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The human society of today has a significantly negative impact on the environment and needs to change its way of living towards a more sustainable path if to continue to live on a healthy planet. One path is believed to be an increased usage of naturally degradable and renewable raw materials and, therefore, attention has been focused on the highly abundant biopolymer cellulose. However, a large drawback with cellulose-based materials is the significant change of their mechanical properties when in contact with water. Despite more than a century of research, the extensively investigated interaction between water and cellulose still possesses many unsettled questions, and if the answer to those were known, cellulose-based materials could be more efficiently utilized. It is well understood that one interaction between cellulose and water is through hydrogen bonds, established between water and the hydroxyl groups of the cellulose. Due to the very similar properties of the hydroxyl groups in water and the hydroxyl groups of the cellulose, the specific interaction-induced effect on the hydroxyl groups at a cellulose surface is difficult to investigate.  Therefore, a method based on 2H MAS NMR spectroscopy has been developed and validated in this work. Due to the verified ability of the methodology to provide site-selective information regarding the molecular dynamics of the cellulose deuteroxyl groups (i.e. deuterium-exchanged hydroxyl groups), it was shown by investigating 1H-2H exchanged cellulose samples that only two of the three accessible hydroxyl groups (on the surface of cellulose fibrils) exchange with water. This finding was also verified by FT-IR spectroscopy, and together with MD simulations we could establish that it is O(2)H and O(6)H hydroxyl groups (of the constituting glucose units) that exchange with water. From the MD simulations additional conclusion could be drawn regarding the molecular interactions required for hydrogen exchange; an exchanging hydroxyl group needs to donate its hydrogen in a hydrogen bond to water. Exchange kinetics of thin cellulose films were investigated by monitoring two different exchange processes with FT-IR spectroscopy. Specific information about the two exchanging hydroxyl/deuteroxyl groups was then extracted by deconvoluting the changing intensities of the recorded IR spectra. It was recognized that the exchange of the hydroxyl groups were well described by a two-region model, which was assessed to correspond to two fibrillary surfaces differentiated by their respective positions in the fibril aggregate. From the detailed deconvolution it was also possible to estimate the fraction of these two surfaces, which indicated that the average aggregate of cotton cellulose is built up by three to four fibrils.                       2H MAS NMR spectroscopy was used to examine different states of water in cellulose samples, hydrated at different relative humidities of heavy water. The results showed that there exist two states of water adsorbed onto the cellulose, differentiated by distinct different mobilities. These two states of water are well separated and had negligible exchange on the time scale of the experiments. It was suggested that they are located at the internal and external surfaces of the fibril aggregates. By letting cellulose nanofibrils undergo an epoxidation reaction with a mono epoxide some indicative results regarding how to protect the cellulose material from the negative impact of water were presented. The protecting effect of the epoxidation were examined by mechanically testing and NMR spectroscopy. It was proposed that by changing the dominant interaction between the fibril aggregates from hydrophilic hydrogen bonds to hydrophobic π-interactions the sensitivity to moisture was much reduced. The results also indicated that the relative reduction in moisture sensitivity was largest for the samples with highest moisture content. / <p>QC 20161229</p>

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<p>This thesis is based on studies, described in four papers, in which the folding energy landscape of MerP was investigated by various techniques. MerP is a water-soluble 72 amino acid protein with a secondary structure consisting of four anti-parallel β-strands and two α-helices on one side of the sheet in the order β1α1β2β3α2β4. </p><p>The first paper describes the use of CD and fluorescence analysis to examine the folding/unfolding process of MerP. From these experiments it was found that the protein folds according to a two-state model in which only the native and unfolded forms are populated without any visible intermediates. With a rate constant of 1.2 s^-1, the folding rate was found to be unusually slow for a protein of this size.</p><p>The studies presented in the second and third papers were based on measurements of native-state amide proton exchange at different temperatures (Paper II) and GuHCl concentrations (Paper III) in the pre-transitional region. In these studies partially unfolded forms were found for MerP which are essentially unrelated to each other. Thus, in the folding energy landscape of MerP, several intermediates seem to occur on different folding trajectories that are parallel to each other. The slow folding rate of MerP might be coupled to extensive visitation of these conformations. Hydrogen exchange in MerP did also reveal structure-dependent differences in compactness between the denatured states in GuHCl and H₂O.</p><p>In the last paper multivariate data analysis was applied to 2-dimensional NMR data to detect conformational changes in the structure of MerP induced by GuHCl. From this analysis it was suggested that regions involved in the most flexible part of the protein structure are disrupted at rather low denaturant concentrations (< 2.1 M GuHCl) while the native structures of the most stable parts are still not completely ruptured at 2.9 M GuHCl.</p><p>Finally, the stability, kinetics, contact order and folding nuclei of six proteins with similar topology (MerP, U1A, S6, ADA2h, AcP and HPr) were compared. In this analysis it was found that their folding properties are quite diverse, despite their topological similarities, and no general rules that have been formulated yet can adequately predict their folding behaviour.</p>

Human thiopurine methyltransferase (TPMT) is an enzyme involved in the metabolism of thiopurine drugs, which are widely used in leukemia and inflammatory bowel diseases such as ulcerative colitis and Crohn´s disease. Due to genetic polymorphisms, approximately 30 protein variants are present in the population, some of which have significantly lowered activity. TPMT *5 (Leu49Ser) is one of the protein variants with almost no activity. The mutation is positioned in the hydrophobic core of the protein, close to the active site. Hydrogen exchange rates measured with NMR spectroscopy for N-terminally truncated constructs of TPMT *5 and TPMT *1 (wild type) show that local stability and hydrogen bonding patterns are changed by the mutation Leu49Ser. Most residues exhibit faster exchange rates and a lower local stability in TPMT *5 in comparison with TPMT *1. Changes occur close to the active site but also throughout the entire protein. Calculated overall stability is similar for the two constructs, so the measured changes are due to local stability. Protein dynamics measured with NMR relaxation experiments show that both TPMT *5 and TPMT *1 are monomeric in solution. Millisecond dynamics exist in TPMT *1 but not in TPMT *5, even though a few residues exhibit a faster dynamic. Dynamics on nanosecond to picosecond time scale have changed but no clear trends are observable.

This thesis is based on studies, described in four papers, in which the folding energy landscape of MerP was investigated by various techniques. MerP is a water-soluble 72 amino acid protein with a secondary structure consisting of four anti-parallel β-strands and two α-helices on one side of the sheet in the order β1α1β2β3α2β4. The first paper describes the use of CD and fluorescence analysis to examine the folding/unfolding process of MerP. From these experiments it was found that the protein folds according to a two-state model in which only the native and unfolded forms are populated without any visible intermediates. With a rate constant of 1.2 s-1, the folding rate was found to be unusually slow for a protein of this size. The studies presented in the second and third papers were based on measurements of native-state amide proton exchange at different temperatures (Paper II) and GuHCl concentrations (Paper III) in the pre-transitional region. In these studies partially unfolded forms were found for MerP which are essentially unrelated to each other. Thus, in the folding energy landscape of MerP, several intermediates seem to occur on different folding trajectories that are parallel to each other. The slow folding rate of MerP might be coupled to extensive visitation of these conformations. Hydrogen exchange in MerP did also reveal structure-dependent differences in compactness between the denatured states in GuHCl and H2O. In the last paper multivariate data analysis was applied to 2-dimensional NMR data to detect conformational changes in the structure of MerP induced by GuHCl. From this analysis it was suggested that regions involved in the most flexible part of the protein structure are disrupted at rather low denaturant concentrations (&lt; 2.1 M GuHCl) while the native structures of the most stable parts are still not completely ruptured at 2.9 M GuHCl. Finally, the stability, kinetics, contact order and folding nuclei of six proteins with similar topology (MerP, U1A, S6, ADA2h, AcP and HPr) were compared. In this analysis it was found that their folding properties are quite diverse, despite their topological similarities, and no general rules that have been formulated yet can adequately predict their folding behaviour.

(βα)8 TIM barrel proteins are one of the most common structural motifs found in biology. They have a complex folding free energy landscape that includes an initial off-pathway intermediate as well as two on-pathway intermediates. The formation of these intermediates is hypothesized to be driven by large clusters of the branched chain amino acids, isoleucine, leucine, and valine (ILV). All-atom MD simulations and circular dichroism experiments on polar mutants of the hydrophobic clusters of α-Trp synthase, a TIM barrel protein, revealed the importance of dehydrating the clusters on intermediate states. Custom, single-piece microfluidic chips were interfaced with small angle x-ray scattering and time resolved FRET experiments to monitor the role of a large ILV cluster on the microsecond timescale in a second TIM barrel protein, sIGPS. Dimensional analysis of the initial misfolded intermediate showed an ILV cluster was responsible for the initiation of structure in the intermediate. Early structure formation in the ILV cluster was confirmed by coarse grained simulations. Native state hydrogen exchange experiments were used to probe the higher energy species that are in equilibrium with the native state. Results from the NMR experiment complement the kinetic studies as the core of stability found by NMR mapped back to the same region of the ILV cluster that was found to initiate folding. When taken together, the results show the importance of hydrophobic clusters on the entire free energy surface of TIM barrel proteins.

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