My research work for PhD has focused on: (i) the development and application of new NMR methodologies to solve challenging problems in structural biology and (ii) studying important biological systems to correlate their structural and functional aspects. I have worked on diverse research projects ranging from NMR methodology development to the study of structure and dynamics of protein-based nanotubes. Chapter 1 of my thesis gives brief introduction to bio-molecular NMR spectroscopy and the different biological systems that I have studied. In recent years, several new methods have emerged for rapid NMR data collection. One class of methods is G-matrix Fourier transform (GFT) projection NMR spectroscopy. GFT NMR spectroscopy involves phase sensitive joint sampling of two or more chemical shifts in an indirect dimension of a multidimensional NMR experiment. Chapter 2 describes a new method based on the principle of GFT NMR for increasing further the speed of data collection. In the current implementations of the GFT method, cosine/sine modulation of all chemical shifts involved in the joint sampling are collected and stored as separate FIDs. A post-acquisition data processing step (application of G-matrix) then separates the different inter-modulations of chemical shifts. Thus, joint sampling of K+1 spins results in 2K combination of chemical shifts (also representing 2K projection angles). One limitation of this approach is that even if only a few of the 2K components of the multiplet (or projection angles) is desired, an entire data set containing information for all 2K shift combinations is collected. We have proposed a simple method which releases this restriction and allows one to selectively detect only the desired linear combination of chemical shifts/projection angles out of 2K combinations in a phase sensitive manner. The method involves selecting the appropriate cosine/sine modulations of chemical shifts and forming the desired linear combination by phase cycling of the radiofrequency pulses and receiver. This will benefit applications where only certain linear combination of shifts are desired or/and are sufficient. Further, G-matrix transformation required for forming the linear combination is performed within the pulse sequence. This avoids the need for any post-acquisition data processing. Taken together, this mode of data acquisition will foster new applications in projection NMR spectroscopy for rapid resonance assignment and structure determination. Chapter 3 describes another GFT NMR-based method for rapid estimation of secondary structure in proteins. This involves the detection of specific linear combination of backbone chemical shifts and facilitates a clear separation and estimation of residues in different secondary structures of a given protein. This methodology named as CSSI-PRO (Combination of Shifts for Secondary structure Identification in PROteins), involves detection of specific linear combination of backbone 1Hα and 13C’ chemical shifts in a two dimensional (2D) NMR experiment. Such linear combination of shifts facilitates editing of residue belonging to α-helical/ β-strand regions into distinct spectral regions nearly independent of the amino acid type. This helps in the estimation of overall secondary structure content of the protein. Comparison of the estimated secondary structure content with those obtained from the respective 3D structures and/or the method of Chemical Shift Index (CSI) was carried out for 254 proteins and gives a correlation of more than 90% and an overall RMSD of 6.5%. The method has high sensitivity and data can be acquired in a few minutes. This methodology has several applications such as for high-throughput screening of proteins in structural proteomics and for monitoring conformational changes during protein folding and/or ligand-binding events. Chapter 4 (Part-A and Part-B) describes an area of my research which involves the study of structure and function in the Insulin-like Growth Factor Binding Protein (IGFBP) family. IGFBPs (six in number; IGFBP1-6) belong to the IGF-system, which plays an important role in growth and development of the human body. This system is comprised of the following components: (i) Two peptide hormones, IGF-1 and -2, (ii) type 1 and type 2 IGF receptors, (iii) six IGF-binding proteins (IGFBP; numbered 1-6) and (iv) IGFBP proteases. IGF-1 and -2 are small signalling peptides (~7.5 kDa) that stimulate action by binding to specific cell surface receptors (IGF-1R) evoking subsequent response inside the cell. Six soluble IGF binding proteins, the IGFBPs, which range in 22-31 kDa in size and share overall sequence and structural homology with each other, regulate the activity of the IGFs. IGFBPs bind strongly to IGFs (KD ~ 300-700 pM) to ensure that all the circulating IGF in the blood stream is sequestered and inhibit the action of IGFs by blocking their access to the receptors. Proteolysis of the IGFBPs dissociates IGFs from the complex, enabling them to bind and activate the cell surface receptors. IGFBPs have been recently implicated in different cancers and HIV/AIDS. However, the nature of their interaction with the ligand: IGF-1 or IGF-2 at a molecular level poorly understood. This is due to the difficulty in over-expressing these proteins in large scale and in soluble amounts which is required for structural studies. We have for the first time developed an efficient method for bacterial expression of full-length human IGFBP-2, a 33 kDa system, in soluble (upto 30 mg/ml) and folded form. Using a single step purification protocol, hIGFBP-2 was obtained with >95% purity and structurally characterized using NMR spectroscopy. The protein was found to exist as a monomer at the high concentrations required for structural studies and to exist in a single conformation exhibiting a unique intra-molecular disulfide-bonding pattern. The protein retained full biologic activity as evident from its strong binding to IGF-1 and IGF-2 detected using surface plasmon resonance (SPR). This study represents the first high-yield expression of wild-type recombinant human IGFBP-2 in E. coli and first structural characterization by NMR. Using different NMR methods, we are now in the process of elucidating the 3D structure of this molecule. Chapter 5 (Part-A and Part-B) describes our discovery of nanotubular structures formed by spontaneous self-assembly of a small fragment from the C-terminal domain of hIGFBP-2. The nanotubular structures are several micrometers long and have a uniform outer diameter of ~35 nm. These structures were studied extensively by NMR and other techniques such as TEM, fluorescence and circular dichroism (CD). The water soluble nanotubes form through intermolecular disulphide bonds due to the presence of three cysteines in the polypeptide chain and exhibit enhanced tyrosine fluorescence. Based on different experimental evidences we have proposed a mechanism for the formation of the nanotubes. This was considered as a breakthrough by the journal ChemComm and featured on the cover-page of the journal. An article highlighting the discovery was also published in RSC news. In recent years, a number of novel polypeptide and DNA based nanotubes have been reported. Our study reveals intrinsically fluorescent self-assembling nanotubes made up of disulphide bonds having the following novel properties: (i) their formation/dissociation can be controlled by tuning the redox conditions, (ii) they do not require the support of any additional chemical agent for self-assembly, (iii) they have high stability due to the involvement of covalent interactions, (iv) the monomer is a small polypeptide chain which can be chemically synthesized or produced using simple recombinant methods and (v) they possess high inherent fluorescence and can thus be easily detected against a background of other proteins. In addition, the presence of an RGD motif in this polypeptide fragment offers avenues for novel biomedical applications. The RGD motif is known to be recognized by integrins. The design of such self-assembling polypeptide fragments containing an RGD motif can be utilized to enhance the efficacy of cancer therapeutics. Towards this end, we have investigated the structural basis of formation of these nanotubular structures by NMR spectroscopy and proposed its application for cancer cell imaging.

The work is focused on the area of the proteins and their domains. It also briefly describes gathering methods of the protein´s structure at the various levels of the hierarchy. This is followed by examining of existing tools for protein´s domains prediction and databases consisting of domain´s information. In the next part of the work selected representatives of prediction methods are introduced.  These methods work with the information about the internal structure of the molecule or the amino acid sequence. The appropriate chapter outlines applied procedure of domains´ boundaries prediction. The prediction is derived from the primary structure of the protein, using a neural network  The implemented procedure and its possibility of further development in the related thesis are introduced at the conclusion of this work.

Protein sequences contain the information in order for a protein to fold to a unique compact, three-dimensional native structure. The forces that drive protein structures to form compact folds are largely dominated by burial of hydrophobic amino acids, which results in non-specific packing of amino acid side-chains. The knob-socket model attempts to organize side-chain packing into tetrahedral packing motifs. This tetrahedral motif is characterized with a three residues on the same secondary structure forming the base of the tetrahedron packing with a side-chain from a separate secondary structure. The base of the motif is termed the socket, and the other side-chain is called the knob. Here, we focus on extending the knob-socket model to understand tertiary and quaternary structure. First, single knobs sometimes pack into more than one socket in real structures. We focus on understanding the topology and amino acid preferences of these tertiary packing surfaces. The main results from the study of tertiary packing surfaces is that they have a preferred handedness, some interactions are ancillary to the packing interaction, there are specific amino preferences for specific positions in packing surfaces, and there is no relationship between side-chain rotamer of the knob packing into the tertiary packing surface. Next, we examine the application of the knob-socket to irregular and mixed packing in protein structure. The main conclusions from these efforts show canonical packing modes between secondary structures and highlight the important of coil secondary structure in providing many of the knobs for packing. Third, we investigate protein quaternary structure with a clique analysis of side-chain interactions. We identify a possible pseudo knob-socket interaction, and compare knob-socket interactions between tertiary and quaternary structure. Lastly, we discuss the workflow used in CASP12 to predict side-chain contacts and atomic coordinates of proteins.

Proteins are molecular machines of life in the truest sense. Being the expressors of genotype, proteins have been a focus in structural biology. Since the first characterization and structure determination of protein molecule more than half a century ago1, our understanding of protein structure is improving only incrementally. While computational analysis and experimental techniques have helped scientist view the structural features of proteins, our concepts about protein folding remain at the level of simple hydrophobic interactions packing side-chain at the core of the protein. Furthermore, because the rate of genome sequencing is far more rapid than protein structure characterization, much more needs to be achieved in the field of structural biology. As a step in this direction, my dissertation research uses computational analysis and experimental techniques to elucidate the fine structural features of the tertiary packing in proteins. With these set of studies, the knowledge of the field of structural biology extends to the fine details of higher order protein structure.

<p>Protein malfunction can cause human diseases, which makes the protein a target in the process of drug discovery. In-depth knowledge of how protein functions can widely contribute to the understanding of the mechanism of these diseases. Protein functions are determined by protein structures and their dynamic properties. Protein dynamics refers to the constant physical movement of atoms in a protein, which may result in the transition between different conformational states of the protein. These conformational transitions are critically important for the proteins to function. Understanding protein dynamics can help to understand and interfere with the conformational states and transitions, and thus with the function of the protein. If we can understand the mechanism of conformational transition of protein, we can design molecules to regulate this process and regulate the protein functions for new drug discovery. Protein Dynamics can be simulated by Molecular Dynamics (MD) Simulations.</p> <p>The MD simulation data generated are spatial-temporal and therefore very high dimensional. To analyze the data, distinguishing various atomic interactions within a protein by interpreting their 3D coordinate values plays a significant role. Since the data is humongous, the essential step is to find ways to interpret the data by generating more efficient algorithms to reduce the dimensionality and developing user-friendly visualization tools to find patterns and trends, which are not usually attainable by traditional methods of data process. The typical allosteric long-range nature of the interactions that lead to large conformational transition, pin-pointing the underlying forces and pathways responsible for the global conformational transition at atomic level is very challenging. To address the problems, Various analytical techniques are performed on the simulation data to better understand the mechanism of protein dynamics at atomic level by developing a new program called Probing Long-distance interactions by Tapping into Paired-Distances (PLITIP), which contains a set of new tools based on analysis of paired distances to remove the interference of the translation and rotation of the protein itself and therefore can capture the absolute changes within the protein.</p> <p>Firstly, we developed a tool called Decomposition of Paired Distances (DPD). This tool generates a distance matrix of all paired residues from our simulation data. This paired distance matrix therefore is not subjected to the interference of the translation or rotation of the protein and can capture the absolute changes within the protein. This matrix is then decomposed by DPD</p> <p>using Principal Component Analysis (PCA) to reduce dimensionality and to capture the largest structural variation. To showcase how DPD works, two protein systems, HIV-1 protease and 14-3-3 σ, that both have tremendous structural changes and conformational transitions as displayed by their MD simulation trajectories. The largest structural variation and conformational transition were captured by the first principal component in both cases. In addition, structural clustering and ranking of representative frames by their PC1 values revealed the long-distance nature of the conformational transition and locked the key candidate regions that might be responsible for the large conformational transitions.</p> <p>Secondly, to facilitate further analysis of identification of the long-distance path, a tool called Pearson Coefficient Spiral (PCP) that generates and visualizes Pearson Coefficient to measure the linear correlation between any two sets of residue pairs is developed. PCP allows users to fix one residue pair and examine the correlation of its change with other residue pairs.</p> <p>Thirdly, a set of visualization tools that generate paired atomic distances for the shortlisted candidate residue and captured significant interactions among them were developed. The first tool is the Residue Interaction Network Graph for Paired Atomic Distances (NG-PAD), which not only generates paired atomic distances for the shortlisted candidate residues, but also display significant interactions by a Network Graph for convenient visualization. Second, the Chord Diagram for Interaction Mapping (CD-IP) was developed to map the interactions to protein secondary structural elements and to further narrow down important interactions. Third, a Distance Plotting for Direct Comparison (DP-DC), which plots any two paired distances at user’s choice, either at residue or atomic level, to facilitate identification of similar or opposite pattern change of distances along the simulation time. All the above tools of PLITIP enabled us to identify critical residues contributing to the large conformational transitions in both HIV-1 protease and 14-3-3σ proteins.</p> <p>Beside the above major project, a side project of developing tools to study protein pseudo-symmetry is also reported. It has been proposed that symmetry provides protein stability, opportunities for allosteric regulation, and even functionality. This tool helps us to answer the questions of why there is a deviation from perfect symmetry in protein and how to quantify it.</p>

No description available.

<p>This dissertation describes the effects of a <i>de novo</i> mutation of CaMKII found in children with learning disabilities and describes its effect on catalytic activity. We develop a malachite green assay for the measurement of CaMKII activation and use it for high-throughput chemical screening to identify CaMKII inhibitors and enhancers. We also propose a new mechanism of regulation of CaMKII activity by ADP.</p><p><br></p>

N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications. The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work. To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs.

N-glycosylation is the most common eukaryotic post-translational modification, impacting on protein stability, folding, and protein-protein interactions. More broadly, N-glycans play biological roles in reaction kinetics modulation, intracellular protein trafficking, and cell-cell communications. The machinery responsible for the initial stages of N-glycan assembly and processing is found on the membrane of the endoplasmic reticulum. Following N-glycan transfer to a nascent glycoprotein, the enzyme Processing α-Glucosidase I (GluI) catalyzes the selective removal of the terminal glucose residue. GluI is a highly substrate-specific enzyme, requiring a minimum glucotriose for catalysis; this glycan is uniquely found in biology in this pathway. The structural basis of the high substrate selectivity and the details of the mechanism of hydrolysis of this reaction have not been characterized. Understanding the structural foundation of this unique relationship forms the major aim of this work. To approach this goal, the S. cerevisiae homolog soluble protein, Cwht1p, was investigated. Cwht1p was expressed and purified in the methyltrophic yeast P. pastoris, improving protein yield to be sufficient for crystallization screens. From Cwht1p crystals, the structure was solved using mercury SAD phasing at a resolution of 2 Å, and two catalytic residues were proposed based upon structural similarity with characterized enzymes. Subsequently, computational methods using a glucotriose ligand were applied to predict the mode of substrate binding. From these results, a proposed model of substrate binding has been formulated, which may be conserved in eukaryotic GluI homologs.