- Biochemical analysis of the interaction between transfer ribonucleic acid and exportin-t (2006)
- In this thesis the ternary complex of tRNA•exportin-t•Ran•GTP was studied by biochemical and biophysical methods. Firstly, photocrosslinking was used to determine the contact sites between tRNA and exportin-t. 4-Thiouridine (s4U) was introduced into tRNAPheT.th by in vitro transcription with T7 RNA polymerase and crosslinked to exportin-t by irradiation at 312 nm. The crosslinking was Ran•GTP dependent and could be competitively inhibited by other tRNA species, showing that the crosslinking was the consequence of the formation of a tRNA•exportin-t•Ran•GTP complex. The crosslinked complex of (s4U)tRNAPheT.th-exportin-t after proteinase K digestion was incubated with a primer complementary to the 3'end of the tRNAPheT.th in the primer extension reaction. The elongation of the reverse transcriptase was forced to halt at s4Us crosslinked to peptides, namely, U55, U47, U33, U20. Among them, U47 and U55 were shown to be the major crosslinking sites. U47A mutation was introduced into tRNAPheT.th to test the role of U47. The mutant tRNAPheT.th transcript was still able to bind exportin-t, though a little weaker than normal tRNAPheT.th transcript. In contrast, s4U containing mutant tRNAPheT.th U47A exhibited a much poorer capability to crosslink exportin-t. It is concluded that U47 may be a major contact site between tRNAPheT.th and exportin-t. Secondly, the binding abilities of different tRNA species in calf liver tRNABulk to exportin-t was examined by affinity chromatography on immobilized exportin-t. With a stepwise elution of 250 mM and 500 mM KCl, tRNABulk was fractionated into 2 peaks. Therefore, Different tRNAs bound to exportin-t with different affinity. Among 7 tRNAs tested, the relative affinity to exportin-t ranked as tRNALeuCAG > tRNASerGCU, tRNALeuCAA, tRNASerUGA > tRNASerAGA, tRNALeuAAG > tRNAArgACG. To interpret the different affinity of tRNAs to exportin-t, it is proposed that exportin-t preferentially exports tRNAs that are required the stronger by the protein synthesis machine. The extent of requirement of a specific tRNA by cells is supposed as the ratio between the tRNA concentration in a cell and its codon frequency in the genome, if all tRNAs are supposed to be aminoacylated and transported to ribosome equally. It was found that among 7 tRNAs identified on the 2D urea PAGE, the theoretical estimation of 5 tRNA species upon their requirement by cell ranked the same as their relative affinity to exportin-t. Finally, atomic force microscopy was used to observe exportin-t and its interaction with tRNA in a native and undisturbed state directly. Exportin-t-esterase, immobilized to trifluoromethyl ketone (TFK) modified mica surface, showed a diameter of 15 ± 2 nm. After binding tRNA and Ran•GppNHp, the diameter of the complex somewhat increased to 16 ± 2 nm.
- Modulation of Incommensurately Modulated Structures Studied by the Maximum Entropy Method (2011)
- In this thesis, modulated properties of incommensurately modulated crystals are studied by the Maximum Entropy Method (MEM). This was done exemplarily on the compounds Rubidium tetrachlorozincate and Chromium pyrophosphate. To do so, the MEM derived (3+1)-dimensional superspace electron density was analysed to receive information on atomic positions and their modulation, and structure refinement by the method of least-squares and interpretation of the difference-Fourier maps were performed to better describe the atomic displacement parameters (ADPs) and to improve the applied structural models. All the MEM calculations are done by the computer program BayMEM. The analysis of this MEM reconstructed density map is done by the computer program EDMA. Our results suggest that the modulation of harmonic ADPs, anharmonic ADPs and its modulation form an intrinsic part of incommensurately modulated structures. We have shown that with a data set of certain resolution and satellite reflections of higher order, the inclusion of modulation of harmonic ADPs, the anharmonic ADPs and the modulation of anharmonic ADPs can significantly improve the fit of the structure model to the diffraction data. Such model then better represents the true nature of the structure under investigation.
- Structural Analysis of Cylindrical Particles by Small Angle X-ray Scattering (2005)
- The objective of this work is to analyze nano-scaled cylindrical particles by small angle X-ray scattering (SAXS). Three systems with cylinder-shaped particles: (1) Laponite particles in aqueous solutions, (2) Poly(carbon suboxide) particles in binary water/DMF solutions, and (3) Suprastructural aggregates of coil-ring-coil block copolymers in cyclohexane, have been studied by SAXS performing either a Kratky-Compact-Camera in our laboratory or ID2 beamline of the European Synchrotron Radiation Facility (ESRF) in Grenoble. The synthetic clay particles, Laponite RD, have been chosen as ideal disc-shaped model particles. In combination of SAXS with static light scattering, the scattering intensities of a concentration serial (volume fraction from 0.0002 to 0.0016) were measured in almost three orders of magnitude of the scattering vector q. Through extrapolation of concentration the scattering intensity at vanishing concentration, i.e. the form factor P(q) of particles was achieved. It shows q-2 decay at intermediate q range, which indicates that the shape of single Laponite particle in aqueous solution is platelet. The plateau of the form factor at low q range implies that there is no aggregate or cluster structure, and the Laponite particles are dispersed completely under the investigated conditions. More detailed structural information was then obtained by fitting of P(q) with disc model. The radii of the discs exhibit a large polydispersity. A radius of 10.5 nm with Schulz-Zimm distribution of Rw/Rn = 1.5 (where Rw and Rn denote weight and number average radius, respectively) was found to fit the form factor perfectly. The thickness of one single platelet was determined to be 0.9 nm. The weight averaged molecular weight and radius of gyration were determined to be 930 kg/mol and 13.4 nm, respectively. The inter-particle interactions of Laponite particles were investigated by the structure factor S(q), from which the effective diameter of interparticle interactions deff was determined for the first time. The strong electrostatic Coulomb repulsion between charged Laponite particles was attributed to the much higher value of deff (= 46 nm), in comparison to 2Rg (= 27 nm). The recently developed multicomponent interaction site model was performed by Harnau to predict these experimental structure factors. An effective potential of interaction, which pays attention to a screened Coulomb interaction as well as an attractive interaction, leads to the best description of the model to the experimental data. By means of SAXS, the size of synthetic polymer carbon suboxide ((C3O2)n) dissolved in binary water/DMF solutions was determined for the first time with radius of gyration Rg = 1.7 nm and molecular weight Mw = 2760 g/mol, which corresponds to a polymerization’s degree of about 40. This value is much larger than literature one (5-10). The form factor of polymer carbon suboxide can be described by a semiflexible chain model. The radius of gyration in cross-section RC and molecular weight per unit length ML were obtained to be 0.3 nm and 350 g/(mol.nm), respectively, which can confirm the fact that the chemical structure of poly(carbon suboxide) is repeated pyronic ring, as suggested in most literatures. Thus the structure and size of polymer carbon suboxide were characterized completely by SAXS. Finally, SAXS was employed to analyze a suprastructural aggregation system derived by self-assembly of coil-ring-coil block copolymers. This is a newly synthesized subclass of rod-coil block copolymers composed of a nanometer-sized shape-persistent macrocycle and two covalently attached polystyrene (PS) coils. The solubility of the rigid ring is largely enhanced due to the attachment of the flexible side groups. With suitable length of the flexible side groups (Mw (PS) = 2500 g/mol) the block copolymers can form colloidal-sized aggregates in selected solvent cyclohexane, which were concluded to be of cylindrical shape with the rigid rings packing densely in a tubular way and the flexible side groups arranging outside of the ring. Such aggregated cylinder brushes can be further confirmed to exist as a mixture of cylinder bundles by analyzing the local structural parameter ML (= 25730 g/(mol.nm), molecular weight per nm length of cylindrical objects). In comparison of this value with M0 (= 6500 g/mol, molecular weight of single coil-ring-coil block copolymer) and d (= 0.6 nm, distance of adjacent densely packed rings), the number fraction of coexisted single cylinder, bi- and tri-cylinder bundles was resulted to be 1:1:2. Through fitting by using approximated circular cylinder model the radius of single cylinders was determined to be 2.6 nm (polydispersity 20 percent) with a hollow inside of radius of 1.2 nm.