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The hallmark feature of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), is the accumulation of cytoplasmic inclusions of key disease-linked proteins. Two of these proteins, TDP-43 and SOD1, represent a significant proportion of sporadic and familial ALS cases, respectively. The population of potentially aggregation-prone partially-folded states on the folding free-energy landscape may serve as a common mechanism for ALS pathogenesis. A detailed biophysical understanding of the folding and misfolding energy landscapes of TDP-43 and SOD1 can provide critical insights into the design of novel therapeutics to delay onset and progression in ALS. Equilibrium unfolding studies on the RNA recognition motif (RRM) domains of TDP-43 revealed the population of a stable RRM intermediate in RRM2, with residual structure localized to the N-terminal half of the domain. Other RRM domains from FUS/TLS and hnRNP A1 similarly populate RRM intermediates, suggesting a possible connection with disease. Mutations, which enhance the population of the RRM2 intermediate, could serve as tools for deciphering the functional and misfolding roles of this partially-folded state in disease models, leading to the development of new biomarkers to track ALS progression. ALS mutations in SOD1 have been shown to destabilize the stable homodimer to result in increased populations of the monomeric and unfolded forms of SOD1. Mechanistic insights into the misfolding of SOD1 demonstrated that the unfolded state is a key species in the initiation and propagation of aggregation, suggesting that limiting these populations may provide therapeutic benefit to ALS patients. An in vitro time-resolved Förster Resonance Energy Transfer assay to screen small molecules that stabilize the native state of SOD1 has identified several lead compounds, providing a pathway to new therapeutics to treat ALS.

Electrostatic interaction has long been proposed to be an important factor for stabilizing protein. Charge-charge interaction may especially be important to the thermostability of protein, as having more surface electrostatic interactions is one of the common structural features found in thermophilic proteins when compared to their mesophilic homologues. In order to quantitatively investigate the electrostatic contribution to protein stability, two complementary approaches, namely the double mutant cycle approach and pKa shift approach, were carried out. / In the double mutant cycle approach, the coupling free energies of two salt bridges (E6/R92 and K46/E62) and one a long range ion pair (E90/R92) were estimated by using circular dichroism, to find out the thermodynamic parameters of the protein model Thermococcus celer L30e and its charge-to-neutral mutants. It was found that the coupling free energy was temperature independent and was about 3 kJ mol-1 per salt bridge. By using a novel analysis of double mutant cycle of DeltaC p, it was also found that the interaction of salt bridge plays an important role in the reduction of DeltaCp. The temperature independency of coupling free energy and the effect of reducing DeltaCp could explain the general observation very well that thermophilic proteins have highly up-shifted protein stability curves is due to its elevated electrostatic interactions when compared with their mesophilic homologs. / In the pKa shift approach, the native state pKa values of acidic residues were obtained by fitting the side chain carboxyl 13C chemical shifts to microscopic model or global fitting of titrational event (GloFTE), whereas the denatured state pKa values were obtained by conventional pH titration of terminal protected 5-residue glycine-based model peptide. It was found that the surface charge-charge interactions, either attractive or repulsive, were strong and complicated because of the high surface charge density of T. celer L30e. However, the fact that most of the acidic residues have significantly downshifted native state pK a values indicated the surface charge distribution of T. celer L30e is optimized for stabilizing the protein. In addition, we have shown that temperature has negligible effect on pKa values in both native state and denatured state, therefore temperature can only marginally amplify the stabilizing effect in linear manner. / To overcome the unwanted crystallization problem of wild-type T. celer L30e in the low ionic strength neutral pH NMR conditions, which were essential for the pKa shift approach, a quintuple Arg-to-Lys variant was designed to dramatically improve the crystalline solubility, while the surface charges, as well as the structural, thermodynamic, and electrostatic properties, were conserved. It has also shown that electrostatic interaction played a critical role in crystallization at low ionic strength conditions, and arginine residue was especially important in crystal packing because of its high ability of forming salt bridges and hydrogen bonds. / Wild-type T. celer L30e has also shown to have no observable residual structure in the guanidine HC1-induced denatured state, indicating that denatured state of T. celer L30e should not have large effect on the overall protein stability. / Chan, Chi Ho. / Adviser: Kam Bo Wong. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 202-218). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

The use of electromagnetic devices such as microwave ovens and mobile phones has certainly brought convenience to our lives. At the same time, the proliferation of said devices has increased public awareness of the potential health hazards. It is generally assumed that there is little or no risk associated with the use of electromagnetic devices, based on the small amount of power associated with those devices. However, case studies on animals indicate that the risk cannot be entirely ruled out. It has long been known that proteins are sensitive to stress, arising from various sources such as temperature, chemical, pressure, and changes in pH condition. In all of these cases, the protein exhibits clear signs of damage and distress, which range from slight unfolding to complete loss of structure. Frequently, the damage to the protein is alleviated by refolding, either by itself or by the aid of molecular chaperones. However, if the damage to the protein is too great, the protein will generally undergo proteolysis. Opinion has been divided over the implication of prolonged use of electromagnetic devices to human health. Studies conducted on animals so far have given conflicting results. The studies on the separate components, electric and magnetic fields, also give inconclusive results. This indicates that our understanding on how electric and magnetic fields interact with biological matter is incomplete. In this project, we use molecular dynamics to explore the behaviour of two forms of insulin chain-B, isolated and monomeric (in the presence of chain-A with all disulfide bonds intact), at ambient conditions and under the influence of various stress. Specifically, we focus our attention to thermal stress and electric field stress. The electric field stress considered in this study takes several forms: static and oscillating with three different frequencies. These fields have strength ranging from 1806 V/m to 109 V/m. By performing molecular dynamics simulations totalling over 500 ns, we have gained valuable insights into the effects of elevated temperature and electric field on insulin chain-B. We observed differences in the damage mechanisms by the application of static electric field and oscillating field. The application of static fields restricts the conformational freedom of a protein, whereas the application of oscillating fields increases the mobility and flexibility of the protein, similar to the effect of thermal stress. Both of these interfere with the normal behaviour of a protein. We have also observed frequency-dependent effects, with low frequency fields having static field-like characteristics in damage mechanism.

Bulk imprinted polymers were synthesized using traditional small molecular weight imprinting techniques for the recognition of bovine serum albumin (BSA). Reproducibility and capacity concerns prompted the use of circular dichroism to investigate the potential effects that conditions commonly employed have on the structure of the protein prior to polymerization. These studies clearly showed a substantial change in the secondary structure of three common model protein templates when in the presence of various monomers and crosslinkers. Molecular docking was used to further examine the interactions taking place at the molecular level. Docking simulations revealed that significant amounts of non-covalent interactions are occurring between the amino acid side chains and ligands; although, the interactions taking place amongst the analyte and polypeptide backbone are responsible for the experimentally observed conformational change. The computational studies also showed that several of the ligands preferentially ‘docked’ to the same amino acids in the protein, indicating that if multiple monomers are employed, this competition for similar binding sites will potentially result in non-specific recognition. These findings are important as they offer insight into the fundamental reasons why recognition of macromolecular templates has proven difficult as well as provide guidance for future success in the field. Using this information, novel surface imprinted polymers were synthesized via a facile technique for the specific recognition of BSA. Thin films based on 2-(dimethylamino)ethyl methacrylate (DMAEMA) as the functional monomer and varying amounts of either N,N’ methylenebisacrylamide (MBA) or poly(ethylene glycol) (400) dimethacrylate (PEG400DMA) as crosslinker were synthesized via UV free-radical polymerization. A clear and reproducible increase in recognition of the template was demonstrated for these systems as 1.6-2.5 times more BSA was recognized by the MIP sample relative to the control polymers. Additionally, these polymers exhibited specific recognition of the template relative to similar competitor proteins with up to 2.9 times more BSA adsorbed than either glucose oxidase or bovine hemoglobin. These synthetic antibody mimics hold significant promise as the next generation of robust recognition elements in a wide range of bioassay and biosensor applications. / text

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.