. We use NMR-metabolomics to assess the effectiveness of probiotics supplementation in the treatment of renal insufficiency in human. For that purpose metabolic profiling of the blood of patients with chronic kidney disease was carried out before and after probiotic supplementation.

Sergei Dukhalin – @DukhalinSergei

Zinc finger proteins play an important role in DNA organization. We have found a new family of D. Melanogaster proteins with an unusual zinc finger domain capable of forming homo- and heterodimers. The 3D structure of one of this family member was determined as homodimer by solution NMR.