Spelling suggestions: "subject:"molecular biophysical""
1 |
Purifacation of Wild Type EcoRI Endonuclease and EcoRI Endonuclease RS187 Crystal Growth of WT EcoRI Endonuclease-DNA comlex and EcoRI EndonucleaseDai, XiaoHu 19 September 2007 (has links)
EcoRI endonuclease is a very useful tool to study the structural mechanism of protein-DNA recognition. In this work, wild type EcoRI Endonuclease and EcoRI Endonuclease mutant RS187 were purified to high purity. Crystals of wild type EcoRI Endonuclease-DNA 13mer complex have been obtained with good size and shape. Some small crystals of EcoRI Endonuclease RS187-DNA 13mer complex were also grown.
|
2 |
Following Dynamics of Protein/Nucleic Acid Interactions in Real-TimeKim, Min Sun 25 September 2008 (has links)
Methyl-CpG binding protein 2 (MeCP2) is a protein associated with transcriptional repression of other proteins and its various mutants are found in Rett syndrome patients, which is a severe neurodevelopmental disease found in 1/15,000 females. It has specific binding affinity to methyl-CpG domain but also bind to architectural DNA structure. Here, its binding dynamics to Holliday junction structure was investigated by using single molecular detections; scanning confocal fluorescence microscope and wide-field evanescent field fluorescence microscope. Through these studies, we could distinguish the difference of transitions of the substrate with and without MeCP2. The effect of this MeCP2 binding to 4WJ will be discussed in terms of the structures and transition times.
|
3 |
Allosteric Modulation of G Protein Coupled Receptors.Yanamala, Naveena VK 13 May 2009 (has links)
Structural coupling between the cytoplasmic (CP), transmembrane (TM) and extracellular (EC) domains of G protein coupled receptors (GPCRs) is crucial for their functioning in signal transfer from the extracellular to the intracellular side of the membrane. The focus of this thesis was to test the hypothesis that ligands can bind in each of the three domains. Depending on the location of the endogenous ligand binding site, the other two sites would become allosteric ligand binding sites. To test this hypothesis, we investigated the binding of accessory ligands to each of the three domains, CP, TM and EC. The major contributions of this thesis are as follows:
I. The anthocyanin Cyanidin-3-glucoside (C3G) and the chlorophyll-derivative chlorin e6 (Ce6), were shown to physically interact with rhodopsin. These studies demonstrated the presence of a novel CP allosteric ligand binding site in rhodopsin. Biophysical evidence indicated differential effects of binding of these ligands on rhodopsin function, structure and dynamics.
II. The allosteric TM ligand binding pocket in metabotropic glutamate receptors (mGluRs) was shown to be analogous in structure and function to the orthosteric TM retinal ligand binding pocket in rhodopsin. Docking of known allosteric modulators to structural models of mGluRs based on rhodopsin conformations was used to predict allosteric modulatory effects. Structural comparison of the mGluR and rhodopsin binding pockets revealed high overlap and preliminary evidence was obtained showing that an mGluR ligand can bind to rhodopsin.
III. Evidence for the existence of an EC ligand binding domain was presented. Rhodopsin was shown to bind the extracellular chemokine ligand, CXCL11, an event which interfered with both rhodopsin and chemokine functions.
IV. As part of the above efforts, it became necessary to develop and improve NMR spectroscopic methodology to study ligand binding of membrane proteins such as GPCRs. Thus, 1H and 19F based NMR methods to screen for novel ligands that bind to GPCRs were developed and applied to rhodopsin.
Collectively, the studies presented in this thesis enhance the understanding of allosteric modulation of GPCRs in general, and of the molecular mechanism of rhodopsin and mGluR activation in the presence of allosteric ligands in particular. The results could help in the identification of new ligands to allosterically modulate receptor structures and in turn their functions at different binding pockets, thus paving new ways to selectively target this pharmacologically important class of receptors.
|
4 |
Processing of Alternative DNA Structures in the Human TelomereNora, Gerald Joseph 16 April 2010 (has links)
Telomeres help maintain the overall genomic stability of an organism, and telomeric homeostasis is critical to navigating between aging and cancer. Telomeric dysfunction is implicated as a contributing factor in numerous aging-related diseases, such as diabetes, impaired hematopoeisis, and atherosclerosis. Telomeric homeostasis is maintained by a shelterin complex of six proteins and an array of telomere-associated proteins that interact with the central shelterin complex, such as the Werner syndrome helicase/exonuclease protein (WRN) or p53. Telomeres also have non-canonical DNA structures that are critical towards their function, especially G-quadruplex DNA (G4 DNA) and Holliday Junctions. The former are pseudoknots that form on the G-rich 3 single-stranded tail of the telomere and may block telomere replication and lengthening, when the 3 telomeric tail is exposed in the open conformation. We found that the shelterin protein protection of telomeres 1 (POT1) competes with and destabilizes G4 DNA on a physiologically realistic telomeric tail substrate, leading to an equilibrium population of diminished G4 DNA coexisting with POT1. While POT1 is a passive binder of DNA, the destabilizing effect of bound POT1 on pre-existing G4 DNA leads to an emergent, de facto cooperativity in G4 DNA unfolding by POT1.
Holliday Junctions (HJ) form when the telomere is in a closed conformation, in which the 3 telomeric tail invades the duplex telomeric DNA, creating a displacement loop (D-loop) and sequestering the end of the chromosome from unwanted DNA damage responses. The D-loop is a homologous recombination intermediate, and we demonstrate that telomere repeat binding factor 2 (TRF2) is necessary to protect HJ DNA from unwanted WRN helicase activity, which has been thought to branch migrate the D-loop into a target for Holliday Junction cleaving enzymes, causing sudden telomere shortening. TRF2 also protects HJ DNA from Holliday Junction cleaving enzymes, and the cleavage protection is due largely to the HJ-binding B-domain on TRF2. In contrast, we found that TRF2-mediated protection against WRN depends on both the B domain and the telomeric-repeat binding Myb domain. We have therefore discovered an overlapping but distinct role for TRF2 in maintaining telomeric stability.
Our work has elucidated novel structural and functional data on the modulation of non-canonical DNA structures by shelterin and telomere-associated proteins. These data help us elucidate the mechanisms underlying cellular and animal models for telomere instability and aging.
|
5 |
Phosphorylation of Dentin Matrix Protein 1 and PhosphophorynDuan, Yuanyuan 02 September 2009 (has links)
Biomineralization, one of the most widespread processes in nature, uses polyanionic proteins to direct oriented crystal growth. In bone and dentin, this process is under precise control of the collagen template and the noncollagenous acidic phosphoproteins. These phosphoproteins function differently depending on their sizes and level of phosphorylation.
The goal of this research is to investigate the in vitro phosphorylation as well as the phosphorylation in mammalian cells of two highly phosphorylated bone/dentin extracellular matrix proteins: dentin phosphophoryn (DPP) and dentin matrix protein 1 (DMP1). This data will be important to the general hypothesis, that the phosphorylation of non-collagenous proteins play a significant role in matrix mediated mineralization.
Our data shows that the in vitro phosphorylation of DPP and DMP1 could be optimized by adjusting the phosphorylation reaction time, calcium concentration, and protein modification by assessing various forms (with or without the C or N terminal end). Following the in vitro phosphorylation, mass spectrometry analysis was used to identify the sites of phoshorylation. In addition, to identify the kinases involved in phosphorylating DMP1, cell lysates from cells that have (MC3T3) and do not have (NIH3T3) the ability to mineralize their matrix and were isolated and analyzed by zymogram. Casein kinase II catalytic subunit was identified in addition to potential novel kinases responsible for DMP1 phosphorylation.
The second goal of this research is to assess if cells that have the ability to form a mineralized matrix will possess specialized kinases that can phosporylate these highly phosphorylated and acidic proteins. To achieve this goal we over-expressed and purified DMP1 from two cell types: 1) cells that have the ability to mineralize their matrix and 2) cells that do not possess the ability to mineralize their matrix. The purified proteins were then analyzed by SDS-PAGE and mass spectrometry to quantify and determine the sites of phoshorylation.
This study has expanded our knowledge on the mechanisms involved in the phosphorylation of DPP and DMP1 and provided the parameters to start assessing the role of phosphorylation on tissue mineralization.
|
6 |
Regulation of Capsid Sizes of Large Tailed BacteriophagesHua, Jianfei 24 June 2010 (has links)
Many bacteriophages and eukaryotic viruses, which share little sequence similarities, have icosahedral protein capsids containing their genetic materials. Generally, an icosahedral viral capsid is assembly of 12 pentamers and a certain number of hexmers of the major capsid protein, following Caspar and Klug¡¯s quasi-equivalence rule. The arrangement of these pentamers and hexmers is characterized by the triangulation (T) number. Questions arise whether viruses have evolved from a few common ancestors, and how the assembly of the icosahedral capsids has been regulated to achieve a defined capsid size and geometry. I present studies of the capsids of several large icosahedral bacteriophages, which broaden our understanding of the regulation of viral capsid assembly.
Bacteriophage SPO1 may share common ancestry with herpesvirus, according to the similarities in their T numbers and in the asymmetric molecules slightly off the local three-fold symmetry positions on the outer surface of both capsids. However, the cryo-EM structure of the SPO1 capsid assembled from the uncleaved major capsid protein show that, unlike the herpesvirus asymmetric molecule, the SPO1 asymmetric protein may not be required for the initial procapsid assembly, suggesting that the two asymmetric molecules may have different origins. Phage P1 is excellent for studying size determination in viral capsid since it produces virions of three sizes. The cryo-EM structures of the three capsids and internal capsid proteins identified suggests a control mechanism for P1 capsids, in which the DarA protein functions as a semi-scaffolding protein to assist the assembly of the P1 big capsid. Jumbo phages have been rarely studied. The structural studies on four jumbo phages showed their T numbers. N3, PAU and 121Q are the first T = 19, 25 and 28 viral capsids found. These results suggest that T-numbers larger than 16 may generally be allowed.
|
7 |
Investigations of Structures and Dynamics of Transmembrane Proteins and Implications in the Action of Inhalational AnestheticsCui, Tanxing 03 September 2010 (has links)
The nicotinic acetylcholine receptor (nAChR) mediates fast signal transduction in peripheral and central nervous systems. It is a pentameric ion channel that belongs to the Cys-loop receptor superfamily. nAChR is one of the plausible targets for general anesthetics. The current concern about nAChR is ambivalent structures of the transmembrane (TM) domain, missing information for the intracellular (IC) domain and elusive mechanisms of the action of general anesthetics. Therefore, this thesis focuses on the following two important aspects: first, the structure and dynamics of the nAChR TM and IC domains; and second, general anesthetic effects on various proteins.
Part I. The secondary structure of human nAChR α7 TM domain and the monomeric tertiary structure of the water-soluble mutant of nAChR α1 TM domain from the Torpedo electrical ray were determined by NMR. The structures among WSA, nAChR α7, and a bacterial analogue of the pentameric channels (GLIC) are similar, but different from the cryo-EM structure of the nAChR. The backbone dynamics analyses of human nAChR α7 with and without the IC domain suggest that the presence of IC domains dramatically affected the intrinsic dynamics of the TM domains.
Part II. The general anesthetic effects on the structure and dynamics of the proteins including two analogues and a real anesthetic target in non-channel and channel-like oligomers were studied. It revealed that general anesthetics prefer amphipathic environments. Compared to the effect on the structures of proteins, general anesthetics show stronger effect on the dynamics. The effect on the dynamics of proteins can manifest directly or allosterically.
Part III. The anesthetic effects on the Na+ flux through the reconstituted channels of nAChR TM domains were investigated by fluorescence microscopy. The anesthetic effects on them are the same as on the full-length nAChR. This indicates that the studies of anesthetic effects on the TM domains of nAChR are functional relevant.
In summary, this dissertation clarifies the current structural ambiguities, provides additional invaluable dynamic information for the TM and IC domain and reveals the possible mechanism of action of general anesthetics.
|
8 |
BIOPHYSICAL AND PHARMACOLOGICAL CHARACTERIZATION OF CYTOPLASMIC DYNEIN HEAVY CHAIN 1Daghestani, Hikmat 07 January 2011 (has links)
The cytoplasmic dynein motor protein complex transports a number of different important cargos along microtubules (MTs) in a retrograde manner. Cytoplasmic dynein plays an important role in many cellular processes and a number of diseases have been associated with defects in its activity. Despite its importance, there are no small molecules that selectively modulate cytoplasmic dynein activity, nor is its atomic structure elucidated. In an effort to identify compounds that target cytoplasmic dynein, hits from a high information content cell-based nuclear translocation assay were further evaluated biochemically. High throughput assays were developed to screen for glucocorticoid ligand competition, MT perturbation, and the ATPase activities of Hsp 70 and 90, cytoplasmic dynein heavy chain 1, and myosin. Several compounds from screening the Library of Pharmacologically Active Compounds (LOPAC1280) were identified to inhibit cytoplasmic dynein, though they had several unattractive pharmacological properties and were generally non-specific. Additional screening of the Molecular Libraries Screening Center Network >220,000-member library showed a number of compounds that specifically inhibited the ATPase activity of cytoplasmic dynein heavy chain 1 with little or no interaction with other proteins involved in cargo complex formation. A novel approach to screen for MT perturbing agents was also developed using biosensors. Thickness, mass, and density measurements from dual polarization interferometry suggested the growth process of MTs on surfaces. Resonant mirror biosensors were used to distinguish MT stabilizers from destabilizers based on rates of MT assembly on the surfaces.
In addition, the structure of the cytoplasmic dynein heavy chain motor domain was characterized by computational and experimental methods. Comparative homology structural modeling was used to predict 15 surface accessible cysteines, which were then correlated experimentally by mass spectrometry. Five cysteines were matched computationally and experimentally to be surface-accessible, suggesting some inadequacy of the proposed model. Finally, attempts to reconstruct a model of cytoplasmic dynein heavy chain 1 by electron microscopy were hindered by the purification of the protein from both a Hi5/baculovirus expression system and bovine brain, although the latter appeared to provide better quality micrographs. Ultimately, structural characterization will assist with the discovery of cytoplasmic dynein heavy chain 1 modulators.
|
9 |
STRUCTURE-FUNCTION STUDIES OF THE METABOTROPIC GLUTAMATE RECEPTOR TYPE 6 (mGluR6) AND COMPARISON WITH RHODOPSINTirupula, Kalyan C 17 May 2011 (has links)
Metabotropic glutamate receptor subtype 6 (mGluR6), a class C G protein coupled receptor (GPCR), plays a key role in visual signal transduction and is also implicated in addiction. Certain mutations in mGluR6 have been reported to cause congenital stationary night blindness. In spite of the importance of mGluR6, knowledge of the molecular basis of its function is lacking. It is imperative to improve the current understanding of its structure-function relationships, so that selective ligands that modulate its activity can be discovered. Furthermore, functional characterization of mGluR6 is also expected to lead to a better understanding of the general principles underlying the activation mechanism of GPCR family. Rhodopsin is the prototypical class A GPCR and serves as a good comparative model to establish general mechanistic patterns of activation of GPCRs. This thesis describes experimental and computational approaches to characterize the structure-function relationship of mGluR6 and its comparison with rhodopsin.
Firstly, inducible stable cell lines with high levels of mGluR6 expression were established. Proper trafficking and folding of mGluR6 in these cell lines were verified. To determine mGluR6 function, existing cell-based and novel membrane-based functional assays were optimized and developed, respectively. These efforts led to the establishment of a robust system that expresses properly folded and functional mGluR6 and enabled structure-function studies to be carried out. Several transmembrane cysteine mutants were created and analyzed with the goal to study the role of the transmembrane domain of mGluR6 in activation mechanism. TM6 of mGluR6 like rhodopsin was found to play a key role in its activation supporting the hypothesis that these two GPCRs may share a general mechanism of activation despite the large sequence divergence. Additional support for this hypothesis was obtained from computational sequence analysis which showed that the highly ranking residues involved in long-range interaction in rhodopsin overlap with the allosteric binding pocket of mGluR6. Finally, with the aim to identify selective ligands for mGluR6, an integrated computational-experimental approach was undertaken. Novel allosteric ligands and possibly selective orthosteric ligands for mGluR6 were identified. Further characterization of these ligands may lead to design of selective ligands for mGluR6.
|
10 |
Fluoren-9-ylidene Hydrazine Inhibitors of HIV-1 Ribonuclease HLaBarge, Krystal Marion 17 August 2011 (has links)
Screening a library of 5,292 hydrazone/hydrazine compounds for inhibition of HIV reverse transcriptase-associated ribonuclease H (RNH) activity identified fluoren-9-yildene hydrazines as highly active inhibitors. The 33 fluoren-9-yildene hydrazines in this library were expanded to 118 compounds, 65 (55%) of which showed validated inhibition of RT RNH activity (IC50 values < 10 uM). These inhibitors were mainly monofunctional for RNH activity, since only 25 (21%) also inhibited RT RNA-dependent DNA polymerase activity. The two most potent RNH inhibitors (RNHIs) were compounds 15 and 25, which inhibited wild type RT-RNH activity with IC50 values of 0.34 ± 0.07 uM and 0.4 ± 0.03 uM, respectively. Similar inhibition was noted with two clinically relevant NNRTI resistant mutants, Y181C and K103N/L100V. Biochemical studies showed that these compounds preferentially inhibited non-directed and DNA 3'-end directed RNH cleavages. These compounds also inhibited the activity of the p15-EC RT RNH domain fragment with IC50 values of 0.43 ± 0.04 uM and 0.032 ± 0.004 uM, respectively. Furthermore, both compounds had antiviral activity against HIV-1 with EC50 values of 10 ± 3 uM and 1.4 ± 0.6 uM for compounds 15 and 25, respectively. Order of addition experiments showed that potent inhibition required pre-incubation of the enzyme with the inhibitor; inhibitory potency substantially decreased if the RNA/DNA substrate was present prior to inhibitor addition. Furthermore, inhibition was competitive with respect to the RNA/DNA substrate, suggesting an active site binding mode. 1H-15N HSQC protein NMR studies with the p15-EC RT RNH domain fragment further suggested that the inhibitor binds to the RNH active site. Molecular docking studies with compound 25 were consistent with an active site binding mode in which the hydrazine functionality hydrogen bonds with essential catalytic metal coordinating residues E52 (RT: 478) and D72 (RT: 498). A sulfonamido-phenyl ring substituent on compound 25 makes edge on-Ï interactions with H127 (RT: 539), another residue essential for RNH catalysis. We therefore propose that the fluoren-9-ylidene hydrazine RNHIs act by preventing access of RNH essential catalytic residues to the RNA/DNA substrate.
|
Page generated in 0.0809 seconds