<p>The study and understanding of Alzheimer’s disease on protein level is fundamentally important in the search for its treatment and there is a demand for proteins that can be used together with candidate drugs in crystallography trials. The refolding time reaching up to three weeks for beta-site APP cleaving enzyme 1 (BACE1), the proposed disease-generating protein, is presently not optimal and new protein constructs are needed. In attempts to shorten the refolding time the six cysteins in BACE1 were substituted to hydrophobic valine or alanine residues. The proteins, both wild type and mutant BACE1, were expressed in <em>Escherichia coli</em>, refolded for one week and purified by ion exchange chromatography and gel filtration. The final products were characterised by measuring stability, homogeneity and enzyme activity. There was significantly lower protein yield for the mutants compared to the wild type BACE1, indicating that generation of the disulphide bonds are important for correctly folded and stable BACE1. Also, it was found that the three different disulphide bonds are not equally important during refolding, with Cys₂₇₈-Cys₄₄₃being the most important and Cys₂₁₆-Cys₄₂₀ and Cys₃₃₀-Cys₃₈₀ being of less importance. The present work shows that one week of refolding is enough for a sufficient protein yield of wt BACE1 and that the current refolding time for wt BACE1 can be shortened. Furthermore the disulphide bridges in BACE1 are important for forming an active protein with correct fold.</p>

The study and understanding of Alzheimer’s disease on protein level is fundamentally important in the search for its treatment and there is a demand for proteins that can be used together with candidate drugs in crystallography trials. The refolding time reaching up to three weeks for beta-site APP cleaving enzyme 1 (BACE1), the proposed disease-generating protein, is presently not optimal and new protein constructs are needed. In attempts to shorten the refolding time the six cysteins in BACE1 were substituted to hydrophobic valine or alanine residues. The proteins, both wild type and mutant BACE1, were expressed in Escherichia coli, refolded for one week and purified by ion exchange chromatography and gel filtration. The final products were characterised by measuring stability, homogeneity and enzyme activity. There was significantly lower protein yield for the mutants compared to the wild type BACE1, indicating that generation of the disulphide bonds are important for correctly folded and stable BACE1. Also, it was found that the three different disulphide bonds are not equally important during refolding, with Cys278-Cys443 being the most important and Cys216-Cys420 and Cys330-Cys380 being of less importance. The present work shows that one week of refolding is enough for a sufficient protein yield of wt BACE1 and that the current refolding time for wt BACE1 can be shortened. Furthermore the disulphide bridges in BACE1 are important for forming an active protein with correct fold.

Voltage-gated ion channels play fundamental roles in neural excitability, they are for instance responsible for every single heart beat in our bodies, and dysfunctional channels cause disease that can be even lethal. Understanding how the voltage sensor of these channels function is critical for drug design of compounds targeting neuronal excitability. The opening and closing of the pore in voltage-gated potassium (Kv) channels is caused by the arginine-rich S4 helix of the voltage sensor domain (VSD) moving in response to an external potential. In fact, VSDs are remarkably efficient at turning membrane potential into conformational changes, which likely makes them the smallest existing biological engines. Exactly how this is accomplished is not yet fully known and an area of hot debate, especially due to the lack of structures of the resting and intermediate states along the activation pathway. In this thesis I study how the VSD activation works and show how toxic compounds modulate channel gating through direct interaction with these quite unexplored drug targets. First, I show that a secondary structure transition from alpha- to 3(10)-helix in the S4 helix is an important part of the gating as this helix type is significantly more favorable compared to the -helix in terms of a lower free energy barrier. Second, I present new models for intermediate states along the whole voltage sensor cycle from closed to open and suggest a new gating model for S4, where it moves as a sliding 3(10)-helix. Interestingly, this 3(10)-helix is formed in the region of the single most conserved residue in Kv channels, the phenylalanine F233. Located in the hydrophobic core, it directly faces S4 and creates a structural barrier for the gating charges. Substituting this residue alters the deactivation free energy barrier and can either facilitate the relaxation of the voltage sensor or increase the free energy barrier, depending on the size of the mutant. These results are confirmed by new experimental data that supports that a rigid ring at the phenylalanine position is the rate-limiting factor for the deactivation gating process, while the activation is unaffected. Finally, we study how the activation can be modulated for pharmaceutical reasons. Neurotoxins such as hanatoxin and stromatoxin push S3b towards S4 helix limiting S4's flexibility. This makes it harder for the VSD to activate and might explain the stronger binding affinities in resting state. All these results are highly important both for the general topic of biological macromolecules undergoing functionally critical conformational transitions, as well as the particular case of voltage-gated ion channels where understanding of the gating process is probably the key step to explain the effects of mutations or drug interactions. / <p>QC 20121115</p>

Previous studies in Dr. Yang’s laboratory have established a grafting, design, and subdomain approach in order to investigate the properties behind Ca2+-binding sites located in Ca2+-binding proteins by employing engineered proteins. These approaches have not only enabled us to isolate Ca2+-binding sites and obtain their Ca2+-binding affinities, but also to investigate conformational changes and cooperativity effects upon Ca2+ binding. The focus of my thesis pertains to optimizing the expression and purification of engineered proteins with tailored functions. Proteins were expressed in E. coli using different cell strains, vectors, temperatures, and inducer concentrations. After rigorous expression optimization procedures, proteins were further purified using chromatographic and/or refolding techniques. Expression and purification optimization of proteins is essential for further analyses, since the techniques used for these studies require high protein concentrations and purity. Evaluated proteins had yields between 5-70 mg/L and purities of 80-90% as confirmed by SDS-PAGE electrophoresis.

The research presented in this dissertation involves the synthesis, characterization, and application of novel anionic and cationic chiral molecular micelles in capillary electrophoresis (CE) for the separation of diverse chiral compounds. Chapter 1 presents brief overview of the surfactants, micelle polymer, CE and micellar electrokinetic chromatography (MEKC). Chapter 2 describes the simultaneous enantioseparation of eight single chiral center â-blockers using two novel leucine and isoleucine based polymeric surfactants. The simultaneous enantioseparation of multichiral center bearing â-blockers, nadolol and labetalol is described in chapter 3. A synergistic approach, using a combination of polysodium N-undecenoxycarbonyl-L-isoleucinate (poly-L-SUCIL) and sulfated â-CD showed dramatic enantioseparation of four stereoisomers of nadolol. On the other hand for labetalol, enantiomeric separation remains unaffected using the dual chiral selector system. Chapter 4 deals with the enantiomeric separation of the binaphthyl derivatives that was found to be influenced by pH, type and concentration of the background electrolyte as well as concentration of the polymeric surfactant. In chapter 5, characterization of five alkenoxy leucine-based surfactants with variations in chain length (C8-C11), polymerization concentration and degree of polymerization showed significant effects on the chiral resolution and efficiency of hydrophobic â-blockers. The synthesis and characterization of two positively charged amino acid derived chiral ionic liquids (ILs) and their corresponding polymers is presented in chapter 6. Chiral separation of two acidic analyte (difficult to resolve with anionic micelles) can be achieved with both monomers and polymers of ILs. In chapter 7, the synthesis and detailed characterization of three pH independent amino acids derived (L-leucinol, L-isoleucinol and L-valinol) sulfated chiral polymeric surfactants is presented. These chiral sulfated surfactants are thoroughly characterized and the morphological behavior of polymeric sulfated surfactants is revealed using cryogenic high-resolution electron microscopy. The work clearly demonstrates for the first time the superiority of chiral separation in MEKC coupled to mass spectrometry at low pH. Finally, in chapter 8, six amino acid derived chiral surfactants with carboxylate and sulfate head groups were compared for enantioseparation of broad range of structurally diverse racemic compounds at neutral and basic pH conditions.

Condensation of water vapor is an everyday phenomenon which plays an important role in power generation schemes, desalination applications and high-heat flux cooling of power electronic devices. Continuous dropwise condensation is a desirable mode of condensation in which small, highly-spherical droplets regularly form and shed off the surface before a thick liquid is formed, thereby minimizing the thermal resistance to heat transfer across the condensate layer. While difficult to induce and sustain, dropwise condensation has been shown to achieve heat and mass transfer coefficients over an order of magnitude higher than its filmwise counterpart. Superhydrophobic surfaces have been extensively studied to promote dropwise condensation with mixed results; often surfaces that are superhydrophobic to deposited droplets formed in the gas phase above the surface do not retain this behavior with condensed droplets nucleated and grown on the surface. Recently, nanostructured superhydrophobic surfaces have been developed that are robust to vapor condensation; however, these surfaces still are not ideal for condensation heat transfer due to the high thermal resistance of the vapor layer trapped underneath the droplets and the reduced footprint of direct contact between the highly-spherical droplets and the underlying substrate. This work has two main objectives. First, a comprehensive free energy based thermodynamic model is developed to better understand why traditional superhydrophobic surfaces often lose their properties when exposed to condensed droplets. The model is first validated using data from the existing literature and then extended to analyze the suitability of amphiphilic (e.g. part hydrophobic and part hydrophilic) nanostructured surfaces for condensation applications. Secondly, one of the promising amphiphilic surfaces identified by the thermodynamic model is fabricated and tested to observe condensation dynamic behavior. Two complementary visualization techniques, environmental scanning electron microscopy (ESEM) and optical (light) microscopy, are used to probe the condensation behavior and compare the performance to that of a traditional superhydrophobic surface. Observations from the condensation experiments are used to propose a new mechanism of coalescence that governs the temporal droplet size distribution on the amphiphilic nanostructured surface and continually generates fresh sites for the droplet nucleation and growth cycle that is most efficient at heat transfer.

Tripeptidyl peptidase II (TPPII) is an exopeptidase which cleaves tripeptides from theN-terminus of peptides. The exact functional role of TPPII is still a matter of investigation. Itis believed that the enzyme is primarily involved in intracellular protein degradation, where itcooperates with the proteasome and other peptidases to degrade proteins into free aminoacids. These amino acids can subsequently be used in the production of new proteins. The aimof this work was to express murine wild type TPPII using E. coli and thereafter purify theenzyme from the bacterial lysate. Methods used for the purification included protein andnucleic acid precipitation, anion exchange chromatography, hydrophobic interactionchromatography and gel filtration. The presence of TPPII was determined using activityassay, western blot and SDS-PAGE. Despite the fact that some modification is still needed,the purification yielded a total of 34μg TPPII with a purity of approximately 60%. Thispurified enzyme can be used for future functional characterization.

This research mainly investigated how the hydrophilic and hydrophobic properties of gas diffusion layer, and the different open ratio of the flowfield may affect the performance of the micro proton exchange membrane fuel cell (£gPEMFC). The flow plate used in this experiment was made through deep UV lithography manufacturing processes and micro-electroforming manufacturing processes. Four different open ratios, 52.8 %, 50.8 %, 75.2 % and 75.75 %, of the flowfield were designed for the flow plate composed of serpentine-parallel and serpentine geometrical micro configurations. Acrylic (PMMA: Polymethylmethacrylate) was used to make the terminal plate placed on both sides of the micro proton exchange membrane fuel cell. By varying values of the hydrophilic and hydrophobic properties of the anode gas diffusion layer, the effects of these two parameters on the polarization curve and power density of the cell were explored. All results obtained in the experiment are presented by P-I curve and V-I curve. The experiment results show that, with 1: 5 flow ratio of anode to cathode, a design with the gas diffusion layer made of the material with hydrophobic factor 20 wt.% and with open ratio of 50.8 % for anode flow channel as well as open ratio of 75.75 % for cathode flow channel may have the best performance.

The aim of this study is to further our understanding of the forces that contribute to protein stability and to investigate how site-directed mutagenesis might be used for increasing protein stability. Eleven proteins ranging from 36 to 370 residues have been studied here. A 36-residue VHP and a 337-residue VlsE were used as model systems for studying the contribution of the hydrophobic effect on protein stability. Mutations were made in both proteins which replaced bulky hydrophobic side chains with smaller ones. All variants were less stable than their wild-type proteins. For VHP, the destabilizing effects of mutations were smaller when compared with similar mutations reported in the literature. For VlsE, a similarity was observed. This different behavior was investigated and reconciled by the difference in hydrophobicity and cavity modeling for both proteins. Therefore, the stabilizing mechanism of the hydrophobic effect appears to be similar for both proteins. Eight proteins were used as model systems for studying the effects of mutating non-proline and non-glycine residues to statistically favored proline and glycine residues in ?-turns. The results suggest that proline mutations generally increase protein stability, provided that the replaced residues are solvent exposed. The glycine mutations, however, only have a stabilizing effect when the wild-type residues have ?, ? angles in the L? region of Ramachandran plot. Nevertheless, this strategy still proves to be a simple and efficient way for increasing protein stability. Finally, using a combination of eight previously identified stabilizing mutations; we successfully designed two RNase Sa variants (7S, 8S) that have both much higher Tms and conformational stabilities than wild-type protein over the entire pH range studied. Further studies of the heat capacity change upon unfolding (?Cps) for both proteins and their variants suggest that residual structure may exist in the denatured state of the 8S variant. An analysis of stability curves for both variants suggests that they achieve their stabilization through different mechanisms, partly attributed to the different role of their denatured states. The 7S variants may have a more rigid denatured state and the 8S variant may have a compact denatured state in comparison with that of wild-type RNase Sa.

Cellulose is a biodegradable, renewable, flexible, inexpensive, biopolymer which is abundantly present in nature. In spite of these inherent advantages, cellulose fibers cannot be used directly in a number of potential industrial applications because of their hydrophilic nature; a surface modification is often required to alter the surface properties of cellulose. This thesis work reports a fabrication method that results in superhydrophobic properties (contact angle (CA) > 150°) on cellulose (paper) surfaces. Superhydrophobicity was obtained by domain-selective etching of amorphous portions of the cellulose fiber in an oxygen plasma, and by subsequently coating the etched surface with a thin fluorocarbon film deposited via plasma enhanced chemical vapor deposition from a pentafluoroethane precursor. Two forms of superhydrophobicity with vastly different degrees of adhesion were obtained by varying the plasma treatment conditions, in particular the duration of oxygen etching: "roll-off" (contact angle (CA): 166.7° ± 0.9° and CA hysteresis: 3.4° ± 0.1°) and "sticky" (CA: 153.4° ± 4.7° and CA hysteresis: 149.8±5.8°) superhydrophobicity. The CA hysteresis could be tuned between the two extremes by adjusting the oxygen etching time to control the formation of nano-scale features on the cellulose fibers. The effects of fiber types (soft vs. hard wood) and paper making parameters on fabricating superhydrophobic paper were also investigated. There were no significant differences in the formation of the nano-scale features created via oxygen etching on paper substrates obtained from different fiber types and paper making parameters. Because "roll-off" superhydrophobicity is primarily determined by the nano-scale roughness, this property is therefore not significantly affected by the fiber types or paper making parameters. While the fiber type does not affect "roll-off" or "sticky" superhydrophobicity, paper making process parameters affect the structure of the paper web on the micro-scale and thus lead to variations in "sticky" superhydrophobicity. Superhydrophobic paper substrates were patterned with high surface energy ink deposited using a commercial desktop printer. The patterns could be used to manipulate the drag and extensional adhesion of water drops on the substrates. Classic 'drag' and 'extensional' adhesion expressions were used to model the behavior of water drops on basic dot and line patterns of variable dimensions. A fundamental understanding of the adhesive forces of water drops as a function of pattern shape and size was thus obtained. Based on this knowledge, patterned paper substrates were then designed and fabricated to perform simple unit operations, such as storage, transfer, mixing and merging of water drops. These basic functionalities were combined in the design of a simple two-dimensional lab-on-paper (LOP) device. Further studies of more complicated pattern shapes led to the generation of patterns that allowed directional mobility and tunable adhesion of water drops. These developments are critical for designing novel components for two-dimensional LOP devices such as flow paths, gates/diodes, junctions and drop size filters.