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Calcium Modulates MGLUR1 Folding in ER in the Trafficking Process and Regulates the Drug Activity Upon the Receptor Expressing on the Cell MembraneJiang, Yusheng 01 August 2012 (has links)
Metabotropic glutamate receptor 1α (mGluR1α) exerts important effects on numerous neurological processes. Although mGluR1α is known to respond to extracellular Ca2+ ([Ca2+]o) and the crystal structures of the extracellular domains (ECDs) of several mGluRs have been determined, the calcium-binding site(s) and structural determinants of Ca2+-modulated signaling in the Glu receptor family remain elusive. Here, we identify a novel Ca2+-binding site (Site 1) in the ECD-mGluR1α using a recently developed computational algorithm. This predicted site (D318, E325, D322 and the bound L-Glu) is situated in the hinge region in the ECD-mGluR1α adjacent to the reported Glu-binding site. Mutagenesis studies indicated that binding of L-Glu and Ca2+ to their distinct but partially overlapping binding sites synergistically modulated mGluR1α activation of intracellular Ca2+ ([Ca2+]i) signaling. Mutating the Glu-binding site completely abolished Glu signaling while leaving its Ca2+-sensing capability largely intact. Mutating the predicted Ca2+-binding residues abolished or significantly reduced the sensitivity of mGluR1α not only to [Ca2+]o and [Gd3+]o but also, in some cases, to Glu. In addition, the Ca2+ effects on drugs targeting mGluR1α were investigated. Ca2+ enhances L-Quis response of the receptor by increasing L-Quis binding to ECD-mGluR1α and promotes the potency of Ro 67-4853, a positive allosteric modulator of mGluR1α. Increasing Ca2+ concentration, the inhibitory effects of a competitive antagonist ((s)-MCPG) and a non-competitive negative allosteric modulator (CPCCOEt), were eliminated. Furthermore, we also identified another potential Ca2+ binding pocket (Site 2) consists of S165, D208, Y236 and D318, which completely overlapped with L-Glu. Thapsigargin (TG) induced ER Ca2+ depletion reduced surface expression of mGluR1α, and D208I and Y236I also decreased the receptor trafficking to plasma membrane suggesting the role of Ca2+ binding in protein folding and trafficking in the ER. Further, to measure ER Ca2+, a series of genetically encoded biosensors were designed by placing a Ca2+ binding pocket at the chromophore sensitive region of red florescent protein mCherry. The designed sensors are able to bind Ca2+ and monitor Ca2+ concentration change both in vitro and in cells. The findings in this dissertation open up new avenues for developing allosteric modulators of mGluR function that target related human diseases.
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Implementation and Evaluation of a RF Receiver Architecture Using an Undersampling Track-and-Hold Circuit / Implementation och utvärdering av en RF-mottagare baserad på en undersamplande track-and-hold-kretsDahlbäck, Magnus January 2003 (has links)
Today's radio frequency receivers for digital wireless communication are getting more and more complex. A single receiver unit should support multiple bands, have a wide bandwidth, be flexible and show good performance. To fulfil these requirements, new receiver architectures have to be developed and used. One possible alternative is the RF undersampling architecture. This thesis evaluates the RF undersampling architecture, which make use of an undersampling track-and-hold circuit with very wide bandwidth to perform direct sampling of the RF carrier before the analogue-to-digital converter. The architecture’s main advantages and drawbacks are identified and analyzed. Also, techniques and improvements to solve or reduce the main problems of the RF undersampling receiver are proposed.
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Fast Stochastic Global Optimization Methods and Their Applications to Cluster Crystallization and Protein FoldingZhan, Lixin January 2005 (has links)
Two global optimization methods are proposed in this thesis. They are the multicanonical basin hopping (MUBH) method and the basin paving (BP) method. <br /><br /> The MUBH method combines the basin hopping (BH) method, which can be used to efficiently map out an energy landscape associated with local minima, with the multicanonical Monte Carlo (MUCA) method, which encourages the system to move out of energy traps during the computation. It is found to be more efficient than the original BH method when applied to the Lennard-Jones systems containing 150-185 particles. <br /><br /> The asynchronous multicanonical basin hopping (AMUBH) method, a parallelization of the MUBH method, is also implemented using the message passing interface (MPI) to take advantage of the full usage of multiprocessors in either a homogeneous or a heterogeneous computational environment. AMUBH, MUBH and BH are used together to find the global minimum structures for Co nanoclusters with system size <em>N</em>≤200. <br /><br /> The BP method is based on the BH method and the idea of the energy landscape paving (ELP) strategy. In comparison with the acceptance scheme of the ELP method, moving towards the low energy region is enhanced and no low energy configuration may be missed during the simulation. The applications to both the pentapeptide Met-enkephalin and the villin subdomain HP-36 locate new configurations having energies lower than those determined previously. <br /><br /> The MUBH, BP and BH methods are further employed to search for the global minimum structures of several proteins/peptides using the ECEPP/2 and ECEPP/3 force fields. These two force fields may produce global minima with different structures. The present study indicates that the global minimum determination from ECEPP/3 prefers helical structures. Also discussed in this thesis is the effect of the environment on the formation of beta hairpins.
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Analysis and Error Correction in Structures of Macromolecular Interiors and InterfacesHeadd, Jeffrey John January 2009 (has links)
<p>As of late 2009, the Protein Data Bank (PDB) has grown to contain over 70,000 models. This recent increase in the amount of structural data allows for more extensive explication of the governing principles of macromolecular folding and association to complement traditional studies focused on a single molecule or complex. PDB-wide characterization of structural features yields insights that are useful in prediction and validation of the 3D structure of macromolecules and their complexes. Here, these insights lead to a deeper understanding of protein--protein interfaces, full-atom critical assessment of increasingly more accurate structure predictions, a better defined library of RNA backbone conformers for validation and building 3D models, and knowledge-based automatic correction of errors in protein sidechain rotamers. </p><p>My study of protein--protein interfaces identifies amino acid pairing preferences in a set of 146 transient interfaces. Using a geometric interface surface definition devoid of arbitrary cutoffs common to previous studies of interface composition, I calculate inter- and intrachain amino acid pairing preferences. As expected, salt-bridges and hydrophobic patches are prevalent, but likelihood correction of observed pairing frequencies reveals some surprising pairing preferences, such as Cys-His interchain pairs and Met-Met intrachain pairs. To complement my statistical observations, I introduce a 2D visualization of the 3D interface surface that can display a variety of interface characteristics, including residue type, atomic distance and backbone/sidechain composition. </p><p>My study of protein interiors finds that 3D structure prediction from sequence (as part of the CASP experiment) is very close to full-atom accuracy. Validation of structure prediction should therefore consider all atom positions instead of the traditional Calpha-only evaluation. I introduce six new full-model quality criteria to assess the accuracy of CASP predictions, which demonstrate that groups who use structural knowledge culled from the PDB to inform their prediction protocols produce the most accurate results. </p><p>My study of RNA backbone introduces a set of rotamer-like "suite" conformers. Initially hand-identified by the Richardson laboratory, these 7D conformers represent backbone segments that are found to be genuine and favorable. X-ray crystallographers can use backbone conformers for model building in often poor backbone density and in validation after refinement. Increasing amounts of high quality RNA data allow for improved conformer identification, but also complicate hand-curation. I demonstrate that affinity propagation successfully differentiates between two related but distinct suite conformers, and is a useful tool for automated conformer clustering. </p><p>My study of protein sidechain rotamers in X-ray structures identifies a class of systematic errors that results in sidechains misfit by approximately 180 degrees. I introduce Autofix, a method for automated detection and correction of such errors. Autofix corrects over 40% of errors for Leu, Thr, and Val residues, and a significant number of Arg residues. On average, Autofix made four corrections per PDB file in 945 X-ray structures. Autofix will be implemented into MolProbity and PHENIX for easy integration into X-ray crystallography workflows.</p> / Dissertation
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Construction, expression, and purification of soluble CD16 in bacteriaSinotte, Christopher Matthew 24 May 2006 (has links)
CD16 is a physiologically essential Fc and #947; receptor III as either a single- pass transmembrane protein (CD16A) or as a glycosylated phosphatidylinositol (GPI) anchored protein (CD16B) on the surface of immune cells that have been implicated in many autoimmune and immune complex-mediated diseases. Its functions include binding and clearing antibody (IgG) coated foreign pathogens, receptor-mediated phagocytosis, and triggering antibody dependent cellular cytotoxicity. It is well established that these functions depend on protein-protein interaction between CD16 and the Fc domain of IgG. However, the molecular details of CD16-IgG interactions are less well defined, but are essential to developing therapeutic compounds to treat many autoimmune and IC diseases. Stable mammalian cell lines expressing wild-type CD16 isoforms and site-specific mutants, including extracellular soluble fragments of CD16 have been established. Soluble forms of wild type CD16A and these CD16 mutants were expressed in a bacterial pathway in order to amass sufficient quantities for x-ray crystallographic studies.
The soluble portions of wild-type CD16A and several site-specific CD16A and CD16B mutants were constructed by PCR amplification and ligation with a pET vector. The proteins were expressed in a prokaryotic pathway, BL21 AI, for 8-10 hours and lysed to obtain inclusion bodies. A hand-held sonicator was used to wash the inclusion bodies, while a Urea solution separated and dissolved the proteins. The target proteins were then refolded by rapid dilution, concentrated with a stir cell, and purified. Wild type sCD16A and four site specific mutants were constructed with good sequencing, while wild type sCD16A, sCD16A F176V, and sCD16A G147D were expressed and refolded to optimal levels. X-ray crystallographic data has been collected from sCD16A F176V as a result of these studies and crystals are currently being grown from wild type sCD16A and sCD16A G147D.
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Optical Properties of Superlattice Photonic CrystalsNeff, Curtis Wayne 22 September 2005 (has links)
Photonic band gap materials, commonly referred to as photonic crystals (PCs), have been a topic of great interest for almost two decades due to their promise of unprecedented control over the propagation and generation of light. We report investigations of the optical properties of a new PC structure based upon a triangular lattice in which adjacent [i, j] rows of holes possess different properties, creating a superlattice (SL) periodicity. Symmetry arguments predicted and quot;band folding and quot; and band splitting behaviors, both of which are direct consequences of the new basis that converts the Brillouin zone from hexagonal (six-fold) to rectangular (two-fold). Plane wave expansion and finite-difference time-domain (FDTD) numerical calculations were used to explore the effects of the new structure on the photonic dispersion relationship of the SL PC. Electron beam lithography and inductively coupled plasma dry etching were used to fabricate 1 mm2 PC areas (lattice constant, a =358 nm and 480 nm) with hole radius ratios ranging from 1.0 (triangular) to 0.585 (r2/r1 = 73.26 nm/125.26 nm) on Silicon-on-insulator wafers. The effects of modifying structural parameters (such as hole size, lattice constant, and SL strength) were measured using the coupled resonant band technique, confirming the SL symmetry arguments and corroborating the band structure calculations. Analysis of the dispersion contours of the static SL (SSL) PC predicted both giant refraction (change in beam propagation angle of 110 for an 8 change in incident angle) and superprism behavior (change in beam propagation angle of 108 for a 12% change in normalized frequency) in these structures. Dynamic control of these refraction effects was also investigated by incorporating electro-optic and nonlinear materials into the SSL PC structure. Wave vector analyses on these structures predicted a change in beam propagation angle and gt;96 when the refractive index inside of the holes of the structure changed from n=1.5 to 1.7. Through this investigation, the first successful measurement of the band folding effect in multidimensional PCs as well as the first explicit measurement of the dielectric band of a 2D PC were reported. In addition, the SL PCs impact on new opto-electronic devices was explored.
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Dissipative Particle Dynamics Simulations Study on Organic Thiol Molecule-Au Nano-particles Aggregation and Protein Folding in Aqueous SolutionJuan, Shen-ching-chi 19 July 2005 (has links)
none
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Investigation of Protein Folding by Using Combined Method of Molecular Dynamics and Monte Carlo SimulationsLiao, Jun-min 10 August 2006 (has links)
We used the combination of molecular dynamics and Monte Carlo method to investigate protein folding problems. The environments of proteins are very big, and often very time-consuming. If simulations are based on traditional methods of molecular simulations, it will cost very long time to accomplish the simulation. We use a special designed method, in which the molecular dynamics is used for determining the soft part of protein, and use Monte Carlo method to move and rotate the bonds of proteins. By removing a lot impossible movements in traditional Monte Carlo method, we shorten simulation time and simulate protein folding process effectively. In this work, we used GBSA solvent model, AMBER force field, and semi-local movements to accelerate the simulations. We obtained good result by this simulation method of a small peptide 1L2Y.
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A comparative study of HPr proteins from extremophilic organismsSyed Ali, Abbas Razvi 12 April 2006 (has links)
A thermodynamic study of five homologous HPr proteins derived from organisms
inhabiting diverse environments has been undertaken. The aim of this study was to further
our understanding of protein stabilization in extremes of environment. Two of the proteins
were derived from moderate thermophiles (Streptococcus thermophilus and Bacillus
staerothermophilus) and two from haloalkaliphilic organisms (Bacillus halodurans and
Oceanobacillus iheyensis); these proteins were compared with HPr from the mesophile Bacillus
subtilus. Genes for three of these homologous HPr proteins were for the first time cloned
from their respective organisms into expression vectors and they were over-expressed and
purified in Escherichia coli. Stability measurements were performed on these proteins under a
variety of solution conditions (varying pH, salinity and temperature) by thermal and solvent
induced denaturation experiments. Stability curves were determined for every homologue
and these reveal very similar conformational stability for these homologues at their
habitat temperatures. The BstHPr homologue is the most thermostable and also has the
highest G25; the stability of other homologues was ranked as Bst>Bh>St>Bs>OiHPr.
Other key thermodynamic parameters, like Cp, have been estimated for all the homologues and it was found that these values are identical within errors of estimation. Also, it was found that the values of TS are very similar for these homologues. Together these observations allow us to propose a thermodynamic mechanism toward achieving higher Tm. The crystal structures of the BstHPr and a single tryptophan-containing variant (BstF29W) of this homologue are also reported here. Also reported is a domain-swapped dimeric structure for the BstF29W variant, together with a detailed investigation into the
solution oligomeric nature of this protein. The crystal structure of BstHPr is analyzed to
enumerate various stabilizating interactions like hydrogen bonds and salt-bridges and these were compared with those for the mesophilic homologue BsHPr. Finally, an analysis of sequence alignments together with structural information for these homologues has allowed design of numerous variants of both Bs and BstHPr. A detailed thermodynamic study of
these variants is presented in an attempt to understand the origins of the differences in
stability of the HPr homologues.
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Reaction coordinates for RNA conformational changesMohan, Srividya 06 April 2009 (has links)
This work investigates pathways of conformational transitions in ubiquitous RNA structural motifs. In our lab, we have developed multi-scale structural datamining techniques for identification of three-dimensional structural patterns in high-resolution crystal structures of globular RNA. I have applied these techniques to identify variations in the conformations of RNA double-helices and tetraloops. The datamined structural information is used to propose reaction coordinates for conformational transitions involved in double-strand helix propagation and tetraloop folding in RNA. I have also presented an algorithm to identify stacked RNA bases. In this work, experimentally derived thermodynamic evaluation of the conformations has been used to as an additional parameter to add detail to RNA structural transitions.
RNA conformational transitions help control processes in small systems such as riboswitches and in large systems such as ribosomes. Adopting functional conformations by globular RNA during a folding process also involves structural transitions. RNA double-helices and tetraloops are common, ubiquitous structural motifs in globular RNA that independently fold in to a thermodynamically stable conformation. Folding models for these motifs are proposed in this work with probable intermediates ordered along the reaction coordinates.
We hypothesize that frequently observed structural states in crystals structures are analogous in conformation to stable thermodynamic â on-pathwayâ folded states. Conversely, we hypothesize that conformations that are rarely observed are improbable folding intermediates, i.e., these conformational states are â off-pathwayâ states. In general on-pathway states are assumed to be thermodynamically more stable than off-pathway states, with the exception of kinetic traps.
Structural datamining shows that double helices in RNA may propagate by the â stack-ratchetâ mechanism proposed here instead of the commonly accepted zipper mechanism. Mechanistic models for RNA tetraloop folding have been proposed and validated with experimentally derived thermodynamic data. The extent of stacking between bases in RNA is variable, indicating that stacking may not be a two-state phenomenon. A novel algorithm to define and identify stacked bases at atomic resolution has also been presented in this work.
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