HIV-1 protease is the most effective target for drugs to treat AIDS, however, the long-term therapeutic efficiency is restricted by the rapid development of drug resistant variants. To better understand the molecular basis of drug resistance, crystallographic and kinetic studies were applied to wild-type HIV-1 protease (PR) and drug-resistant mutants, PRV82A, and PRI84V, in complex with substrate analogues, the current drug saquinavir and the new inhibitor UIC-94017 (TMC-114). UIC-94017 was also studied with mutants PRD30N and PRI50V. The drug-resistant mutations V82A, I84V, D30N and I50V participate in substrate binding. Eighteen crystal structures were refined at resolutions of 0.97-1.60A. The high accuracy of the atomic resolution crystal structures helps understand the reaction mechanism of HIV-1 PR. Different binding modes are observed for different types of inhibitors. The substrate analogs have more extended interactions with PR subsites up to S5-S5', while the clinical inhibitors maximize the contacts within S2-S2'. Hydrophobic interactions are the major force for saquinavir binding since it was designed with enhanced hydrophobic groups based on substrate side-chains. In contrast, the new clinical inhibitor UIC-94017 was designed to mimic the hydrogen bonds between substrates and PR. UIC-94017 forms polar interactions with the PR main-chain atoms of Asp29/30, which have been proposed to be critical for its potency against resistant HIV. The mutants showed different structural and kinetic effects, depending on the inhibitor and location of the mutations. The observed structural changes were consistent with the relative inhibition data. Both PRI84V and PRI50V lost favorable hydrophobic interactions with inhibitor compared with PR. Similarly, in PRD30N the UIC-94017 had a water-mediated interaction with the side-chain of Asn30 rather than the direct interaction observed in PR. However, PRV82A compensated for the mutation by shifts of the backbone of Ala82. Furthermore, the complexes of PRV82A showed smaller shifts relative to PR, but more movement of the peptide analog, compared to complexes with clinical inhibitors. The structures suggest that substrate analogs have more flexibility than the drugs to accommodate the structural changes caused by mutation, which may explain how HIV can develop drug resistance while retaining the ability of PR to hydrolyze natural substrates.

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The main focus of this work is the preparation, chemical and structural characterization along with the investigation of physical properties of intermetallic clathrates. Starting from the history of clathrate research, classification of clathrate types, their structural properties and possible application areas are evaluated in chapter 2. The methodologies of sample preparation and materials characterization as well as quantum chemical calculations are discussed in chapter 3. The complete characterization of Ba8Ge433 ( is a Schottky-symbol standing for vacancies),12-14 which is a parent compound for the variety of ternary variants, is the subject of chapter 4. Ba8Ge433 is a high temperature phase,12 which was prepared for the first time as single phase bulk material in this work.15, 16 In this way, the intrinsic transport properties could be investigated without influence of grain boundary and impurity effects. The transport behavior is analyzed at low and high temperatures and referred to the former results. In addition, crystal structure and vacancy ordering in terms of the reaction conditions are discussed. Chemical bonding in Ba8Ge433 is investigated by topological analysis of the electron localizability indicator and the electron density. Chapter 5 deals with the preparation, phase analysis, crystal structure and physical properties of BaGe5, which constitutes a new clathrate type oP60.17, 18 So far, two clathrate types were known in the binary system Ba – Ge, namely the clathrate cP124 Ba6Ge25,19-21 and the clathrate-I Ba8Ge433. Originally, BaGe5 was detected by optical and scanning electron microscopy within the grains of Ba8Ge433.12 Once the preparation of phase-pure Ba8Ge433 was achieved, it became possible to make detailed investigations of its decomposition along with the formation of BaGe5. A detailed theoretical and experimental analysis on the relation between crystal structure and physical properties of BaGe5 is presented. In chapter 6, a thorough structural characterization and the physical properties of clathrates in the system Ba – Ni – Ge is presented based on the subtle relation between the crystal structure containing vacancies and the thermoelectric properties. During the investigations in this system, a large single crystal was grown by Nguyen et al. 22, 23 from the melt with the composition Ba8Ni3.5Ge42.10.4. A systematic reinvestigation of the phase relations in this system was performed and the influence of different Ni content to the crystal structure and physical properties is evaluated. The Si-based ternary clathrate with composition Ba8–δNixySi46–x–y is the subject of chapter 7. The phase relations and the homogeneity range are established. The crystal structure taking into account vacancies in the framework is discussed. Physical properties of bulk pieces are analyzed and the results are related to the sample composition. In addition, first-principles electronic structure calculations are carried out to assess variations in the electronic band structure, phase stability and chemical bonding.24 Chapter 8 reports on the intermetallic compound Ba3Si4,25, 26 which was encountered during the investigations on the Ba – Ni – Si phase diagram. The discussion covers issues related to preparation, crystal structure, phase diagram analysis, electrical and magnetic properties, NMR measurements, quantum mechanical calculations and oxidation to nanoporous silicon with gaseous HCl. Besides my contributions to the NoE CMA, I studied under the Priority Program 1178 of Deutsche Forschungsgemeinschaft “Experimental electron density as the key for understanding chemical interactions” with the project of “Charge distribution changes by external electric fields: investigations of bond selective redistributions of valence electron densities”. Chapter 9 deals with the preparation of chalcopyrites ZnSiP2 and CuAlS2 for experimental charge density analysis. Both phases show semiconducting properties and have non-centrosymmetric structures with high space group symmetry as needed to investigate the structural changes induced by external electric field. In this chapter, I describe the preparation and the crystal structure analyses of ZnSiP2 and CuAlS2 including issues related to the data collection as well as the results of NMR investigation.

The SUMO protein was named Small Ubiquitin-like MOdifier because its 3-D structure was similar to Ubiquitin. In human, three SUMO proteins were discovered, namely, SUMO-1/2/3. The recombinant ¡µ1-8, 94-95 SUMO-2 protein with 10 histidine residues at its N-terminus was expressed using E. coli. BL-21(DE3), purified at 4 oC and crystallized at room temperature. The surface properties of human SUMO-1/2/3 proteins and 3-D structure of ¡µ1-8, 94-95 SUMO-2 protein were analyzed using computer modeling and X-ray diffraction technology respectively. The two-step purification by immobilized metal ion affinity chromatography(IMAC) was developed to yield ¡µ1-8, 94-95 SUMO-2 protein that reached 60 mg/ml for crystallization. On protein expression, 120 mg protein was obtained from 6 L bacterial growth broth. Crystals of ¡µ1-8, 94-95 SUMO-2 were obtained by the hanging-drop vapor diffusion method and many different crystal forms were observed. One of single crystal with triangular plate polyhedron form diffracted to 1.6 Å resolution, the other one with rectangular polyhedron form diffracted to 1.2 Å. Analysis of the diffraction pattern suggests the crystals belong to R3 space group, the former one owned unit cell parameters a= b=75.3 Å, c=29.2 Å, £\=90¢X, £]=90¢X,£^=120¢X, and the later one owned unit cell parameters a= b=74.9 Å, c=33.2 Å and the same angles respectively. The R factor and Rfree of refinement are 0.133 and 0.190 with highly precise phase on 3-D structure of SUMO-2 protein. Comparison of crystal structure between human SUMO-2 and yeast SMT3 showed that the r.m.s. deviation of C£\ coordinate is 1.054 Å. In addition, comparison of SUMO-1 NMR structure and SMT3 crystal structure showed that the r.m.s. deviation of C£\ coordinates is 2.736 Å. Hence, the structures of SUMO-2 and SMT3 are more similar each other than those of SUMO-1and SMT3.

A molecule can crystallise in more than one crystal structure, a common phenomenon in organic compounds known as polymorphism. Different polymorphic forms may have significantly different physical properties, and a reliable prediction would be beneficial to the pharmaceutical industry. However, crystal structure prediction (CSP) based on the knowledge of the chemical structure had long been considered impossible. Previous failures of some CSP attempts led to speculation that the thermodynamic calculations in CSP methodologies failed to predict the kinetically favoured structures. Similarly, regarding the stabilities of co-crystals relative to their pure components, the results from lattice energy calculations and full CSP studies were inconclusive. In this thesis, these problems are addressed using the state-of-the-art CSP methodology implemented in the GRACE software. Firstly, it is shown that the low-energy predicted structures of four organic molecules, which have previously been considered difficult for CSP, correspond to their experimental structures. The possible outcomes of crystallisation can be reliably predicted by sufficiently accurate thermodynamic calculations. Then, the polymorphism of 5- chloroaspirin is investigated theoretically. The order of polymorph stability is predicted correctly and the isostructural relationships between a number of predicted structures and the experimental structures of other aspirin derivatives are established. Regarding the stabilities of co-crystals, 99 out of 102 co-crystals and salts of nicotinamide, isonicotinamide and picolinamide reported in the Cambridge Structural Database (CSD) are found to be more stable than their corresponding co-formers. Finally, full CSP studies of two co-crystal systems are conducted to explain why the co-crystals are not easily obtained experimentally.

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No description available.

Structure I and II gas hydrates were observed in the same sediment cores of a mud volcano in the Kukuy Canyon, Lake Baikal. The sII gas hydrate contained about 13-15% of ethane, whereas the sI gas hydrate contained about 1-5% of ethane and placed beneath the sII gas hydrate. We measured isotopic composition of dissociation gas from both type gas hydrates and dissolved gas in pore water. We found that ethane δD of sI gas hydrate (from -196 to -211 ‰) was larger than that of sII (from -215 to -220 ‰), whereas methane δ13C, methane δD and ethane δD in both hydrate structures were almost the same. δ13C of methane and ethane in gas hydrate seemed several permil smaller than those in pore water. These results support the following idea that the current gas in pore water is not the source of these gas hydrates of both structures. Isotopic data also provide useful information how the “double structure” gas hydrates formed.

A new synthetic methodology for the combinatorial preparation of C-terminus-modified Green and Red Fluorescent Protein chromophores is described. This method involves the modification of the previously reported [2+3] cycloaddition reaction scheme to incorporate new R2 groups in the imidate used in the final step. This is achieved through two primary routes: (a) the imidation of nitriles using hydrochloric acid gas and (b) the O-alkylation of amides using a variant of Meerwein's Salt to provide conjugated imidates. The preparation of fluorescent microcrystals and nanofibers from Green Fluorescent Protein chromophore derivatives via the reprecipitation method is also demonstrated. The properties of these microcrystals and nanofibers, especially in relation to the powder obtained from organic solvents, are also explored. Additionally, it is demonstrated that the size and shape of the microcrystals and nanofibers can be modulated with varying experimental conditions for RP. A new class of AIE-active GFP chromophores is reported. These chromophores contain a benzoxazole group on the phenyl ring and varying lengths of alkyl chains on the imidazolidinone nitrogen. These benzoxazole-based chromophores exhibit unique properties in the solid state not previously observed for GFP chromophore derivatives, namely, a broadening of the excitation spectrum and red-shifting of the emission, likely caused by excimer formation. The crystal structure also reveals a unique "hot-dog" stacking motif. Additionally, some projects which require further work are discussed at the end of the thesis. These include a stress-responsive GFP-based polymer and DNA-binding fluorophores.