Nuclear Magnetic Resonance (NMR) spectroscopy is a quantitative, non-invasive and non-destructive technique useful in biological studies. By manipulating the magnetization of nuclei with non-zero spin, NMR gives insights into atomic level details. Application of NMR as a tool for discovering structure, understanding dynamics of bio-molecules such as proteins, metabolites, DNA, RNA and their interactions constitutes the field of bio-molecular NMR. In this thesis, new methods for rapid data analysis of NMR spectrum of proteins and metabolites are proposed. The first computational method, PROMEB (Pattern Recognition Based Assignment in Metabolomics) is useful for the identification and assignments of metabolites. This is an important step in metabolomics and is necessary for the discovery of new biomarkers. In NMR spectroscopy based studies, the conventional approach involves a database search, wherein chemical shifts are assigned to specific metabolites by use of a tolerance limit. This is inefficient because deviation in chemical shifts associated with pH or temperature variations, as well as missing peaks, impairs a robust comparison with the database. These drawbacks are overcome in PROMEB, which is a method based on matching the pattern of peaks of a metabolite in 2D [13C, 1H] HSQC NMR spectrum, rather than conventionally used absolute tolerance thresholds. A high success rate is obtained even in the presence of large chemical shift deviations such as 0.5 ppm in 1H and 3 ppm in 13C and missing peaks (up to 50%), compared to nearly no assignments obtained under these conditions with existing methods that employ a direct database search approach. The pattern recognition approach thus helps in identification and assignment of metabolites in-dependent of the pH, temperature, and ionic strength used, thereby obviating the need for spectral calibration with internal or external standards. Another computational method, ChemSMP(Chemical Shifts to Metabolic Path-ways), is described which facilitates the identification of metabolic pathways from a single two dimensional (2D) NMR spectrum. Typically in other approaches, this is done after relevant metabolites are identified to allow their mapping onto specific metabolic pathways. This task is daunting due to the complex nature of cellular processes and the difficulty in establishing the identity of individual metabolites. ChemSMP uses a novel indexing and scoring system comprised of a uniqueness score and a coverage score. Benchmarks show that ChemSMP has a positive prediction rate of > 90% in the presence of decluttered data and can sustain the same at 60 − 70% even in the presence of noise, such as deletions of peaks and chemical shift deviations. The method tested on NMR data acquired for a mixture of 20 amino acids shows a success rate of 93% in correct recovery of metabolic pathways. The third method developed is a new approach for rapid resonance assignments in proteins based on amino acid selective unlabeling. The method involves choosing a set of multiple amino acid types for selective unlabeling and identifying specific tripeptides surrounding the labeled residues from specific 2D NMR spectra in a combinatorial manner. The methodology directly yields sequence specific resonance assignments, without requiring a contiguously assigned stretch of amino acid residues to be linked, and is applicable to deuterated proteins. The fourth method involves a simple approach to rapidly identify amino acid types in proteins from a 2D NMR spectrum. The method is based on the fact that 13Cβ chemical shifts of different amino acid types fall in distinct spectral regions. By evolving the 13C chemical shifts in the conventional HNCACB or HN(CO)CACB type experiment for a single specified delay period, the phase of the cross peaks of different amino acid residues are modulated depending on their 13Cβ chemical shift values. Following this specified evolution period, the 2D HN projections of these experiments are acquired. The 13C evolution period can be chosen such that all residues belonging to a given set of amino acid types have the same phase pattern (positive or negative) facilitating their identification. This approach does not re-quire the preparation of any additional samples, involves the analysis of 2D [15N,1H] HSQC-type spectra obtained from the routinely used triple resonance experiments with minor modifications, and is applicable to deuterated proteins. Finally, the practical application of these methods for laboratory research is presented. PROMEB and ChemSMP is used to study cancer cell metabolism in previously unexplored oncogenic cell line. PROMEB helped in assigning a differential metabolite present at high concentration in cancer cell line compared to control non-cancerous cell line. ChemSMP revealed active metabolic pathways responsible for regulating energy homeostasis of cancer cells which were previously reported in literature. The two methods developed for rapid protein resonance assignments can be used in applications such as identifying active-site residues involved in ligand binding, phosphorylation, or protein-protein interactions. The phase modulated experiments will be useful for quick assignment of signals that shift during ligand binding or in combination with selective labeling/unlabeling approaches for identification of amino acid types to aid the sequential assignment process. Both the methodology was applied to two proteins: Ubiquitin (8 kDa) and L-IGFBP2 an intrinsically disordered protein (12 kDa), for demonstrating rapid resonance assignment using only set of 2D NMR experiments.

abstract: Coronaviruses are the causative agents of SARS, MERS and the ongoing COVID-19 pandemic. Coronavirus envelope proteins have received increasing attention as drug targets, due to their multiple functional roles during the infection cycle. The murine coronavirus mouse hepatitis virus strain A59, a hepatic and neuronal tropic coronavirus, is considered a prototype of the betacoronaviruses. The envelope protein of the mouse hepatitis virus (MHV-E) was extensively screened with various membrane mimetics by solution state nuclear magnetic resonance spectroscopy to find a suitable mimetic, which allowed for assignment of ~97% of the backbone atoms in the transmembrane region. Following resonance assignments, the binding site of the ion channel inhibitor hexamethylene amiloride (HMA) was mapped to MHV-E using chemical shift perturbations in both amide and aromatic transverse relaxation optimized spectroscopy (TROSY) spectra, which indicated the inhibitor binding site is located at the N-terminal opening of the channel, in accord with one of the proposed HMA binding sites in the envelope protein from the related SARS (severe acute respiratory syndrome) betacoronavirus. Structure calculation of residues M1-K38 of MHV-E, encompassing the transmembrane region, is currently in progress using dihedral angle restraints obtained from isotropic chemical shifts and distance restraints obtained from manually assigned NOE cross-peaks, with the ultimate aim of generating a model of the MHV-E viroporin bound to the inhibitor HMA. This work outlines the first NMR studies on MHV-E, which have provided a foundation for structure based drug design and probing interactions, and the methods can be extended, with suitable modifications, to other coronavirus envelope proteins. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2020

Synopsis My research work is focused on: (i) development of novel Fast NMR methods in solution state and their application to metabolomics and small molecules. (ii) NMR based metabolic study of human IVF to assess embryo viability for implantation. The major components of the embryo growth media were identified for evaluating the embryo quality. Described below are the projects carried out towards the dissertation of my PhD. Chapter 1 describes NMR methods which are the foundation stones for new Fast NMR methods developed. Typical 1D and 2D NMR experiments used in metabolomics and statistical methods for analysis are described. A few applications of metabolomics are also covered in the chapter. Chapter 2 describes a new Fast NMR method based on polarization sharing and parallel acquisition using the dual receiver system. The method developed helps in acquiring simultaneously three 2D NMR spectra: 2D [13C-1H] HETCOR, 2D [1H-1H] TOCSY and 2D [13C-1H] HSQC-TOCSY in a single data set. This method achieves a time saving of about two fold. All the experiments are acquired on molecules with natural abundance of 13C. The method was used to assign the side chain atoms (1H and 13C) of two important peptides. i) 12 amino acid residue peptide, which is a part of central linker domain of Human Insulin like Growth Factor Binding Protein-2 known to play a vital role in the IGF system and ii) a 18 amino acid residue peptide which acts as an antimicrobial agent. Chapter 3 describes extension of the Fast NMR method described in chapter 2. The method is combined with G-matrix Fourier Transform NMR spectroscopy. In this method we have acquire simultaneously two 2D NMR experiments and one reduced dimensional 3D experiment. The three experiments are 2D [13C-1H] HETCOR, 2D [1H-1H] TOCSY and GFT (3,2)D [13C-1H] HSQC-TOCSY, which provide complementary information for rapid assignments. GFT (3,2)D [13C-1H] HSQC-TOCSY gives 3D correlations in a 2D manner facilitating high resolution and unambiguous assignments. The experiments were applied for complete assignment of 21 unlabeled metabolite mixtures corresponding to the Innovative Sequential medium (ISM1) used for culturing human embryos for IVF. Further, a 13C multiplicity edition block is added to the method to simplify the resonances assignment in GFT (3,2)D [13C-1H] HSQC-TOCSY. Taken together, experiments provide time gain of order of magnitudes compared to conventional data acquisition. Chapter 4 of the thesis describes a metabolomics study of Human in-vitro fertilization to assess viable embryos of implantation potential using NMR as non-invasive tool. NMR study included the analysis of 127 embryo culture media (Innovative Sequential Media-1) and 29 controls (culture media without embryo) of both day-2 and day-3 transferred. The embryos were divided into 3 categories 1) implanted (successful) 2) transferred not-implanted (unsuccessful) 3) not transferred based on morphological studies. All NMR experiments were acquired with CPMG (T2 filter) incorporated in 1D 1H presaturation pulse scheme. The study was based on estimation of lactate, pyruvate and alanine levels in the embryo culture media (ISM1). The study reveals higher uptake of pyruvate and high pyruvate/alanine ratios in case of implanted embryos compared to one which failed to implant. Present study provides pyruvate/alanine ratio as a biomarker to select the embryos with high implantation potential. The method combined with morphology based assessment or with other biomarkers can be serve as a powerful tool to assess the embryo quality. Chapter 5 describes a novel NMR method for rapid characterization of translation diffusion of molecules in solution either in mixture or pure form. Unlike acquisition of several 2D [13C-1H] HSQC experiments with varying gradients to get diffusion measurement, a single 2D [13C-1H] HSQC is sufficient to measure the diffusion coefficients which is in the linewidths of peaks. The method uses the idea of accordion NMR spectroscopy, wherein gradients are linearly co-incremented with 13C chemical shift evolution period during t1. The methodology speeds up the acquisition by replacing series of 2D [13C-1H] HSQC with single 2D constant time [13C-1H] HSQC. The method was used to monitor the diffusion of metabolites in a time-resolved manner during polymerization of SDS-PAGE gel. Using this method, it was possible to detect the presence of oligomers of diphenylalanine (FF) during its self assembly to form nanotubular structures.