Biological NMR
“My roommate fought off the intruders!” – Characterising the virus-repressing effect of Wolbachia in Drosophila melanogaster
Sarah Walsh - @SarahW29329
Wolbachia, a common endosymbiont of insects, exerts significant antiviral effects. This phenomenon is valuable in reducing the spread of arboviruses, such as dengue. Employing NMR metabolomics, we uncover that in virus-infected Drosophila melanogaster Wolbachia drives host metabolism in a way that inhibits RNA virus replication both directly and indirectly.
NMR-Based Structural Insight into DNA G-Quadruplex
Julia Pakuła - @x_yuliya_x
Emerging as a promising area in anticancer research, noncanonical DNA structures such as G-quadruplexes are studied using NMR spectroscopy. NOESY spectra reveals crucial interactions between ligands and G-quadruplexes, helping to understand non-covalent binding mechanisms. This knowledge is essential for rational drug design targeting these structures.
Understanding the functional dynamics of PV-3C(D) and RNA interactions
Somnath Mondal - @somomsom
The picornaviral 3C(D) protein is a vital protease that cleaves viral polyproteins and host cell defense proteins, interacting with replication elements to control replication and translation. Its interaction with RNA, causing liquid-liquid phase separation, could influence viral apoptosis and infection in the host cells.
Hot and bothered: understanding the metabolic effects of heat stress in feedlot cattle using NMR-based metabolomics and clinical biochemistry
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.
Probing Dynamics of an Intact Virus Using Pseudo 3D REDOR-based MAS Experiments
Orr Lusky - @ItIsLusk
We present a robust method using ssNMR to characterise the protein dynamics in an intact virus using pseudo 3D experiments. We show an automated protocol to quantify site-specific order parameters in the backbone, and demonstrate it on the fd-Y21M bacteriophage.
Structural Insights into gp120 Glycan Recognition by 2G12 Antibody: A Paramagnetic NMR approach
Mayra Paola Oquist Phillips - @oquist_paola
Glycoprotein 120 (gp120) shields the HIV virus using high mannose glycans, which are difficult to analyze structurally due to their high isochronicity causing NMR signal collapse. Novel NMR methods, including paramagnetic probes, now enable detailed study of gp120 and its interaction with the neutralizing antibody 2G12.
Unveiling Charge-Pair Salt-Bridge Interaction Between GAGs and Collagen Protein in Cartilage: Atomic Evidence from DNP-Enhanced ssNMR at Natural Isotopic Abundance
Bijaylaxmi Patra - @BijaylaxmiNMR
Glycosaminoglycans (GAGs) and protein interactions are essential in numerous biochemical processes involving ion-pair interactions. However, there is no evidence of this, in native cartilage. Using dynamic nuclear polarization (DNP)-enhanced magic-angle spinning (MAS) NMR, we detected these interactions, revealing charge-pair salt-bridges and potential hydrogen bonding, offering new structural insights.
Protein hydraulics of AsLOV2
Shiny Maity - @shiny_maity
Water dynamics near AsLOV2's surface decrease upon light activation, suggesting expulsion of wrap water and leaving a tightly bound hydration shell. Under high pressure, AsLOV2 shifts from folded to unfolded states, mimicking light's effect. 17O NMR reveals distinct water populations. We aim to identify light's impact on these water populations.
Unusial Zn-finger protein
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.
NMR Metabolomics for Assessing the Probiotics Efficacy in the Treatment of Renal Insufficiency
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.
NMR spectroscopy – based metabolomics in variants of Non Alcoholic - Steatohepatitis (NASH) and Cryptogenic Cirrhotic patients
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.
Identify the initial pinning sites of tau to seeding-competent fibrils and the role of structural water
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.
Alterations in metabolomic profile after β-Hydroxybutyrate supplementation
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.
Solid State NMR Characterizations of Biomolecules and Materials
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.
Ester hydrolysis of calcein-AM in a lipid bilayer demonstrates the concept of lipozyme catalysis
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
Functional insight and Mechanistic Study of UBA domain of E2-25K in Lys-48 Ubiquitin Chain Elongation
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.
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.