The appropriate lipid environment is crucial for the proper function of membrane proteins. There is a tremendous variety of lipid molecules in the membrane and so far it is often unclear which component of the lipid matrix is essential for the function of a respective protein. Lipid molecules and proteins mutually influence each other; parameters such as acyl chain order, membrane thickness, membrane elasticity, permeability, lipid-domain and annulus formation are strongly modulated by proteins. More recent data also indicates that the influence of proteins goes beyond a single annulus of next-neighbor boundary lipids. Therefore, a mesoscopic approach to membrane lipid-protein interactions in terms of elastic membrane deformations has been developed. Solid-state NMR has greatly contributed to the understanding of lipid-protein interactions and the modern view of biological membranes. Methods that detect the influence of proteins on the membrane as well as direct lipid-protein interactions have been developed and are reviewed here. Examples for solid-state NMR studies on the interaction of Ras proteins, the antimicrobial peptide protegrin-1, the G protein-coupled receptor rhodopsin, and the K+ channel KcsA are discussed.

Inflammatory bowel disease is a serious condition with an incomplete etiology and pathogenesis. In this thesis, a mouse model of sodium dextran sulfate-induced inflammation was used to study different changes in the metabolism of germ-free and conventionally raised mice due to the development of the inflammatory process. NMR metabolomics of fecal, urine and serum samples, combined with uni- and multivariate statistical analysis, were used to characterize the changes. It was shown that the metabolic signature differs between germ-free and conventional mice. In germ-free mice, significant amounts of carbohydrates were found in feces. Their levels decreased during inflammation as they were excreted in urine. In contrast to conventional mice, germ-free mice also excreted large amounts of amino acids in feces during the developing inflammation. Disorders of sugar and protein metabolism found in germ-free mice indicate severe malnutrition caused by inflammation. The results show that the presence of a microbiome represents a protective mechanism against significant disruption in the body. A stability study of fecal extracts of healthy conventionally colonized mice confirmed that none of the identified and quantified metabolites showed significant systemic changes in several consecutively collected...

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Arctic soils contain a large fraction of our planets terrestrial carbon (C) pool. When tundra soils become warmer and permafrost thaws, non-native geoengineering earthworms can enter these soils and ingest organic matter accumulated over long timescales. Previous studies have found that earthworms increase mineralization rates of soil organic matter into carbon dioxide (CO2) when introduced. Yet, this initial mineralization boost seems transient with time and it has been hypothesized that earthworms stimulate the formation of persistent C forms. In this study, I investigated how non-native, geoengineering earthworms affected the relative proportions of seven carbon forms in the O and A1 horizon of tundra soil and if their effect induced a change in pH. I used Nuclear Magnetic Resonance (NMR) spectroscopy to understand what happens to soil carbon compounds in two different tundra vegetation types (heath and meadow), that had been subjected to earthworm treatment for three summers. I found that O-aromatic C increased from 7.22% ± 0.24 (mean ± stderr) in the meadow soil lacking earthworms to 8.98% ± 0.30 in the meadow exposed to earthworms, and that aromatic C increased from 8.71% ± 0.23 to 9.93% ± 0.25. In similar, the result suggested that alkyl C decreased in this vegetation type from 20.43% ± 0.38 to 18.70% ± 0.25 due to earthworm activities. I found no effect on the chemical properties in the heath. I conclude that geoengineering earthworms affect the two vegetation types differently and that earthworms seem to enhance the accumulation of recalcitrant aromatic C forms.

No description available.

While Li-ion batteries (LIBs) are the prevailing electrochemical energy storage technology, development of batteries using earth abundant alkali metals (e.g., Na and K) is necessary to alleviate LIB supply chain concerns. K-ion batteries (KIBs) offer a compelling advantage over Na via their compatibility with commercialized graphite anodes, and therefore may be more readily adopted within existing battery production lines. K-ions present some inherent advantages as well, such as rapid diffusion and low energy barriers to desolvation in the battery electrolyte that may enable fast charging. Presently, research on KIBs is in early stages and it is unclear if the same battery design principles produced by decades of study on LIBs apply to KIBs. Here, I examine structure-performance relationships in KIB anodes and electrolytes to propose broad design rules. In the first chapter, I summarize the motivations and prominent advancements in materials used for KIBs, providing commentary on the direction of the field. I begin by summarizing present concerns over materials criticality facing LIBs and how KIBs address these concerns but do not necessarily achieve lower costs. I continue with a summary of popular materials choices for KIB anodes, cathodes, and electrolytes. I place particular emphasis on the discovery and development of graphite anodes and the advantages of using a weak Lewis acid such as K-ions in batteries. Finally, I discuss the challenges presented by using highly reactive K metal anodes in research. In the second chapter, I examine the mechanisms of potassiation/depotassiation of two high-capacity tin phosphide anodes, Sn₄P₃ and SnP₃, and discuss possible failure modes. Ex situ 31P and 119Sn solid-state nuclear magnetic resonance (NMR) analyses reveal that both Sn₄P₃ and SnP₃ exhibit phase separation of elemental P and the formation of KSnP-type environments (which are predicted to be stable based on DFT calculations) during potassiation, while only Sn₄P₃ produces metallic Sn as a byproduct. In both anode materials, K reacts with elemental P to form K-rich compounds containing isolated P sites that resemble K₃P, but K does not alloy with Sn during potassiation of Sn₄P₃. During charge, K is only fully removed from the K3P-type structures, suggesting that the formation of ternary regions in the anode and phase separation contribute to capacity loss upon reaction of K with tin phosphides. The third chapter addresses the use of fluorinated electrolyte additives in KIBs. Fluoroethylene carbonate (FEC) is a well-known additive used in Li-ion electrolytes, because the products of its sacrificial decomposition aid in forming a stable solid electrolyte interphase (SEI) on the anode surface. Here, we show that FEC addition to KIBs containing hard carbon anodes results in a dramatic decrease in capacity and cell failure. Using a combination of 19F solid-state NMR spectroscopy, X-ray photoelectron spectroscopy (XPS), and electrochemical impedance spectroscopy (EIS), we show that FEC decomposes during galvanostatic cycling to form insoluble KF and K₂CO₃ on the anode surface, which correlates with increased interfacial resistance in the cell. Our results strongly suggest KIB performance is sensitive to accumulation of an inorganic SEI, likely due to poor K transport in these compounds. The fourth chapter presents a nonflammable electrolyte mixture for use in KIBs. In this report, we show that a low-concentration (1 M) KPF6 electrolyte combining ethylene carbonate (EC), propylene carbonate (PC), and triethyl phosphate (TEP) is nonflammable, retains high ionic conductivity, and is compatible with graphite. Notably, we then show that this electrolyte is only usable in KIBs; the analogous Li electrolyte fails immediately due to the incompatibility of Li, PC, and graphite. We continue the study by characterizing the impact of TEP on the graphite interphase using a combination of EIS, XPS, and 1D and 2D NMR spectroscopy. We show that, compared to using EC/PC alone, the addition of TEP reduces resistance of the SEI layer, lessens reductive decomposition of carbonates to soluble organic species, and produces inorganic phosphate salts (that we posit contribute to passivation in lieu of fluorination in the SEI). The fifth chapter concludes by summarizing the design strategies learned in each of the preceding three chapters and makes recommendations for future studies. The proposed research emphasizes the need for fundamental studies on materials properties in KIBs, contradicting the current push towards optimizing capacity and longevity of KIBs to prove their relevance. Doing so will not only inform how to design high-performance batteries, but potentially uncover distinct advantages of KIBs that complement existing LIB technologies.

Résumé : Les personnes atteintes de la maladie d’Alzheimer présentent une diminution de la capture cérébrale du glucose, qui semble être impliquée dans le développement des problèmes cognitifs associés à la maladie. Toutefois, il n’y a encore aucun consensus quant à savoir si la capture cérébrale du glucose est diminuée chez les personnes âgées cognitivement saines. En condition de déficit de glucose, les cétones sont le substrat énergétique alternatif pour le cerveau. La diète cétogène, induisant une cétose légère, améliore les fonctions cognitives chez les modèles animaux et chez l’homme. Notre premier objectif était d’évaluer l’effet du vieillissement sain et de la diète cétogène sur la capture cérébrale du glucose et des cétones chez le rat. Pour cela, la capture cérébrale de radiotraceurs analogues au glucose et aux cétones a été mesurée par tomographie par émission de positons. Nos résultats montrent que la capture des deux principaux substrats énergétiques du cerveau est globalement similaire chez des rats sains jeunes et âgés, mais est plus élevée suite à la diète cétogène. L’induction d’une cétose légère pourrait corriger la diminution de capture cérébrale du glucose subvenant au cours de la maladie d’Alzheimer. Le second objectif était de déterminer l’effet d’une diète cétogène sur le métabolisme cérébral du glucose et des cétones chez le rat. Pour cela, les différents intermédiaires des voies métaboliques du glucose et des cétones ont été mesurés par spectroscopie par résonance magnétique nucléaire. Les résultats démontrent que le métabolisme du glucose et des cétones dans les cellules du cerveau est plus élevé suite à la diète cétogène. Le contenu en acide [gamma]-aminobutyrique, le principal neurotransmetteur inhibiteur, est aussi plus élevé suite à la diète cétogène, ce qui pourrait contribuer à l’effet antiépileptique de la diète cétogène. Le troisième objectif était d’évaluer l’impact d’une restriction calorique à long terme, pouvant induire une cétose légère, sur le métabolisme cérébral chez des rats âgés sains. Nos résultats montrent que, couplée à une diète à haute teneur en sucrose et faible en acides gras oméga-3, la restriction calorique à long terme chez les rats âgés ne modifie pas le profil des métabolites et des acides gras du cerveau. La déficience en acides gras oméga-3 et la surcharge de sucrose pourraient empêcher une grande partie des effets bénéfiques de la restriction calorique au cerveau.//Abstract : Alzheimer’s disease is associated with a reduction of brain glucose uptake, which may be involved in the development of the cognitive problems associated with the disease. It is however unclear whether brain glucose uptake is decreased in the cognitively healthy elderly. Under conditions of glucose deficit, ketones are the alternative brain energy substrate. A mild ketosis, induced by the ketogenic diet, improves cognitive functions in animal models and humans. Our first objective was to evaluate the effect of healthy aging and of a ketogenic diet on brain glucose and ketone uptake in the rat. Brain uptake of radiotracers analogous to glucose and ketones was measured by positron emission tomography. Our results show that the uptake of the brains two main energy substrates is generally similar in healthy young and aged rats, but is higher under the ketogenic diet. The induction of a mild ketosis may compensate the reduction of brain glucose uptake occurring in Alzheimer’s disease. The second objective was to assess the effect of a ketogenic diet on brain glucose and ketone metabolism in the rat. Metabolic pathway intermediates of glucose and ketones were measured by nuclear magnetic resonance spectroscopy. Results show that glucose and ketone metabolism in brain cells is higher under the ketogenic diet. Content of [gamma]-aminobutyric acid, the main inhibitory neurotransmitter, is also higher under the ketogenic diet, which could contribute to the antiepileptic effect of the ketogenic diet. The third objective was to evaluate the effect of a long-term calorie restriction, which may induce a mild ketosis, on brain metabolism in healthy aged rats. Our results show that, in conjunction with a diet enriched in sucrose and low in omega-3 fatty acids, long-term calorie restriction in aged rats does not change brain metabolite and fatty acid profiles. Omega-3 fatty acid deficiency and an overload of sucrose may prevent the beneficial effects associated with calorie restriction in the brain.

The objectives of the research were to examine the effects of high dietary levels of fibrous feeds, and of supplementation with Roxazyme® G2 (RX), on the digestive metabolic and physiological responses of growing pigs fed maize-soybean diets. The nutrient and dietary fibre (DF) composition, the swelling and water-binding capacities of maize (MM), its hominy chop (HC) and cobs (MC), dehulled soybean (dSBM) and the hulls (SH), brewer’s grains (BG), lucerne hay (LH) and wheat bran (WB) were evaluated using standard procedures. Feed fibre fractions were isolated by simulating upper tract digestion in an Ankom® DaisyII Incubator, whereby each feed was digested in pepsin (porcine, 200 FIP-U/g, Merck No, 7190), followed by pancreatin (porcine, grade IV, Sigma No P-1750), with recovery of the fibrous residues. In a third step to complete the simulated pig gastro-intestinal digestion, the pepsin-pancreatin fibre extracts were digested by RX or Viscozyme L ® V2010 (VZ). Enzyme activity was measured as the coefficients of partial degradability (solubilisation) of the washed fibre extracts. The kinetics and products of fermentation of the DF were evaluated in an AnkomRF gas production system, using buffered faecal inoculum. Among the feed ingredients, dissimilar, fibre source-dependent activities between RX (0.02 to 0.12) and VZ (0.04-0.33) were observed. The lowest RX activities were observed on the maize and soybean derived fibres, with similarly low VZ activity on MC fibre. Variation in the activity of faecal microbial enzymes was similarly indicated by the variable production of fermentation gas (51.8-299.4 mL g-1 DM) and short chain fatty acids (SCFA) (2.3-6.0 mMol g-1 DM). Soy hull, dSBH, MM and HC fibres were highly fermentable, with low fermentability of BG, MC and WB fibres. The fibres differed in the composition of fermentation SCFA, whereby SH, LH and MC shifted fermentation to Ace, and BG, dSBM, WB, MM, HC favoured Pro, while MM and HC favoured But production. The same nutritional properties were similarly evaluated in complete diets which were formulated from the ingredients for growth, and metabolic trials. For the growth trial, a standard (STD) (control), 141 g total dietary fibre (TDF) kg-1 dry matter (DM) maize-soybean growing pig diet, and five iso-nutritive, 246 g TDF kg-1 DM nutritionally balanced diets were formulated. The high DF was achieved by partial replacement of the MM and dSBM in the STD diet with MC, SH, BG, LH or WB. The differences in RX and VZ activities and in the fermentation characteristics which were observed on the fibre extracts from the high fibre ingredients were reflected in the DF from the respective complete diets in which they were included. However, the fibre from the basal dietary ingredients reduced the absolute values and the variation in the activities of RX (0.03-0.06) and VZ (0.16-0.22), and similarly reduced the variation in gas (126.6-187.6 mL g-1 DM) and SCFA (4.1-5.4 mMol g-1 DM) production of the DF from the fibrous diets. Enzyme activities on the STD DF were low for RX (0.03) and high for VZ (0.25). The STD DF produced 205.3 mL gas g-1 DM, which was similar to SH DF, and higher than all the other diets. The STD DF produced 5.0-mMol SCFA g-1 DM, which was quantitatively, and not statistically higher than the other fibres. The composition of SCFA was similar across all diets, except for the high percent Ace, with low Pro by the SH DF. Compared to the STD, the high DF diets increased percent Ace, with reduced Pro and But. The STD, MC, SH, BG, LH and WB diets were each prepared in duplicate mixes, one of which was fortified with 200 mg RX kg-1 feed (as fed). Seventy-two intact Large White X Landrace, male, 32.0 ± 5.6 kg live weight (LW) pigs were allocated to the diets in two completely randomised weight blocks in a 2 (fibre source) X 2 (enzyme) factorial arrangement. The pigs were fed ad libitum for 10 weeks. Cumulative LW gain and feed intake were measured at different stages of growth, and at slaughter. Apparent total tract digestibility (ATTD) of nutrients was estimated at 65-70 kg LW, using 0.2% (as fed) chromium oxide as the indigestible marker. Ileal tissue was sampled 50 cm above the ileo-caecal valve, on which villi height and area, and crypt depth were evaluated by computerised image analysis. Blood was sampled at slaughter from the severed vena jugularis, 16 hours after feeding. Serum urea, creatinine, triglycerides, glucose, and total cholesterol were analysed chemically. The serum metabolome was further explored using Proton Nuclear Magnetic Resonance Spectroscopy (1H -NMRS). There was fibre X RX interaction for villi height, whereby the enzyme reduced the villi height in pigs on the SH, STD and WB diets, with an opposite effect on pigs on the MC, BG, LH diets. The soluble fibre content was negatively correlated with crypt depth. Chemical analysis did not detect differences in metabolite concentration between the STD and the high fibre diets. However, more serum cholesterol was observed in pigs fed the WB compared to the LH and MC diets. 1H-NMRS indicated that feeding pigs the WB diet increased serum Cys and His, while supplementation of RX increased serum formate, glucose, and urea. There was diet X enzyme interaction for fructose, glucose, Arg, Cys, Ser, and Trp, whereby RX increased the levels in pigs on MC and WB, with an opposite effect in pigs on the other diets. There was large DF source-dependent variation among diets in ATTD of DM (0.80-0.85), organic matter (OM) (0.81-0.87), gross energy (GE) (079-0.85) and CP (0.81-0.85), whereby, relative to the STD diet, high DF reduced the ATTD of DM (all diets except SH), organic matter (OM) and energy and CP (all diets except the MC). Positive correlation was observed between fermentability and the ATTD digestibility of DM, OM, energy, ADF, NDF, and fat. Negative correlation was observed between the swelling capacity and the ATTD of DM, OM, energy and protein, between DF solubility and DM, OM, protein, ADF and NDF, and between water binding capacity and ATTD of DM and OM, energy and NDF. At slaughter, there was similarly large, and DF source-dependent variation among the high fibre diets in feed intake (2.31-2.71 kg as fed day-1), live weight gain (0.75-0.86 kg day-1), and feed: gain ratio (2.73-3.00). Corresponding values for the STD diet were 2.44 kg day-1, 0.83 kg day-1and 2.86 kg day-1, respectively. Relative to the STD, LH reduced feed intake and live weight gain, and MC increased the feed: gain ratio. Predictions based on the in vitro fermentability of DF and feed intake suggested that due to poor fermentability, and or restriction of feed intake, relative to a standard fibre diet, high dietary levels of MC, WB and BG may reduce fermentation in the lower gut, while similar dietary levels of SH and LH may result in substantial increases in fermentation. At 50 kg LW, the fermentability of DF was positively correlated with feed intake and with weight gain, while water binding capacity and solubility of DF were negatively correlated with feed intake. At slaughter, the solubility of DF was negatively correlated with feed intake and feed: gain ratio. Large variation among the high fibre diets was also observed in the slaughter weight (89.2-96.8 kg), dressing % (68.6-76.4), meat colour (80.4-82.3), lean % (69.5-71.2), and fat % (10.1-12.6). In comparison, pigs on the STD diet scored 94.7 kg slaughter weight, 75.1% dressing, 81.6 cm carcass length, 82.5 meat colour, 68.4% lean, and 15.0% fat. Relative to the STD, LH reduced dressing and fat %. Lucerne hay and WB increased the lean%. For the metabolic trial, two iso-nutritive, mixed high fibre (319 g TDF kg-1 DM), nutritionally balanced diets were formulated to contain DF of high (HF) versus low (LF) fermentability. The diets had similar content of soluble DF and similar swelling and water binding capacities. Viscozyme was more active than RX on both the HF (0.20 versus 0.04) and the LF (0.17 versus 0.07) DF. The combination of RX and VZ statistically increased the enzyme activity on the HF (0.25) and quantitatively increased enzyme activity on the LF (0.18) DF, suggesting additive or synergistic effects. More gas was produced by the HF (159.5 mL g-1 DM) compared to the LF DF (96.6 mL g-1 DM). More SCFA were produced by HF (5.0 mMol g-1 DM), compared to the LF DF (3.6 mMol g-1 DM). Compared to the STD, HF DF increased percent Ace, with reduced Pro and But. The LF DF increased percent Ace, with quantitative, and not statistical reduction of Pro and But. In a metabolic trial, the HF and LF diets, and their duplicates containing 0.270 g RX kg-1 DM of feed (as fed) were fed ad libitum to eight ileum T-cannulised, intact Large White X Landrace male pigs weighing 65.0 ± 5.1 kg. The diets were allocated to the pigs in a duplicate 4 x 4 Latin Square design, in a 2 (enzyme) x 2 (fermentability) factorial arrangement. Each period consisted of two weeks of adaptation followed by five days of sampling. The ileal digesta was collected in each period and was similarly subjected to the fermentation test. Apparent ileal digestibility (AID) and ATTD were determined using 0.2% (as fed) chromium oxide as the indigestible marker. N excretion in faeces and urine were measured, and N retention was calculated. Blood was sampled by vena jugularis puncture on the last day of each period. Two blood samples were collected, the first 15 hours after removal from feed (15-hour serum), and the second 3 hours after re-introduction to feed (3-hour serum). Serum metabolites were evaluated by both chemical analyses and by 1H-NMRS, as described for the growth trial. Roxazyme did not affect the fermentation characteristics of the ileal digesta. In similar proportion to the fermentability of the PP digesta, the HF ileal digesta was more fermentable (65.4 mL gas g-1 DM and 6.1 mMol SCFA g-1 DM) than the LF ileal digesta (46.7 mL gas g-1 DM and 4.4 mMol SCFA g-1 DM SCFA). Prediction based on the in vitro fermentability of DF and feed intake suggested the HF diet could support one half times more fermentation in the lower gut compared to the LF diet. The HF diet had higher AID of DM (62.5 vs. 58.6), OM (65.6 vs. 62.1), energy (64.4 vs. 61.0), fat (85.8 vs. 81.7) and ash (41.8 vs. 32.7). The AID of HO-Pro, Met and Val were higher for the LF diet. There was diet X enzyme interaction on the AID of Met, whereby the RX reduced the AID of met in the LF diet, and not that of the HF diet. The ATTD was higher for the HF diet for DM (74.2 vs. 68.4), NDF (64.7 vs. 57.4), and ADF (35.1 vs. 21.0). There was positive correlation between the fermentability of DF and the AID DM, OM, ash, ash, fat and energy. The solubility of DF was negatively correlated with the AID of DM, OM, ash, fat, ADF and energy, and with the ATTD of DM, OM, ash, fat, energy, NDF, and ADF. Negative correlation was also observed between the swelling capacity of DF and the AID of protein, Trp and Lys. The solubility of DF was positively correlated with Ser, Ala, Val, Iso-Leu and His. There was diet X enzyme interaction for urea in the 15- hour serum, whereby RX tended to reduce the urea in the LF diet, while it increased that of the HF diet. Fermentability negatively correlated with urea in the 15- hour serum, and positively correlated with serum glucose in the 3-hour serum. In the 3-hour sample, 1H-NMRS indicated higher fucose, Pro and cholesterol in the LF diet. 1H-NMRS also indicated fermentability x RX interaction for Ser, Tyr, Lys, creatine, and possibly, glucose or fructose, glycerol or Gly and His or Arg, whereby RX increased the levels in the LF diets, with opposite effect in the HF diet. In conclusion, enzyme activities and fermentability were higly variable among different DF sources, and the effects were evident in the fibrous complete diets. The results of the in vitro studies supported the application of the methods to formulate fermentable insoluble fibre-rich, maize-soybean-mixed co-product diets. Correlation analyses suggested that DF fermentability, and solubility, swelling and water binding capacities explained significant proportions of the variances of the metabolic and physiological responses of the pigs to different feeds. Predictions based on the in vitro fermentability of DF and feed intake suggested that a strategy whereby pig diets are enriched in DF after the feedstuffs are screened on DF fermentability could substantially increase fermentation in the lower gut. Overall, the results suggested that productivity can be maintained in growing pigs fed diets containing up to twice the standard levels of DF, provided producers target co-product feeds that contain highly fermentable DF. The use of RX to improve nutrient digestion and to stimulate gut fermentation was not justified. / Environmental Sciences / Ph.D. (Environmental Sciences)

The HIV-1 pandemic affects millions of people worldwide with approximately 70% of those affected residing in sub-Saharan Africa (SSA) relying on traditional medicines for treatment. The key aim of the study was to isolate and characterise an active phyto-pharmaceutical ingredient (API) from L. trigonus for use as a vaginal microbicide. The aerial parts of L. trigonus were oven-dried at 80°C, ground and then extracted with boiling water for 30 minutes. Aqueous extracts were screened using an HIV-1 neutralization assay in TZM bl cells. Chromatographic and spectroscopic techniques were used to purify, isolate and identify the API. The API (BP36-117-26464C) was identified as a polymeric macromolecule with IC50 = 0.04 μg/ml against HIV-1 HXB 2 subtype B. This activity is comparable to the ARV drug, enfuvirtide (IC50 = 0.02 μg/ml). The API consists of galacturonic acid polymer and a mixture of seven compounds. Its mode of action may involve inhibiting virus attachment. The activity of this precipitate (BP36-117-26464C) tested against HIV-1 subtype C pseudovirions and shown to compare favorably with that of enfuvirtide (T20). The water-soluble nature of this API and its mode of action identified it as a potential microbicide. In the current form, the precipitate (API) would be difficult to develop as an oral treatment for HIV, as high-molecular weight agents often have poor bioavailability following oral administration. However, large molecules with potent anti-HIV activity are ideal for topical use and potent development as a microbicide. / Life & Consumer Sciences / M.Sc (Life Sciences)

The human α-lactalbumin (α-LA) molten globule formed at low pH is a model for the study of protein folding intermediates. The molten globule lacks native-like side-chain interactions, resulting in a fluctuating ensemble of tertiary structures, characterisation of which has been precluded by severe line-broadening in NMR spectra and a lack of long-range NOEs. Paramagnetic relaxation enhancements (PREs) have been measured in a variant of α-LA in which all native cysteines have been mutated to alanine (all-Ala α-LA). Cysteine residues have been mutated into regions of interest and spin labelled with MTSL. These measurements have confirmed that all-Ala α-LA forms a compact molten globule. Transient, long-range interactions that are stabilising the compact fold have also been identified using PREs measured in urea-denatured states. This has identified several interactions formed by hydrophobic residues from both the α- and β-domain, which could be important for initiating and driving folding. The molten globule’s 3D topology has been probed by measuring long-range distances between MTSL pairs using Double Electron-Electron Resonance (DEER). Broad distance distributions have been identified between elements of secondary structure, indicative of a fluctuating but compact fold. By contrast, a narrower distance distribution has been measured within one of the major helices, indicative of native-like secondary structure. The surface accessibility of all-Ala α-LA and that of two other variants ([28-111] α-LA and 4SS α-LA) has been probed using solvent PREs obtained using TEMPOL, a paramagnetic co-solute. This has revealed differences in the solvent-exposure of hydrophobic residues due to the removal of disulphide bonds. This method has also identified buried hydrophobic residues that contribute to forming the molten globule’s stable, native-like core.