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Biophysical characterization of heterocyst differentiation regulators, HetR and PatS, from the cyanobacterium, Anabaena sp. strain PCC 7120 and structural biology of bacterial proteins from the Northeast Structural Genomics ConsortiumFeldmann, Erik A. 25 July 2012 (has links)
No description available.
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Structure and Dynamics of the Y145Stop Variant of the Human Prion Protein Studied by Magic-Angle Spinning Solid State NMRHelmus, Jonathan Jaye 06 September 2011 (has links)
No description available.
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Variable pressure NMR analyses to assess compressive motion in PETNR and catalytically germane PETNR:Ligand complexesGuerriero, Andrew January 2012 (has links)
The involvement of dynamical fluctuations in driving enzymatic processes is widely accepted. With respect to NQM tunnelling enzymes, the role of promoting motions in facilitating hydrogenic transfers is well studied. Few studies have however, specifically attributed, dedicated dynamical fluctuations characterised by their timescales and magnitudes, as a function of a reaction coordinate, to specific groups in a protein system. An effectively full suite of backbone resonance assignments were obtained for PETNR and on relevant ligand complexes. This provided an essential platform on which residue specific, backbone amide fluctuations were assessed. This thesis documents the application of pressure up to 1500 bar, in tandem with high resolution TROSY based NMR analysis, as a means of studying residue specific, conformer exchange perturbations. Residue specific amide compression profiles of the PETNR:FMN free enzyme system, and complexes with progesterone and tetrahydropyridine dinucleotides have been obtained. The binding of progesterone appears to induce conformational tightening of residues within the active site vicinity. The complexation of PETNR:FMN with tetrahydropyridine dinucleotides, appears to stimulate conformational shifts towards intermediate, and in some cases, slow exchange regimes in multiple residues about the active site vicinity. This is evidenced by extensive intensity attenuation of 1H-15N TROSY resonances, on the binding of tetrahydropyridine dinucleotides at 1 bar pressure, and on going from 1 bar to 1500 bar pressure. Multiple regions of sequence, spatially clustering about the active site vicinity within a 10 Å sphere of the FMN binding pocket, display appreciable sensitivity to ligand binding. Differential responses of residues to the application of high pressure between complexes was noted within segments of these regions. A region of sequence, named the β-hairpin flap displays significant differential compression profiles between the PETNR:FMN free enzyme system, and associated progesterone and tetrahydropyridine dinucleotide complexes. A role in mediating ligand engagement is proposed for R130 and R142 in the β-hairpin flap. A central hydrogen bonding network, perhaps constituting a putative proton wire in the active site of the PETNR:FMN:Progesterone complex, has been identified that could enable the shuttling of protons following catalytic protonation of oxidative substrate. The resonance response behaviour of G185 acts as a sensitive reporter on the formation of these interactions, revealed by an interrogation of the differences in chemical shift changes on progesterone binding, and in response to high pressure. The recruitment of high resolution crystallographic data sets readily supported a structural and dynamical interpretation of the observed chemical shift responses to ligand binding at 1 bar pressure, and on the application high pressure. A definitive atomistic identification of fast motion contribution to activation barrier compression was not obtained. Nevertheless, detailed, residue specific amide compression profiles, and shifts in backbone amide conformational exchange regimes in response to ground state ligand binding, and at high pressure, have been catalogued in the PETNR:FMN free enzyme system. These dynamical profiles in the free enzyme are contrasted against comparative, residue specific observations in analogue complexes of the oxidative and reductive half reactions of PETNR.
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Correlated and Further Dynamics in Proteins by NMR Spectroscopy / Korrelierte und weitere Dynamik in Proteinen mittels NMR SpektroskopieWalter, Korvin 15 September 2011 (has links)
No description available.
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Development Of NMR Methods For Metabolomics And Protein Resonance AssignmentsDubey, Abhinav 15 May 2016 (has links) (PDF)
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.
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Structural and Dynamic Studies of Protein-Nanomaterial InteractionsMondal, Somnath January 2016 (has links) (PDF)
My thesis is divided into five chapters, starting with a general introduction in first chapter and sample preparation and protein-NMR assignment techniques in second chapter. The remaining three chapters focus on three different areas/projects that I have worked on.
Chapter 1: Introduction to nanomaterials and all the experimental techniques
This chapter reviews different kinds of nanomaterials and their application utilized for protein-nanomaterial interaction in our study, along with the introduction to different spectroscopy and microscopy techniques used for the interaction studies. Starting with introduction of nanomaterials and all the experimental techniques, which constitute the arsenal for structural studies of the protein-nanomaterial interaction, different steps enroute to structural and dynamic interaction are outlined in detail.
Chapter 2: Preparation and Characterization of Proteins used for nanomaterial interaction studies Proteins are generally of three kinds- globular (structured), intrinsically disordered and membrane bound. These proteins have different functions in living organisms and play a major role to maintain metabolism and other important factors. To probe protein-nanomaterial interactions, we have chosen different protein/peptides. This chapter describes the protocol/procedure used for purifying the proteins. For studying a globular protein, ubiquitin was chosen. Nanomaterial-IDP interaction was investigated using the intrinsically disordered central linker domain of human insulin like growth factor binding protein-2 (L-hIGFBP2). The hydrophobic membrane interacting part of the prion protein was chosen as a representative membrane protein. The characterization of the proteins by NMR spectroscopy is also described.
Chapter 3: A nanomaterial based novel macromolecular crowding agent Carbon quantum dots (CQD) are nanomaterials with size less than 10 nm, first obtained in 2004 during purification of single-walled carbon-nanotubes. Since then CQDs have been used in a wide range of applications due to their low cost of preparation and favorable properties such as chemical inertness, biocompatibility, non-toxicity and solubility in aqueous medium.
One of the applications of CQDs has been their use for imaging and tracking proteins inside cells, based on their intrinsic fluorescence. Further, quantum dots exhibit concentration dependent aggregation while retaining their solubility. Fluorescent carbon quantum dots (CQD) induce macromolecular crowding making them suitable for probing the structure, function and dynamics of both hydrophilic and hydrophobic peptides/ proteins under near in-cell conditions. We have prepared hydrophilic and hydrophobic quantum dots to see the crowding effect. After characterization of CQD, we tested the property of proteins with CQD and found that CQD behaves as a macromolecular crowding agent by mimicking near in-cell conditions. In our study, we have chosen a globular protein, an intrinsically disordered protein (IDP) and one hydrophobic membrane peptide. We have also compared the crowding property of CQD with ficoll which is widely used commercial crowding agent. The overall study tells that the CQD acts like crowding agent and can be used for the study of macromolecular crowding effect. This makes them suitable for structural and functional studies of proteins in near in-cell conditions.
Chapter 4: Ubiquitin-Graphene oxide interactions
Described here is the interaction of human ubiquitin with GO using NMR spectroscopy and other techniques such as Fluorescence spectroscopy, isothermal titration calorimetry (ITC), UV-Visible spectroscopy, dynamic light scattering (DLS), zeta potential measurements and transmission electron microscopy (TEM). The globular protein ubiquitin interacts with GO and undergoes a dynamic and reversible association-dissociation in a fast exchange regimen as revealed by NMR spectroscopy. The conformation of the protein is not affected and the primary interaction is seen to be electrostatic in nature due to the polar functional groups present on the protein and GO sheet surface. For the first time we have shown that the interaction between ubiquitin and GO is dynamic in nature with fast and reversible adsorption/desorption of protein from the surface of GO. This insight will help in understanding the mechanistic aspects of interaction of GO with cellular proteins and will help in designing appropriate functionalized graphene oxide for its biological application.
Chapter 5: Section A: Interaction of an intrinsically disordered protein (L-HIGFBP2) with graphene oxide
The interaction between intrinsically disordered linker domain of human insulin-like growth factor binding protein-2 (L-hIGFBP2) with GO was studied using NMR spectroscopy and other techniques such as isothermal titration calorimetry (ITC), dynamic light scattering (DLS), zeta-potential measurements. The study revealed that the disordered protein L-hIGFBP2 interacts with GO through electrostatic interaction and undergoes a dynamic and reversible association-dissociation in a fast exchange regime. The conformation of the protein is not affected.
Section B: Stability of an Intrinsically disordered protein through weak interaction with Silver nanoparticles
Using NMR spectroscopy and other techniques we probed the mechanism of L-hIGFBP2–AgNP interactions which render the IDP stable. The study reveals a mechanism which involves a relatively fast and reversible association–dissociation of L-hIGFBP2 (dynamic exchange) from the surface of AgNP. The AgNP–L-hIGFBP2 complex remains stable for more than a month. The techniques employed in addition to NMR include UV-Visible spectroscopy, dynamic light scattering (DLS), zeta potential measurements and transmission electron microscopy (TEM) to probe the protein-AgNP interaction here in this section.
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Study Of Structure, Dynamics & Self-Assembly Of Human Insulin-Like Growth Factor Binding Protein-2 By Novel NMR And Biophysical MethodsSwain, Monalisa 07 1900 (has links) (PDF)
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.
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