It is well known that methane hydrate undergoes several phase transformations at high pressures. At room temperature and low to moderate pressure, methane and water form a stable cubic structure I (sI) hydrate that is also known as MH-I. The structure is transformed to a hexagonal phase (sH/MH-II) above 1.0GPa. Another phase transformation occurs above 1.9GPa where the filled ice structure (MH-III) is stable up to 40 GPa before a new high pressure phase transition occurs. Experiments at such high pressures have to be performed in a diamond anvil cell (DAC). Our main interest, though, is to form sH methane hydrate at a lower pressure than reported in previous studies but with some methane in the large cages consequently increasing the methane content. This can be accomplished by introducing the molecules of the large hydrate forming substance (tert-butyl methyl ether/TBME) at a concentration slightly below the stoichiometric amount as suggested by molecular dynamics simulations. In this study we have synthesized mixed methane hydrate of sI and sH and loaded the clathrate with methane into several DACs. Raman spectra were collected at room temperature and pressures in the range of 0.1 to 11.3 GPa. The existence of sH methane hydrate was observed down to 0.2 GPa. However, the existence of methane in the large cages was visible only at pressure higher than 1.0 GPa. The excess methane in the system apparently destabilizes the sH clathrate at pressure below 1.0 GPa as it transforms to sI clathrate.

13C NMR spectroscopic analysis was carried out to clarify the formed hydrate structure in specific conditions on hydrate phase diagram of ternary methane, neohexane, and water system. The obtained NMR spectra at three different conditions suggested that both structure I and H were formed simultaneously and coexisted at 273.6 K and 50 bar. But, for both conditions of 273.6 K, 25 bar and 283.1 K, 50 bar the formed hydrate was identified as structure H only. These results showed that the pure CH4 hydrate of structure I was formed and coexisted with mixed CH4+neohexane hydrate of structure H in low temperature and high pressure region after passing through the phase boundary of pure CH4 hydrate. We have examined the structure coexistence at 273.6 K and 50 bar with other structure H formers of isopentane, methylcyclopentane, and methylcyclohexane. In case of isopentane, the obtained NMR spectrum showed that structure I and H coexisted and the amount of methane molecules in structure I was two times as many as in cages of structure H. However, there were no resonance lines of structure I when methylcyclohexane formed structure H with methane molecules.

The presence of structure H (sH) methane hydrate in natural environments, in addition to the well-known structure-I (sI) and II (sII) hydrates, has recently been documented. Methane in the presence of condensates (C5-C7) forms sH hydrate at lower pressure than the sI hydrate. Thus, the occurrence of sH methane hydrate is likely to have both beneficial and negative practical implications. On the negative side, in the presence of condensate, sH hydrate may form and plug gas transmission pipelines at lower pressures than sI hydrate. On the other hand, sH hydrate can be synthesized at lower pressures and exploited to store methane. The existence of natural hydrates containing sH hydrate may also be expected in shallow offshore areas. There are at least 26 large guest molecules known as sH hydrate formers and each of them produces a sH hydrates with different properties. The hydrate stability, the cage occupancies and the rates of hydrate formation depend on the type of large molecule selected. Consequently, it is essential to understand how the host and the guest molecules interact. Studies at the molecular-level are therefore indispensable in providing information that is not obtainable from experiments or too costly to acquire. Free energy calculations are performed to determine the relative stability among different sH hydrate systems and the preferable cage occupancy. The latter would give indications of how much methane gas can be stored in the hydrate. The interaction of guest molecule inside the hydrate cage is also investigated. The results are related to the physical and chemical properties of gas hydrates observed from the experiments or reported in the literature.

Among the three common gas hydrate structures, structure-H (sH) hydrate has been regarded as forming only in the laboratory since it was first reported in 1987. However, natural gas hydrate samples obtained from the Cascadia margin showed that sH hydrate can form naturally. Not only was the sH hydrate found in natural samples, but it was also discovered that n-alkanes such as n-pentane and n-hexane, considered to have too large molecular size to be sH hydrate formers, can act as co-guests of sH hydrates in mixtures with other sH hydrate formers. In this study, thermodynamic measurements and spectroscopic analysis of powder X-ray diffraction and 13C solid-state NMR methods, were performed for synthetic hydrate samples in order to identify the accommodation of n-alkanes with five or more carbon atoms. In addition, some new hydrate guests were found to form sH hydrates. From the present results, it is clear that, so far, our understanding of gas hydrates and guest enclathration needs to be revised and expanded in order to explain new findings.

Some proteins require presence of their specific ligand, cofactor or prosthetic group for their activity. Binding of this specific molecule can cause conformational changes which permit to perform their function. In some occasions the identification of conformational changes could be really challenging task. In this thesis we describe the novel approach for monitoring structural changes in proteins using chemical cross-linking and high resolution mass spectrometry and its application on model calmodulin system. It is demonstrated that analysis using isotope-labelled cross-linking agents enabled us to get insight into the structural rearrangement caused by presence or absence of the protein ligand. However, it is shown that the method has potential drawback due to limited enzymatic proteolysis. The novel approach that also makes it possible to quantify the changes in protein structure was used together with other methods for characterization of the neutral trehalase Nth1 in complex with Bmh1 protein (yeast isoform of protein 14-3-3). The results revealed that Bmh1 induce structural rearrangement of Nth1 molecule with changes within the EF- hand like motif which is essential for the activation process.