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Fluorescence spectroscopy and imaging have a very broad spectrum of applicationswithin the life sciences, in particular for detection and characterization ofbiomolecular dynamics and interactions in different environments. This thesis comprisesprojects that strive to further expand the information content extracted fromthe detected fluorescence, leading to sensitive readout parameters for studies ofbiomolecular dynamics and interactions. Two major strategies are presented toachieve this aim. The first strategy is based on the expansion of the availablereadout parameters beyond the "traditional" fluorescence parameters: intensity,wavelength, polarization and fluorescence lifetime. The additional parameters arebased on blinking properties of fluorescent labels. In particular on transitions betweensinglet and triplet states, and transitions between the trans- and cis-isomersof fluorophores. Two publications in the thesis are based on this strategy (paperI and IV). The second strategy is based on the utilization of fluorescence intensityfluctuations in order to detect the oligomerization mechanisms of fluorescentlylabeled peptides and proteins. This strategy combines the intensity fluctuationanalysis and the readout of distance dependent energy transfer between fluorescentmolecules together with the correlation analysis of fluorescence from two labeledproteins emitting at different wavelengths. Another two publications presented inthe thesis are based on the second comprehensive strategy (papers II and III).The work presented in this thesis shows that the blinking kinetics of fluorescentlabels contain significant information that can be exploited by a combination of fluctuationsanalysis with distance dependent excitation energy transfer between thefluorescent molecules, or by analysis of fluorescence covariance between moleculesthat emit at different wavelengths. These fluorescence-based methods have a significantpotential for molecular interaction studies in the biomedical field. / <p>QC 20160527</p>

In this work, multinuclear solid-state nuclear magnetic resonance (NMR) spectroscopy is used to investigate a range of inorganic materials, often in combination with DFT (density functional theory) studies. Solid-state NMR is particularly suited to the study of aluminophosphates (AlPOs), as the basic components of their frameworks have NMR active isotopes (²⁷Al, ³¹P, ¹⁷O), as do many of the atoms that comprise the structure directing agent (¹³C, ¹H, ¹⁵N), and the charge-balancing anions (OH⁻, F⁻). A study of the AlPO STA-15 (St Andrews microporous solid-15) provides an introduction to using solid-state NMR spectroscopy to investigate AlPOs. More in-depth studies of AlPO STA-2 (St Andrews microporous solid-2) and MgAPO STA-2 (magnesium-substituted AlPO) examine charge-balancing mechanisms in AlPO-based materials. A range of scandium carboxylate metal-organic frameworks (MOFs), with rigid and flexible frameworks, have been characterised by multinuclear solid-state NMR spectroscopy (⁴⁵Sc, ¹³C and ¹H). The materials studied contain a variety of metal units and organic linkers. ¹³C and ¹H magic-angle spinning (MAS) NMR were used to study the organic linker molecules and ⁴⁵Sc MAS NMR was used to study the scandium environment in the MOFs Sc₂BDC₃ (BDC = 1,4-benzenedicarboxylate), MIL-53(Sc), MIL-88(Sc), MIL-100(Sc) and Sc-ABTC (ABTC = 3,3`,5,5`-azobenzenetetracarboxylate). Functionalised derivatives of Sc₂BDC₃ and MIL-53(Sc) were also studied. The ⁴⁵Sc MAS NMR spectra are found to be strongly dependant on the Sc³⁺ coordination environment. ²⁷Al and ²⁵Mg MAS NMR have been used to study Ti-bearing hibonite samples (of general formula Ca(Al, Ti, Mg)₁₂O₁₉), and results compared to a recent complementary neutron powder diffraction study, in order to investigate the substitution sites for Ti³⁺/⁴⁺ and Mg²⁺. A DFT investigation was also carried out on the aluminium end member, CaAl₁₂O₁₉, due to debate in the literature on the ²⁷Al NMR parameters for the trigonal-bipyramidal site. The substitution of Mg onto the tetrahedral site (M3) and Ti primarily onto one of the octahedral sites (M4) is supported.

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<p>The Amyloid beta peptide (Ab) is related to Alzheimer’s disease and is suggested to be the molecular pathogenic species of the disease, probably through the neurotoxic effect of Ab oligomers. Here the results from biophysical studies of Ab and fragments thereof, are presented. Pulsed field gradient NMR diffusion experiments show that Ab exists mainly as an unfolded monomer. In addition, the hydrodynamic radius of Ab suggests that Ab has residual secondary structure propensities. CD experiments reveal that Ab has a high propensity to adopt a polyproline type II (PII) helix at low temperature. NMR diffusion measurements as well as the 3JHNH values show that increasing the temperature from 4 C induces a structure transition from PII propensity to a beta strand propensity around 15 C and to a random coil conformation at higher temperature. The small hydrodynamic radius at low temperature may be explained by the presence of a population of a hairpin conformation as was suggested by MD simulations. 15N relaxation and secondary chemical shifts suggest that Ab consists of 6 structural regions, two regions with high PII propensity are separated by a highly mobile region located in the N-terminal part of the peptide. In the C-terminal part two regions with a propensity to adopt b-strand are located, separated by a mobile region. The structural propensities of soluble monomeric Ab agree well with the structure of the peptide in fibril aggregates as well as in SDS micelles. Ab binds zinc specifically and with high affinity. This interaction was studied using heteronuclear correlation experiments. The metal ligands were determined to be three histidines, 6,13 and 14 and the N-terminus. The Ab peptide also binds b-cyclodextrin and the combined use of NMR diffusion experiments and induced chemical shifts show that Ab has at least two binding sites for b-cyclodextrin, and the dissociation constants of these binding sites were determined.</p>

The shikimate pathway is responsible for the biosynthesis of essential aromatic metabolites and, as such, its enzymes are targets for the design of potential antimicrobial and herbicidal agents. The enzyme 3-dehydroquinate dehydratase (dehydroquinase, DHQase) catalyses the conversion of 3-dehydroquinate to 3-dehydroshikimate, the third step of the shikimate pathway. There are two types of DHQase, unrelated structurally and mechanistically. Type I DHQase catalyses the rection by via a covalently attached imine intermediate. Type II DHQase catalyses the reaction by way of an enolate intermediate. This thesis describes the synthesis of a series of potential inhibitors of type II DHQase. Inhibitors with C and N at C-3 and with both sp2 and sp3 character at this position were prepared. A potential type I DHQase inhibitor was also prepared. The biological evaluation of these inhibitors against type II DHQases from Mycobacterium tuberculosis and Streptomyces coelicolor and type I DHQase from Salmonella typhi is described. Inhibitors were evaluated by spectrophotometric assay. However, this proved inappropriate for some inhibitors with the S. coelicolor enzyme. The development of an alternative 1H NMR assay and its application to the evaluation of S. coelicolor DHQase inhibitors is therefore also described. Some insights into the structure activity relationships of type II DHQases, obtained from the results of these studies, are discussed.

NMR studies have long been used as a tool to derive structural and dynamic information. Such information has a wide range of applications, and notably is used in the study of structure-activity relationships. The aims of this work were to use NMR spectroscopy to derive structures of the molecules inhibiting the activation of the alternative pathway of the complement portion of the innate immune system (namely, the N-terminus of factor H (FH) and two small peptides, Compstatin 10 and Compstatin 20) and to consider the interdomain dynamics of proteins consisting of three modules theoretically (in silico) and experimentally (for the three N-terminal domains of FH). We focused on the three N-terminal complement control protein (CCP) domains of the important complement regulator, human factor H (i.e. FH1-3). Its three-dimensional solution structure was derived based on nuclear Overhauser effects and residual dipolar couplings (RDCs). Each of the three CCP modules in this structure was similar to the corresponding CCP in the previously derived C3b-bound structure of FH1-4, but the relative orientations of the domains were different. These orientations were additionally different from the interdomain orientations in other molecules that interact with C3b, such as DAF2-4 and CR1-15-17. The measured RDC datasets, collected under three different conditions in media containing magnetically aligned bicelles (disk-like particles formed from phospholipids), were used to estimate interdomain motions in FH1-3. A method in which the data was fitted to a structural ensemble was used to analyze such interdomain flexibility. More than 80% of the conformers of this predominantly extended three-domain molecule exhibit flexions of < 40°. Such segmental flexibility (together with the local dynamics of the hypervariable loop within domain 3) could facilitate recognition of C3b via initial anchoring, as well as eventual reorganization of modules into the conformation captured in the previously solved crystal structure of a C3b complex with FH1-4. The NMR study of the Compstatin analogues revealed unique structural features that had not before been observed in this group of peptides. These features included two b-turns per peptide, neither of which was located in the ‘canonical’ regions in which b-turns were observed in previous molecular dynamics and NMR studies. The structures of Compstatin 10 and Compstatin 20 derived here were consistent with the isothermal calorimetry (ITC) and surface plasmon resonance (SPR) data recorded previously. In the in silico study of interdomain motion of three-domain proteins carried out here, the domains were represented as vectors attached to one another in a linear fashion. They were allowed to undergo Brownian motion biased by the potentials between the sequential vectors. The resulting trajectories were analyzed using model-free and extended model-free formalism. The degree of coupling of the interdomain motion with overall motion was determined, along with a representation of the overall motion. The similarity between the trajectories of the vectors transformed to this overall motion frame and the results obtained from the model-free analysis was determined.

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