Thesis (Ph.D.)--McMaster University (Canada), 1993. / Source: Dissertation Abstracts International, Volume: 54-12, Section: B, page: 6168. Adviser: R. F. Childs.

Würzburg, Univ., Diss., 2009

Universiẗat, Diss., 2000--Freiburg (Breisgau).

Solid-state Nuclear Magnetic Resonance (NMR)is one of the premiere biophysical methods that can be applied for addressing the structure and dynamics of biomolecules, including proteins, lipids, and nucleic acids. It illustrates the general problem of determining the average biomolecular structure, including the motional mean-square amplitudes and rates of the fluctuations. Lineshape and relaxtion studies give us a view into the molecular properties under different environments. To help the understanding of NMR theory, both lineshape and relaxation experiments are conducted with hexamethylbezene (HMB). This chemical compound with a simple structure serves as a perfect test molecule. Because of its highly symmetric structure, its motions are not very difficult to understand. The results for HMB set benchmarks for other more complicated systems like membrane proteins. After accumulating a large data set on HMB, we also proceed to develop a completely new method of data analysis, which yields the spectral densities in a body-fixed frame revealing internal motions of the system. Among the possible applications of solid-state NMR spectroscopy, we study the light activation mechanism of visual rhodopsin in lipid membranes. As a prototype of G-protein-coupled receptors, which are a large class of membrane proteins, the cofactor isomerization is triggered by photon absorption, and the local structural change is then propagated to a large-scale conformational change of the protein. Facilitation of the binding of transducin then passes along the visual signal to downstream effector proteins like transducin. To study this process, we introduce 2H labels into the rhodopsin chromophore retinal and the C-terminal peptide of transducin to probe the local structure and dynamics of these two hotspots of the rhodopsin activation process. In addition to the examination of local sites with solid-state 2H NMR spectroscopy, wide angle X-ray scattering (WAXS) provides us the chance of looking at the overall conformational changes through difference scattering profiles. Although the resolution of this method is not as high as NMR spectroscopy, which gives information on atomic scale, the early activation probing is possible because of the short duration of the optical pump and X-ray probe lasers. We can thus visualize the energy dissipation process by observing and comparing the difference scattering profiles at different times after the light activation moments.

An in vitro phagocytosis assay system was developed and characterized for studying the specific receptor-mediated phagocytosis of bovine ROS by bovine RPE cells. The phagocytosis of ROS was detected qualitatively by electron microscopy and quantitatively by treating RPE cells with radioiodinated ROS or by probing ROS-treated RPE cells with a radiolabeled antirhodopsin monoclonal antibody. The binding sites for various antirhodopsin monoclonal antibodies were localized as an essential step in their application as immunochemical probes for analysis of the structure and function of rhodopsin. Five monoclonal antibodies raised against rhodopsin have been shown to be directed against the N-terminal regions on the basis of their reactivity to an immunoaffinity purified 2-39 glycopeptide, a 2-16 tryptic glycopeptide and a 1-16 synthetic peptide as measured by radioimmune competition assays. Limited proteolysis, immunogold-dextran labeling and competitive inhibition studies identified two antirhodopsin monoclonal antibodies which bound to internal cytoplasmic loop regions of rhodopsin. Finally, the binding sites for these and other C-terminal specific antirhodopsin monoclonal antibodies were used to elucidate the proposed transmembrane helical model of rhodopsin. An antirhodopsin monoclonal antibody (rho 4D2), which bound to rhodopsin in glutaraldehyde-fixed ROS plasma membranes, was employed as an immunocytochemical probe in studying the possible role of rhodopsin in the binding and phagocytosis of rod outer segments. An immunoaffinity purified 2-39 N-terminal rhodopsin glycopeptide, a synthetic 1-16 peptide analogue of rhodopsin and phospholipid vesicles reconstituted with rhodopsin were all found to be ineffective in inhibiting the phagocytosis of ¹²⁵I-labeled ROS by RPE cells. In essence, these results provided compelling evidence that rhodopsin in the ROS plasma membrane does not function as the ligand for recognition by RPE cells. The molecular properties of the ROS cell surface ligand(s), which are involved in recognition by bovine RPE cells, were studied by limited-proteolytic digestion in conjunction with quantitative phagocytosis assays. Mildly trypsin-treated ROS were found to be less effectively phagocytized than untreated ROS by bovine RPE cells. Moreover, the glycopolypeptides (34kD and 24kD) released from the ROS cell surface by trypsin were capable of inhibiting ROS phagocytosis. The ROS plasma membrane specific, ricin-binding, 230kD glycoprotein was observed by SDS-gel electrophoresis and western blotting to be highly trypsin sensitive under these conditions. Hence, ricin affinity chromatography and immunoaffinity chromatography were employed in an attempt to purify this 230kD glycoprotein from ROS membranes. Enriched preparations of the 230kD glycoprotein were reconstituted into phospholipid vesicles and effectively used to inhibit the phagocytosis of ROS by RPE cells. In summary, a ROS plasma membrane specific, 230kD glycoprotein has been identified and isolated; this protein may act as a ligand in specific ligand-receptor interactions between ROS and RPE cells. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Vision requires the photoreceptors in the eye to rapidly respond to changes in light intensity. These processes are accomplished within rod photoreceptors by the visual pigment rhodopsin that initiates a downstream signaling cascade called phototransduction. Rhodopsin is composed of an apoprotein opsin that is covalently bonded with light sensitive 11-cis retinal. Rhodopsin is activated when 11-cis retinal is photoisomerized into all-trans retinal. This isomerization initiates the phototransduction cascade that culminates in a change in current at the plasma membrane. Rhodopsin, once activated ("bleached"), can no longer absorb photons to activate phototransduction, and must be regenerated through the visual cycle. To enable the photoreceptors to respond to rapid changes in light intensities, phototransduction must terminate in a timely manner. Deactivation involves phosphorylation of activated rhodopsin by rhodopsin kinase, and then binding of visual arrestin. Exposing rods to daylight bleaches a large proportion of rhodopsin molecules. This exposure leads to desensitization of the photoreceptors and phosphorylation of bleached rhodopsin. Full recovery of receptor sensitivity is achieved when rhodopsin is recycled and regenerated through a series of steps to its ground state. The last step in this process is the dephosphorylation of rhodopsin. This dissertation focuses on how rhodopsin dephosphorylation affects rod sensitivity. I exploited a novel observation; mouse retinae when isolated from the retinal pigment epithelium (and eye cup), display blunted rhodopsin dephosphorylation. Isoelectric focusing followed by Western blot analysis of retinal homogenate from bleached isolated retinae showed little dephosphorylation of rhodopsin for up to four hours in darkness, even under conditions when rhodopsin was completely regenerated. Microspectrophotometric measurements of rhodopsin spectra show that regenerated phospho-rhodopsin has the same molecular photosensitivity as unphosphorylated rhodopsin and that flash responses measured by trans-retinal electroretinogram or single cell suction electrode recording displayed dark-adapted kinetics. Single quantal responses displayed normal dark-adapted kinetics, but rods were only half as sensitive as those containing exclusively unphosphorylated rhodopsin. I propose a revised model in which light-exposed retinae contain a mixed population of phosphorylated and unphosphorylated rhodopsin. Moreover, complete dark-adaptation can only occur when all rhodopsin has been dephosphorylated, a process that requires more than three hours in complete darkness.

No description available.

G-protein–coupled receptors are the largest superfamily in the human genome, and involved in critical cellular signaling processes in living cells. Protein structural fluctuations are the key for GPCR function that is driven and modulated by a variety of factors that are not well understood. This dissertation focusses on understanding the activation of GPCRs using the visual receptor, rhodopsin as the prototype. Rhodopsin is an ideal candidate for this study, as it represents the largest class of GPCRs, and is known to demonstrate more noticeable structural changes upon activation compared to the other GPCRs. What structural fluctuations occur, the role of water, and how the retinal cofactor regulates the protein dynamics during rhodopsin activation are specific research problems addressed in this work. Hypothesizing an ensemble activation mechanism, experiments were conducted using a variety of techniques to probe structural and dynamical fluctuations of rhodopsin in native membranes, as well as in membrane mimetics such as detergent micelles. Time-resolved wide-angle X-ray scattering (TR-WAXS), small-angle neutron scattering (SANS), quasielastic neutron scattering (QENS), and electronic spectroscopy are among the prominent techniques used to gain insights into the photo-intermediates that are key to understanding the rhodopsin activation process. The small-angle neutron scattering (SANS) experiments revealed a volumetric expansion of the protein molecule upon photoactivation of rhodopsin. Electronic spectroscopy together with the differential hydration study revealed the crucial role of water in rhodopsin signaling process and signal amplification by water. The quasielastic neutron scattering study conducted on powdered rhodopsin probed the changes in the local dynamics that are regulated by the retinal cofactor of the rhodopsin molecule. The increased local steric crowding in the ligand-free opsin is consistent with collapsing of the apoprotein structure in the absence of the retinal chromophore leading to inactive opsin conformation. Finally, a time-resolved wide-angle X-ray scattering study was conducted using the X-ray free electron laser at the SLAC national laboratory to probe the early structural fluctuations in rhodopsin photoactivation. The preliminary pump-probe experiments conducted on rhodopsin in CHAPS detergent micelles revealed a light-triggered protein quake that occurs during the early activation stages of rhodopsin photoactivation. Thus the protein fluctuations underlying the GPCR function are revealed by neutrons, X-rays, and other photons in a combined implementation of both spectroscopic and scattering techniques as applied to the investigation of rhodopsin activation.

Yeung Kwun Yan. / Thesis submitted in: December 2000. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 106-122). / Abstracts in English and Chinese. / Acknowledgements --- p.iv / Table of Contents --- p.v / List of Tables --- p.viii / List of Figures --- p.ix / Abbreviations --- p.x / Conference Presentations --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Retinitis Pigmentosa (RP) --- p.1 / Chapter 1.1.1 --- Molecular genetics --- p.1 / Chapter 1.1.2 --- Clinical features --- p.2 / Chapter 1.1.3 --- Clinical classifications of RP --- p.3 / Chapter 1.2 --- Molecular Biology of Rhodopsin --- p.4 / Chapter 1.2.1 --- Anatomy and functions of human retina --- p.4 / Chapter 1.2.2 --- Physiology of rhodopsin --- p.5 / Chapter 1.2.3 --- Rhodopsin cycle and the visual transduction cascade --- p.7 / Chapter 1.2.4 --- The human rhodopsin gene (RHO) --- p.8 / Chapter 1.2.5 --- RHO mutations --- p.8 / Chapter 1.2.6 --- Frequencies & phenotypes ofmutations --- p.10 / Chapter 1.2.7 --- Findings of in vitro experiments --- p.11 / Chapter 1.2.8 --- Findings in animal models --- p.12 / Chapter 1.2.9 --- Findings in human --- p.14 / Chapter 1.3 --- Molecular Biology of RP1 --- p.15 / Chapter 1.3.1 --- RP1 gene in animals --- p.16 / Chapter 1.3.2 --- Mutations in RP1 --- p.16 / Chapter 1.3.3 --- Phenotypes & frequencies of RP1mutations --- p.17 / Chapter 1.4 --- Mutation Pattern of RHO & RP1 in Chinese --- p.18 / Chapter 1.5 --- Methods for Detecting Mutations in RHO and RP1 --- p.18 / Chapter 1.6 --- Management of RP --- p.20 / Chapter Chapter 2 --- Study Objectives --- p.31 / Chapter Chapter 3 --- Methodology --- p.32 / Chapter 3.1 --- Study Subjects --- p.32 / Chapter 3.2 --- Clinical Data Sheet --- p.32 / Chapter 3.3 --- "Chemicals, Reagents, and Kits" --- p.35 / Chapter 3.4 --- Solutions and Buffers --- p.36 / Chapter 3.5 --- Enzymes --- p.37 / Chapter 3.6 --- Equipment --- p.37 / Chapter 3.7 --- Software --- p.38 / Chapter 3.8 --- "Oligonucleotide Primers for PCR, CSGE and Sequencing" --- p.38 / Chapter 3.9 --- DNA Extraction --- p.38 / Chapter 3.9.1 --- DNA extraction from blood samples --- p.39 / Chapter 3.9.2 --- DNA extraction from buccal swab --- p.39 / Chapter 3.9.3 --- DNA quantitation --- p.39 / Chapter 3.10 --- Polymerase Chain Reaction (PCR) --- p.40 / Chapter 3.10.1 --- Amplification of RHO --- p.40 / Chapter 3.10.2 --- Amplification of RP1 --- p.40 / Chapter 3.11 --- Gel Electrophoresis --- p.40 / Chapter 3.11.1 --- Agarose gel electrophoresis --- p.41 / Chapter 3.11.2 --- Conformation sensitive gel electrophoresis (CSGE) --- p.41 / Chapter 3.11.3 --- DNA sequencing --- p.42 / Chapter 3.12 --- Statistical Methods --- p.43 / Chapter Chapter 4 --- Results --- p.51 / Chapter 4.1 --- Study Subjects --- p.51 / Chapter 4.1.1 --- RP index patients --- p.51 / Chapter 4.1.2 --- Family members of index patients --- p.51 / Chapter 4.1.3 --- Controls --- p.51 / Chapter 4.2 --- Genetic subtypes of RP in our study --- p.52 / Chapter 4.3 --- PCR --- p.52 / Chapter 4.4 --- Conformation Sensitive Gel Electrophresis (CSGE) --- p.53 / Chapter 4.5 --- Direct DNA Sequencing --- p.53 / Chapter 4.5.1 --- Sequence alterations in RHO --- p.54 / Chapter 4.5.2 --- Sequence alterations in RP1 --- p.56 / Chapter 4.6 --- Family Studies --- p.60 / Chapter Chapter 5 --- Discussion --- p.77 / Chapter 5.1 --- The Expected Frequencies of RHO & RP1 Mutationsin Chinese RP Patients --- p.82 / Chapter 5.2 --- The Mutation Screening Technique in this Study --- p.84 / Chapter 5.3 --- Mutations and Sequence Alterations Identified in RHO --- p.86 / Chapter 5.3.1 --- Novel mutation: 521 ldelC --- p.86 / Chapter 5.3.2 --- Reported mutation: Pro347Leu --- p.90 / Chapter 5.3.3 --- Novel nonpathogenic missense change: Ala299Ser --- p.92 / Chapter 5.3.4 --- Novel silent sequence alterations --- p.93 / Chapter 5.3.5 --- Other polymorphisms in RHO --- p.93 / Chapter 5.4 --- Mutation and Sequence Alterations Detected in RP1 --- p.94 / Chapter 5.4.1 --- Mutation found in Chinese: Arg677ter --- p.95 / Chapter 5.4.2 --- Novel nonsense sequence alteration: Arg l933ter --- p.96 / Chapter 5.4.3 --- Novel missense and non-coding changes in RP1 --- p.97 / Chapter 5.4.4 --- Reported polymorphisms --- p.98 / Chapter 5.5 --- Possible Functions of RP1 --- p.99 / Chapter Chapter 6 --- Conclusion --- p.105 / Chapter Chapter 7 --- References --- p.106

Die zur Gruppe der mikrobiellen Rhodopsine gehörenden lichtaktivierbaren Ionenkanäle Channelrhodopsin 1 (ChR1) und Channelrhodopsin 2 (ChR2) aus dem Augenfleck von C. rheinhardtii sind Bestandteile des visuellen Systems und an der Phototaxis beteiligt. Sie bestehen aus einem zytosolisch gelegenen C Terminus, dessen Funktion noch ungeklärt ist und einem, für die Kanalaktivität verantwortlichen, N terminalen Bereich aus sieben Transmembranhelices. Der lichtsensitive Kofaktor all trans Retinal ist kovalent an einen Lysinrest (K257) der siebten Transmembranhelix gebunden. Bei einer Belichtung mit Blaulicht isomerisiert das Chromophor zur 13 cis Form, was eine Konformationsänderung und das Öffnen des Kanals zur Folge hat. Im Zuge dessen strömen ein und zweiwertige Kationen in die Zelle und eine Depolarisation findet statt. Um einen tieferen Einblick in Struktur und funktionelle Mechanismen zu bekommen, wurden Wildtyp und Mutanten von Ch1 und ChR2 heterolog in Oozyten von X. laevis exprimiert. In Bakteriorhodopsin bilden die Seitenketten von T90 und D115 eine für Stabilität und Funktion wichtige Wasserstoffbrücke aus. Durch elektrophysiologische, fluoreszenzmikroskopische und biochemische Verfahren wurden Mutanten der entsprechenden Reste in ChR2 (C128, D156) untersucht. Diese zeigten eine deutlich verlangsamte Kinetik und eine 10 bis 100fache Erhöhung der Lichtempfindlichkeit. Die identischen Auswirkungen von Mutationen beider Reste deuten auf eine Bindung mit funktioneller Bedeutung zwischen C128 und D156 hin. Im Falle von ChR2 C128T, C128A und D156A konnte der Kanal nach Anregung mit Blaulicht, durch grünes und violettes Licht vorzeitig geschlossen werden. Diese Lichtqualitäten entsprechen den Absorptionswellenlängen zweier Intermediate des Photozyklus von ChR2 (P390 und P520). Durch Veränderung des externen pH-Wertes konnten Hinweise auf eine protonenabhängige Gleichgewichtsreaktion dieser Intermediate gefunden werden. Auch in dem für Protonen höher leitfähigen ChR1 konnten Hinweise auf eine Interaktion zwischen den Resten C167 und D195 gefunden werden. Elektrische Messungen von Mutanten zeigten eine deutliche Erhöhung des Photostroms bei verhältnismäßig geringem Anstieg der Schließzeit. Der Einfluss dieser Mutationen auf die Kinetik war somit weniger ausgeprägt als bei ChR2. Einen besonderen Stellenwert unter allen Channelrhodopsin Mutanten nehmen ChR2 D156C und ChR1 D195C ein. Mit einem Photostrom von 5 µA bei ChR1 D195C und bis zu 50 µA bei ChR2 D156C konnten für diese die höchsten Photoströme aller bisher charakterisierten ChR1 bzw. ChR2 Varianten nachgewiesen werden. Durch fluoreszenzmikroskopische Quantifizierung konnte für alle im Rahmen dieser Arbeit erstellten ChR1 und ChR2 Mutanten eine erhöhte Proteinmenge sowohl in Anwesenheit als auch Abwesenheit zusätzlichen all trans Retinals während der Inkubation nachgewiesen werden. Die Fluoreszenzintensitäten korrelierten hierbei mit der Höhe der Stromamplituden und erreichten ein Maximum bei ChR2 D156C. Biochemische Experimente mit der Gesamtmembranfraktion von ChR2 exprimierenden Oozyten lieferten Hinweise auf eine dimere Quartärstruktur von Channelrhodopsinen, was durch die Kristallstruktur einer Chimäre aus ChR1 und ChR2 von (Kato et al., 2012) bestätigt wurde. Unter der Annahme, dass die Poren in den Proteomeren gebildet werden, konnte eine gegenseitige Beeinflussung der Regionen in heterodimeren Kanälen aus ChR2 Wildtyp und Mutanten aufgrund kinetischer Unterschiede bei kurzer und langer Belichtung oder der Verwendung von unterschiedlichen Lichtintensitäten nachgewiesen werden. Eine Voraussetzung für diesen Effekt ist eine synchrone Anregung beider Untereinheiten. Die Interaktion von Channelrhodopsin Untereinheiten konnte in vivo mithilfe der bimolekularen Fluoreszenzkomplementation nachgewiesen werden. Dabei zeigte sich, dass die Wechselwirkung nicht nur auf identische Untereinheiten in Homodimeren beschränkt ist, sondern auch bei Heterodimeren aus verschiedenen ChR2 Untereinheiten und sogar zwischen ChR2 und ChR1 möglich ist. / The microbial type rhodopsins Channelrhodopsin 1 (ChR1) and Channelrhodopsin 2 (ChR2) are located in the eyespot of the green algae Chlamydomonas rheinhardtii. They are light activated cation channels and play an important role in phototaxis. They are comprised of a cytosolic C terminal part of unknown function and a transmembranal N terminal part responsible for channel activity. The latter consists of seven transmembrane helices and the chromophore all trans Retinal, which is bound covalently as a Schiff base to a lysine residue. When activated with blue light, the chromophore isomerizes to the 13 cis state, followed by a conformational change of the protein and opening of the channel. The resulting influx of mono and divalent cations leads to a depolarization of the cell. To get a deeper insight in structure and function of ChR1 and ChR2, wildtype and mutants have been heterologously expressed in oocytes of Xenopus laevis. In bacteriorhodopsin, a hydrogen bond between the amino acids T90 and D115 is vital for protein stability and proper pump function. Mutants of the corresponding amino acids in ChR2 (C128, D156) were generated and analyzed electrophysiologically. They displayed a significant deceleration of closing time and a 10 100fold increase in light sensitivity. The identical impact of these mutations on kinetics suggests an interaction between these residues, probably also by the formation of a hydrogen bond. In the case of ChR2 C128T, C128A and D156A closing could be accelerated via green and violet light application. These wavelengths correspond to the absorption wavelengths of the photointermediates P390 and P520 in the photocycle of ChR2. Furthermore, a possible proton-dependent equilibrium between these intermediates was identified by varying external proton concentrations. Electrophysiological analyses of C167 and D195 mutants in ChR1 hinted towards an interaction similar to the one between the homologous residues C128 and D156 in ChR2. Both, ChR1 C167 and ChR1 D195 displayed increased current amplitudes, accompanied by only a small increase in closing time. The impact of these mutations on channel kinetics was less pronounced than in ChR2. The mutants ChR2 D156C and ChR1 D195C are of particular importance. Photocurrent amplitudes of 5 µA for ChR1 and 50 µA for ChR2 at 100 mV render these mutants the ones with the highest current amplitudes of all channelrhodopsin variants characterized up till now. In comparison to wildtype channelrhodopsins, quantification by fluorescence microscopy revealed an increased protein amount in oocytes expressing ChR1 and ChR2 mutants, both in absence and presence of additional all trans Retinal during incubation. The fluorescence intensities correlated to the increased photocurrent amplitudes, reaching a maximum in the ChR2 D156C mutant. Immunoblot experiments with total membrane fractions of oocytes expressing ChR2, bacteriorhodopsin and halorhodopsin hinted towards a dimeric quartenary structure of the channel. This has been confirmed by the recently published crystal structure of a chimaeric ChR1/ChR2 protein (Kato et al., 2012). Assuming the pore in a channel dimer is formed by the protomers, interference or crosstalk between the subunits has been identified. Oocytes expressing mixtures of channelrhodopsins demonstrated differences in kinetics when illumination length and light intensity was varied. A prerequisite for this effect is a simultaneous excitation of both subunits. The interaction of channelrhodopsin-subunits in vivo has been demonstrated by bimolecular fluorescence complementation assays. It was shown, that oligomerization is not restricted to identical subunits in a homodimer but also possible between different ChR2 variants in a heterodimer and even between ChR1 and ChR2.