Structural Aspects of Proteins and Protein-Protein Assemblies at Single Molecule Level using Scanning Probe Microscopy Approach

The research work embodied in the present thesis entitled “Structural Aspects of Proteins and Protein-Protein Assemblies at Single Molecule Level using Scanning Probe Microscopy Approach” deals with proteins/protein-protein complexes that are known to be important in biological events like DNA transcription, DNA packaging etc., but whose tertiary structures are not known yet. One of such protein is p300, which is a transcriptional coactivator having important role in different cellular processes. Scanning probe microscopy has been applied to probe the structural features of this protein and the complexes between p300 and some of its important target proteins, e.g., p53, histone and HMGB1. The thesis work has been presented in six chapters. Chapter 1 provides a general background on functions of proteins and structure-function correlation, established biophysical techniques to probe the protein structure, and necessity of the single molecule approaches for studying biomolecular structure and behaviour. In addition, this chapter presents basic information on the proteins employed, and the basic operational principles of the biochemical/biophysical techniques applied in the present thesis. Chapter 2 describes the applicability of scanning probe microscopy approach for investigating biomolecules at the single molecule level. The capability of this approach has been exemplified in several specific cases, i.e., in probing mechanical and electronic properties of the iron-storage protein ferritin, in eliciting the precise DNA structural changes that are induced by binding of platinum anticancer drugs/agents in the presence of HMGB1 protein, and in delineating the structural aspects of the complex between DNA polymerase klenow fragment and dsDNA. These confidence-building exercises have effectively served towards establishing our AFM single molecule detection setup, prior to performing experiments on protein/protein- protein complexes of unknown structure(s). Chapter 3 presents the structural features of human histone acetyltransferase p300 and its complex with p53, as revealed by atomic force microscopy. In this chapter, the overall shape and major structural signatures of the full-length p300 protein have been discussed. It is found that p300 FL is almost prolate ellipsoidal in shape, having several bulges. Functionally important N-terminal and C-terminal regions have been identified by applying domain-specific antibodies. Furthermore, the binding region of p53 protein on p300 FL has been located. Chapter 4 provides information on the binding location of the chromatin protein histone on p300 FL. In this chapter, the plausible location of bromo and HAT domain of p300 FL has been identified. The interactions between p300 FL and octameric histone/histone H3 in both acetylating and non-acetylating conditions have been probed by applying single molecule atomic force spectroscopy. Chapter 5 discusses the binding of HMGB1 protein on p300 FL. It is observed that HMGB1 binds at the central region of p300 FL. Force of interaction operating between these two proteins both in acetylating and non-acetylating medium has been probed by force spectroscopic method. The mechanism of unbinding process between them has been monitored by dynamic force spectroscopy. Chapter 6 provides a general conclusion and scopes for more advanced experiments.Research was conducted under the supervision of Prof. Rupa Mukhopadhyay of Biological Chemistry division under SBS [School of Biological Sciences]Research was carried out under CSIR fellowshi

Molecular characterization of glyceraldehyde-3-phosphate dehydrogenase from normal and malignant cells

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme of glycolysis and increased glycolysis is a hallmark of malignancy. Previously we showed that methylglyoxal (MG), a normal metabolite, inhibits glycolysis. Moreover GAPDH, purified exclusively from different malignant cells, is a heterodimer (~90 kDa) containing two subunits of 33±2 and 55±3 kDa and is strongly inactivated by MG suggesting a distinct alteration of GAPDH in malignant cells, whereas GAPDH from normal sources is a homotetramer (~144 kDa), each subunit of 36 kDa. Here, by MALDI-TOF analysis, we report that the 55 kDa subunit is either glucose-6-phosphate isomerase (GPI) or pyruvate kinase M2 (PKM2) isoform which is another important biomarker of malignancy and GAPDH is associated with these two proteins separately to form a protein complex which is confirmed by immunoprecipitation analysis. MG can alter the secondary structure of purified GAPDH protein complex may be due to the glycation at PKM2 and GAPDH as detected in CD spectroscopy and mass analysis. We also use biocompatible surface functionalized Quantum Dots (QDs) to modulate the enzyme activity of GAPDH from cancer cells. Cumulative kinetic studies reveal that both reversible and irreversible inhibition mechanisms owing to the site specific interactions enabling control over the inhibition kinetics of GAPDH activity. This study indicates a different molecular association of GAPDH in cancer cells and tissues, and experimental approach to inhibit its enzymatic activity in cancer cells.Research was conducted under supervision of Prof. S S Jana of the Biological Chemistry division under SBS [School of Biological Sciences] and under Prof. Manju Ray of BI [Bose Institute]Research was carried out under DBT & DST gran

Magnetic properties of transition metal based intermetallic alloys

The present thesis deals with unique magnetic behaviour of Heusler based metamagnetic shape memory alloys. Transition metal based alloys and compounds are formed by combining magnetically important d-block element with nonmagnetic metals (such as Al, Ga, Zn, In) or sp elements (such as Si, Ge). The alloys and compounds can be binary (containing two elements), ternary (containing three elements) or even quaternary (containing four elements). A major part of the present thesis work is devoted to Huesler based metamagnetic shape memory alloys of general formula Ni2Mn1+xZ1−x (where Z is a sp element such as In, Sn or Sb) via suitable doping at the different sites. A brief summary of all the chapters including the plan and procedure of this work, the results from the systems and control experiments, and discussion on the results have been put forward in eight chapters of this thesis. A general introductory view of the field of research is discussed in the introduction. Basic properties and application of Heusler based metamagnetic shape memory alloys, electrical resistivity and various magnetofunctional properties of metamagnetic shape memory alloys are also added in this chapter. Various experimental techniques which were followed during the investigation are the basic contents of the next chapter. It also includes sample synthesis and annealing procedure, characterization methods and various measurement techniques. Chapter 3 is based on studying the effect of Ga doping at the In site of Ni2Mn1.32In0.68 metamagnetic shape memory alloy by transport and dc magnetization measurements. The parent sample Ni2Mn1.32In0.68 does not show any structural instability, however small Ga substitution at the In site suddenly induces martensitic transition in it. The studied alloys are all found to be ferromagnetic below about room temperature and also undergo the martensitic phase transition at different temperature depending upon Ga doping. Samples showing thermally driven maretnsitic transition are also found to be susceptible to the applied magnetic field as they show large magnetoresistance and magnetocaloric effect. Clear indication of magnetic field induced transition and kinetically arrested state are also observed. A detailed investigation on the effect of excess Ni doping at the expense of Mn and Sn in Ni2Mn1.4Sn0.6 is presented by means of studying the magnetic and structural properties are present in Chapter 4. The parent alloy undergoes martensitic type structural transition below approximately 180 K. However, doping of Ni at the Mn site keeping Sn unaltered (Ni2+xMn1.4−xSn0.6) leads to non-monotonous variation of lattice parameter, martensitic transition temperature, magnetic moment, ferromagnetic Curie point, magnitude of magnetoresistance etc. Approximately up to the concentration x = 0.08, all the physical parameters vary more or less in a uniform manner. But beyond this concentration, there is a change in the trend of variation. Clear signatures of spin-glass like behaviour, large exchange bias effect and thermoremanent magnetization at low temperature are observed for the x = 0.04 sample. On the other hand, for doping of Ni at the Sn site keeping Mn unchanged (Ni2+xMn1.4Sn0.6−x) shifts the martensitic transition temperature close to 290 K for x = 0.04. Magnetization study reveals the presence of multiple magnetic transitions in the sample. Apart from that, the magnetofunctional properties also get enhanced as evident from the observation of large magnetoresistance and magnetocaloric effect around 290 K. Chapter 5 deals with the magnetic character of Ni1.84Mn1.64In0.52 and Ni2.048Mn1.312In0.64 alloys through transport and magnetization measurements. Both the samples undergo long range ferromagnetic ordering below Curie point followed by the martensitic transition. The second composition is also subjected to another transition from a paramagnetic- like state to a ferromagnetic state upon further cooling below martensitic transition. Large negative magnetoresistance and inverse magnetocaloric effect are observed across the region of martensitic instability for both the samples. However for the second composition, the martensitic transition temperature is very close to room temperature and the magnitude of magnetofunctionality is quite large. Almost 16.7 J/kg-K (for H0 = 50 kOe) of magnetocaloric effect and -45% of magnetoresistance at 300 K are observed. The sample also shows a maximum of 2.25 J/kg-K (for H0 = 50 kOe) conventional entropy change across the second magnetic transition in the martensite along with the observation of -4% magnetoresistace for H = 80 kOe around 195 K. Chapter 6 is devoted to the study of the effect of external pressure on the magnetic character of Ni2.04Mn1.4Sn0.56 and Ni2.048Mn1.312In0.64 samples via magnetization measurements. For the first alloy, martensitic start temperature increases in presence of pressure and almost 2 K shift per 1 kBar pressure is observed. The ferromagnetic Curie temperature in the austenite also moves towards higher temperature with a rate of about 1 K increase per 1 kBar pressure. The ground state saturation moment and exchange bias effect get enhanced with applied pressure. Magnitude of inverse magnetocaloric effect increases from 12.36 J/kg-K to 16 J/kg-K for 0 to 8 kBar pressure change at 50 kOe magnetic field and the peak temperature moves to 300 K. However, for the second sample, effect of pressure is quite opposite to some extent. Though for this case also the martensitic start temperature increases with pressure, but the ground state saturation moment and magnetocaloric effect get suppressed too. Magnitude of inverse magnetocaloric effect decreases from 16.7 J/kg-K to 14.06 J/kg-K for 7 kBar applied pressure at 50 kOe of field. Detailed investigation on the polycrystalline sample of Mn11Ge8 via magnetic, transport and heat capacity measurements is presented in chapter 7. The sample orders ferromagnetically below 274 K, which occurs only due to the ordering of small atomic moment (∼ 0.05 μB/Mn) along the b direction of the crystallographic axis and on further cooling below 150 K, it undergoes a spin reorientation type transition to a non-collinear antiferromagnetic state. The development of antiferromagnetic correlation below 150 K occurs through a first order structural instability, where lattice parameters show discontinuous change keeping the overall crystal symmetry unchanged. Drastic change in resistivity is observed below 150 K. Both the positive and negative magnetoresistance are obtained depending upon the measurement temperature and strength of applied field. The sample also shows large inverse magnetocaloric effect having the magnitude of 2.8 J/kg-K for H = 90 kOe around 140 K. Concluding remarks and summary of the whole work with the inference have been presented next. Some plans of future works are also included in chapter 8.Research was carried out under the supervision of Prof. Subham Majumdar of Solid State Physics division under SPS [School of Physical Sciences]Research was conducted under DST gran

Synthesis, Characterization And Potential Applications Of Organosilicon Based And Related Mesoporous Molecular Sieves

Mesoporous materials having high surface area with tunable pore size distribution in nano scale dimensions have attracted widespread interest in recent times. These special properties make mesoporous materials focus of great research interests in catalysis, adsorption, sensing, separation, drug delivery and controlled release of bioactive pharmaceuticals. During the past two decades a prevalent interest and incredible resonance as a fundamental and technological challenge to chemists, physicists and engineers has been gained by organic-inorganic hybrid materials and macromolecular building blocks of nanoscale dimensions. Hybrid mesoporous materials are unique and molecular control of their intrinsic topological and chemical characteristics can be explained through self-assembly and nanochemistry techniques. Post synthesis and in-situ synthesis both the techniques have been adopted to develop the organic-inorganic hybrid mesoporous materials. Furfural imine functionalized mesoporous silica has been synthesized by using CTAB as structure directing agent by applying in-situ grafting technique. This furfural imine functionalized mesoporous SBA-15 has been also developed by employing post-grafting approach. Then these two different mesoporous materials have been subjected to react with Cu(OAc)2 to deliver Cu-grafted mesoporous organosilica. After Cu grafting different crystalline phases have been generated for the two different mesoporous silica materials. Also In another case –NH2 functionalized mesoporous organoslica has been developed by applying in-situ grafting technique. Surface of mesoporous SBA-15 has been functionalized by reaction with 3-aminopropyltriethoxy silane. Then –CO2H functionalized mesoporous SBA-15 has been synthesized by applying Schiff-base condensation reaction. Fe3O4 nanoparticle has been grafted into the mesopore channel of cysteine functionalized mesoporous SBA-15 material. Thiol-ene click chemistry has been adopted to prepare cysteine functionalized mesoporous SBA-15 material.The research was carried out under the supervision of Prof. Asim Bhaumik of the Material Science division under SMS [School of Materials Science]The research was carried out under CSIR fellowship and research gran

Synthesis of Carbohydrate Functionalized Nanomaterials for Biomedical Application

Carbohydrate functionalized nanomaterials have emergent application potential in the field of biological science. Carbohydrates are the most abundant biomolecules in the living body and are present in the form of glycoprotein, glycolipid and various other complex structures. They play an important role in cell–cell communication, cell signaling and metastasis of cancer cell. Some carbohydrate receptors are over expressed in certain cancer cells. These receptors can be used for selective cancer targeting and therapy by using carbohydrate functionalized nanomaterials. Carbohydrate functionalized nanomaterials are used in protein detection, pathogen detection, sensing of biomolecule, specific and non specific cellular labeling, targeted drug delivery, therapy and diagnosis and in the study of various biological events in vitro and in vivo. Preparation of good quality carbohydrate functionalized nanomaterials is very challenging for the scientific community. Most of the carbohydrate functionalized nanomaterials, reported in the literature, were prepared using thiol based ligand exchange of thiolated carbohydrates. This approach is most popular as it produces monodispersed particles of small hydrodynamic diameter. But this approach needs the synthesis of thiolated carbohydrates and stabilization of carbohydrate functionalized nanoparticles in physiological conditions, particularly in the presence of other thiol based biomolecules, is a critical issue. Other approaches of preparation of carbohydrate functionalized are less popular due to multiple steps, complexity associated with conjugation chemistry, poor conjugation efficiency, destruction of the carbohydrate structure during conjugation with nanomaterials, large hydrodynamic diameter of the resultant particle and limited colloidal stability of the final products. Therefore appropriate conjugation methods are highly desirable that can be used for preparation of different high quality carbohydrates functionalized nanomaterials useful for different biomedical application.Research was conducted under the supervision of Prof. N R Jana of CAM under SPS [School of Physical Sciences]Research was carried out under CSIR fellowship and gran

Structural and Magnetic Properties of Novel Oxides

Transition-metal and rare earth based oxides belong to an attractive and challenging field of research in condensed matter physics. This class of oxide possesses a wide range of intriguing properties and reveals novel phenomena from insulator to superconductor and ferroelectricity to ferromagnetism. The competition and coexistence of different types of ground states gives rise to complex electronic and magnetic phases. This thesis entitled “Structural and Magnetic Properties of Novel Oxides” mainly concentrates on investigation of structural, optical, dielectric, electric and magnetic properties of some novel oxides such as BaTiO3, NiFe2O4, SrRuO3 and LaCrO3. Lattice distortion, defects and chemical composition are the key parameters to modify basic interactions to induce new behavior in oxides. Reduction of size to fundamental length scale i.e. coherence and cooperative length also significantly influences long range order in ferroelectric and ferromagnetic oxides.Research was conducted under the supervision of Prof. S K De of the Materials Science division under SPS [School of Physical Sciences]Research was carried out under CSIR fellowship and also CSIR & DST travel gran

Study of magnetic and dielectric properties of 3d transition metal oxides

Transition metal compounds present a unique class of solids with complex and diverse thermodynamic properties. 3d transition metal oxides exist with a great variety of crystal structures, which are reflected in the richness of their physical properties. The orbital states of 3d electrons are to a large extent responsible for the complex relationship between the electronic properties and crystal structure. This complexity and diversity are the indication of strong interplay between electronic, lattice, orbital and spin degrees of freedom. The magnetic ground states of these oxides depend strongly on the environment surrounding the transition metal and also on the exchange interaction pathways between two magnetic ions, which is often mediated through the 2p levels of oxygen. The thesis entitled “Study of magnetic and dielectric properties of 3d transition metal oxides” is devoted to the experimental investigations focusing the magnetic and electric properties of some exotic transition metal oxides with fascinating crystal structures. Where necessary, the X-ray photo-electron spectroscopy, temperature dependent XRD have also been studied to have a comprehensive understanding of the systems. In many cases experimental results are fitted to the existing theoretical models to clarify the analysis. All the results and analyses based on the investigations performed during this tenure have been included in this thesis along with the theoretical background and experimental methodologies.The research was carried out under the supervision of Prof. Subham Majumdar of Solid State Physics division under SPS [School of Physical Sciences]The research was conducted under IACS fellowshi

Designing Functional Coordination Polymers - A Crystal Engineering Approach

Chapter 1 briefly describes the basic introduction on coordination polymer (CP), coordination polymeric gel or metallogel, discrete coordination compound (metal-organic polyhedron), their applications in diverse fields and the role of crystal engineering and supramolecular chemistry in designing such materials. Chapter 2 gives an overview of coordination polymers and metallogels derived from a bis-pyridyl-bis-urea ligand and CuII metal salts. The coordination polymer obtained from Cu(SO4)2, capable of separating anion (sulphate) via in situ competitive crystallization technique. An attempt has been made to design metallogelator based on crystal engineering approach. Chapter 3 deals with metal-organic polyhedron (MOP) synthesized from a C3-symmetric trimesic acid and Cu(NO3)2. Such nano sized MOP molecules have been seen under transmission electron microscopy (TEM). Interestingly, this CuII based MOP shows catalytic property towards oxidation of benzyl alcohol to benzaldehyde. Chapter 4 consists of CPs derived from pyridyl-carboxylate ligands equipped with amide functionality and different transition metals. The environmentally toxic CuII metalcation has been separated in the form of the corresponding CuII coordination polymer following in situ crystallization technique. Chapter 5 deals with metallogels derived from silver coordination polymers of C3- symmetric tris(pyridylamide) tripodal ligands. Upon exposure to visible light, these silver metallogels produce silver nanoparticles (AgNPs), which are characterized by different physicochemical techniques. These NPs effectively catalyze the reduction of 4-nitrophenolate to 4-aminophenolate without the use of any exogenous reducing agent. Chapter 6 consists of bioactive CPs derived from a series of nonsteroidal antiinflammatory drugs (NSAIDs) and a biogenic MnII metal salt. A nontoxic bis-pyridyl molecule has been used as a co-ligand. Two of such CPs display intriguing photoluminescence. These CPs have been applied in cell imaging performed on a murine macrophage cell line RAW 264.7. A conscious effort was made to incorporate most of the significant literature considering the limitation imposed by the space constraint and relevance to the current focus of topic.Research was carried out under the supervision of Prof. P. Dastidar of the Organic Chemistry division under SCS [School of Chemical Sciences]Research was conducted under DST, CSIR & DBT gran

Studies on Coordination Complexes Metal Organic Framework (MOFs) and Network using Salicylic Acid Derivatives and Pyrazole based Ligand

The whole work is divided in three parts. The 1st part deals with synthesis if series of metal organic framework from bis-pyrazole ligands and various aromatic carboxylic acids and their potential application. the 2nd part contains synthesis and investigation of energy parametres of supramolecular framework resulting from the combination between a bispyrazole ligand and inorganic anions. The 3rd part and the final part contains synthesis of diverse metal organic framework and metal complexes based on chloro-salicyclic acid.Research was carried out under the supervision of Dr. Raju Mondal of the Inorganic Chemistry division under SCS [School of Chemical Sciences]Research was conducted under DST grant and CSIR fellowshi

Chiral Synthons from Carbohydrates towards the Synthesis of Bioactive Natural Products

Construction of diverse molecular architecture containing carbocyclic frameworks, conceived and created by nature, continues to be the most exhilarating and challenging task to the practitioners of organic synthesis. A large number of natural products which are isolated from natural resources possess carbocyclic skeletons as the primary structural unit and show a wide range of biological activities. Among the natural products, terpenoid class of natural products are well suited for developing, testing and implementing new synthetic strategies towards the construction of carbocyclic frameworks. Thus, a combination of potential applications and the intellectual challenges associated with their synthesis have brought natural products into the center stage of contemporary organic chemistry-research. Investigation embodied in this dissertation entitled “Chiral Synthons from Carbohydrates towards the Synthesis of Bioactive Natural Products” directed towards the development of synthetic methodologies for fused and bridged tricycle present in natural productThe research was conducted under the supervision of Prof. Subrata Ghosh of the Organic Chemistry division under the SCS [School of Chemical Sciences]The research was conducted under CSIR project and fellowshi