Fractional deuteration applied to biomolecular solid-state NMR spectroscopy

Solid-state Nuclear Magnetic Resonance can provide detailed insight into structural and dynamical aspects of complex biomolecules. With increasing molecular size, advanced approaches for spectral simplification and the detection of medium to long-range contacts become of critical relevance. We have analyzed the protonation pattern of a membrane-embedded ion channel that was obtained from bacterial expression using protonated precursors and D2O medium. We find an overall reduction of 50% in protein protonation. High levels of deuteration at Hα and Hβ positions reduce spectral congestion in (1H,13C,15N) correlation experiments and generate a transfer profile in longitudinal mixing schemes that can be tuned to specific resonance frequencies. At the same time, residual protons are predominantly found at amino-acid side-chain positions enhancing the prospects for obtaining side-chain resonance assignments and for detecting medium to long-range contacts. Fractional deuteration thus provides a powerful means to aid the structural analysis of complex biomolecules by solid-state NMR

Structural and functional studies of a prokaryotic cyclic nucleotide-gated channel

Ion channels gated by cyclic nucleotides have crucial roles in cardiac and neuronal excitability and in signal transduction of sensory neurons. On binding cyclic nucleotides these channels are activated, which results an increase in membrane conductance. Although a lot of information is available on the function of these channel proteins, the molecular events that relay ligand binding to channel activation is not well understood. Here, I studied ligand binding of prokaryotic cyclic nucleotide-activated K$^{+}$ channels. One of them, the mlCNG channel from the nitrogen-fixing bacterium Mesorhizobium loti was suitable for biophysical characterization. One of the key questions that I worked on was how gating of the channel affects its ligand binding properties? I performed ligand binding studies on the tetrameric mlCNG protein and its isolated cyclic nucleotide-binding domain (CNBD). Affinity of cyclic nucleotides to the full-length mlCNG protein and to the CNBD was determined using spectroscopic methods. Both, the mlCNG channel and the CNBD bind cAMP in a non-cooperative manner with similar binding affinity. These results indicate that either no appreciable binding energy is required for activation, or the conformational change in the CNBD is the activation step itself. Crystallography experiments were performed on the mlCNG channel. Two-dimensional crystals were obtained in which the channel proteins were ordered in a square lattice. The channel proteins were assembled as tetramers and were arranged in a head-to tail fashion. The crystal diffracts to 15 Å. This is an excellent starting condition for future work to eventually obtain a structure at atomic resolution

A metabolomics footprint approach to understanding the benefits of synbiotics in functional foods and dietary therapeutics for health, communicable and non-communicable diseases

Gut microbiota have been shown to affect various cellular and host response elements such as immunological, neurological, energy, storage, etc. In recent years, this has led to rapid expansion in dietary products containing probiotics, prebiotics and combination thereof in synbiotics. While benefits of consuming functional foods derived from probiotics strains have been demonstrated for various metabolites, a detailed analysis of the biochemical footprints and their benefits remain under-studied. Herein, using a combination of NMR metabolomics, microbial techniques and cell-culture assays, we have characterized metabolite profiles of probiotic viz. Lactobacillus delbruekii ATCC 9649, Lactobacillus casei ATCC 335, Lactobacillus plantarum NRC 716 and Bacillus coagulans ATCC 12425 cultures in fermented milk. We identified predominance of sugars, small chain fatty acids, organic acids and branched chain amino acids from natural abundance 13C NMR studies. Additionally, we identified myriad metabolites and their respective pathways using 1H NMR spectroscopy. Based on our findings, synbiotic fermented dairy products were customized with co-cultures and complemented with pro- and pre- biotics. Furthermore, we demonstrate epithelial cell interaction and anti-microbial activity of L. plantarum based ferment against a range of bacterial pathogens highlighting possible biochemical mechanisms for anti-microbial activity, quorum sensing, gut colonization and other beneficial factors that may be crucial. Furthermore, we propose plausible explanation against non-communicable diseases such as tumor-inhibitory, anti-proliferative and pro-apoptotic effects which has direct implications for dietary therapeutics

Kinetics of ligand-receptor interaction reveals an induced-fit mode of binding in a cyclic nucleotide-activated protein

Many receptors and ion channels are activated by ligands. One key question concerns the binding mechanism. Does the ligand induce conformational changes in the protein via the induced-fit mechanism? Or does the protein preexist as an ensemble of conformers and the ligand selects the most complementary one, via the conformational selection mechanism? Here, we study ligand binding of a tetrameric cyclic nucleotide-gated channel from Mesorhizobium loti and of its monomeric binding domain (CNBD) using rapid mixing, mutagenesis, and structure-based computational biology. Association rate constants of ∼10(7) M(−1) s(−1) are compatible with diffusion-limited binding. Ligand binding to the full-length CNG channel and the isolated CNBD differ, revealing allosteric control of the CNBD by the effector domain. Finally, mutagenesis of allosteric residues affects only the dissociation rate constant, suggesting that binding follows the induced-fit mechanism. This study illustrates the strength of combining mutational, kinetic, and computational approaches to unravel important mechanistic features of ligand binding

Subunits act independently in a cyclic nucleotide-activated K+ channel

Ion channels gated by cyclic nucleotides have crucial roles in neuronal excitability and signal transduction of sensory neurons. Here, we studied ligand binding of a cyclic nucleotide-activated K+ channel from Mesorhizobium loti and its isolated cyclic nucleotide-binding domain. The channel and the binding domain alone bind cyclic AMP with similar affinity in a non-cooperative manner. The cAMP sensitivities of binding and activation coincide. Thus, each subunit in the tetrameric channel acts independently of the others. The binding and gating properties of the bacterial channel are distinctively different from those of eukaryotic cyclic nucleotide-gated channels

Conformational heterogeneity coupled with β-fibril formation of a scaffold protein involved in chronic mental illnesses

Chronic mental illnesses (CMIs) pose a significant challenge to global health due to their complex and poorly understood etiologies and hence, absence of causal therapies. Research of the past two decades has revealed dysfunction of the disrupted in schizophrenia 1 (DISC1) protein as a predisposing factor involved in several psychiatric disorders. DISC1 is a multifaceted protein that serves myriads of functions in mammalian cells, for instance, influencing neuronal development and synapse maintenance. It serves as a scaffold hub forming complexes with a variety (~300) of partners that constitute its interactome. Herein, using combinations of structural and biophysical tools, we demonstrate that the C-region of the DISC1 protein is highly polymorphic, with important consequences for its physiological role. Results from solid-state NMR spectroscopy and electron microscopy indicate that the protein not only forms symmetric oligomers but also gives rise to fibrils closely resembling those found in certain established amyloid proteinopathies. Furthermore, its aggregation as studied by isothermal titration calorimetry (ITC) is an exergonic process, involving a negative enthalpy change that drives the formation of oligomeric (presumably tetrameric) species as well as β-fibrils. We have been able to narrow down the β-core region participating in fibrillization to residues 716–761 of full-length human DISC1. This region is absent in the DISC1Δ22aa splice variant, resulting in reduced association with proteins from the dynein motor complex, viz., NDE-like 1 (NDEL1) and lissencephaly 1 (LIS1), which are crucial during mitosis. By employing surface plasmon resonance, we show that the oligomeric DISC1 C-region has an increased affinity and shows cooperativity in binding to LIS1 and NDEL1, in contrast to the noncooperative binding mode exhibited by the monomeric version. Based on the derived structural models, we propose that the association between the binding partners involves two neighboring subunits of DISC1 C-region oligomers. Altogether, our findings highlight the significance of the DISC1 C-region as a crucial factor governing the balance between its physiological role as a multifunctional scaffold protein and aggregation-related aberrations with potential significance for disease