Copper-containing nitrite reductase (NiR) is a homotrimer of two cupredoxin domains and catalyzes the single electron reduction of NO2- to NO during dissimilatory denitrification. To investigate the evolution of NiR, methods of mutagenic library generation and high-throughput variant screening from E. coli colonies were developed. These methods allow for facile screening of 105 mutants for folding efficiency or substrate specificity. Initial proof of principle studies yielded several variants that oxidized the artificial substrate ο-dianisidine up to 8 times faster than wild type NiR, suggesting that this methodology has the potential to engineer NiR to acquire other reductase functions. A crystal structure was solved for a putative multicopper oxidase (MCO) and NiR homologue from Arthrobacter sp. (AMMCO) to 1.8 Å resolution. The overall folds of AMMCO and NiR are very similar (r.m.s.d. of 2.0 Å over 250 Cα atoms); Like NiR, AMMCO is a trimer with type-1 Cu sites in the N-terminal domain of each monomer; however, the active site of AMMCO contains trinuclear Cu site characteristic of MCOs instead of a the mononuclear type-2 Cu site found in NiR. Detailed structural analysis supports the theory that two-domain MCOs similar to AMMCO were intermediaries in the evolution of NiR and the more common three-domain MCOs. The physiological function of AMMCO remains uncertain, but genomic, crystallographic and functional analysis suggests that the enzyme is involved in metal regulation. Considering the extensive similarity between AMMCO and NiR, particularly at the active site, engineering a trinuclear cluster into NiR appears feasible with a modest number of alterations to the polypeptide chain. With the aid of my newly developed high-throughput screening technique and site-directed mutagenesis, the mononuclear NiR active site was remodelled into a trinuclear Cu site similar to that of MCO. A crystal structure of this variant was solved to 2.0 Å and the presence of three copper atoms at the engineered cluster was confirmed by Cu-edge anomalous diffraction data. Although the trinuclear copper cluster is present and catalyzes the reduction of oxygen, achieving rates of catalysis seen in native MCOs has proven more difficult. With the framework provided, further engineering NiR into a robust MCO is likely to provide further insights into the structural basis of oxygen reduction by trinuclear copper sites.

Graduation date: 1992

In studies of serum, in the early 1930s, from patients with pneumonia, a factor was found. It could agglutinate certain pneumococcal species. This factor, which later became known as C-reactive protein (CRP), increased sharply during the early and middle stages of the disease. CRP was identified as an acute phase protein and found especially in bacterial infections. CRP is synthesized in the liver by stimulation of interleukin 6 (IL-6), which is produced by the monocytes, and consists of five non-covalently bound subunits. The aim of this study was to compare two different analytical instruments (Vitros 5.1 FS Ortho-Clinical Diagnostics and QuikRead CRP Orion Diagnostica) for CRP analysis. In this study, Vitros 5.1 FS is used as a reference instrument. QuikRead CRP is a small instrument intended for patient-near testing. Vitros 5.1 FS (Ortho-Clinical Diagnostics a Johmon-Johmon Company, Rochester, NY, U.S.A) is a fully automated instrument for measuring various analytes of clinical importance in body fluids. For analysis of CRP, plasma is used. The amount of CRP is obtained by measuring turbidity at a specific wavelength in an Immuno-turbidimetric reaction. QuikRead CRP (Orion Diagnostica, Espoo, Finland) is an immuno-turbidimetric test in which micro-particles coated with anti-human CRP are used to measure the amount of CRP in whole blood, plasma or serum. Comparison between Vitros 5.1 FS and QuikRead CRP for the P-CRP analysis shows a good correlation (R= 0.997) of the mean value from the analysis I, II and III.  An intercept of -8.52 shows a decrease in the values of CRP for QuikRead comparatively Vitros 5.1 FS.  Bland-Altman-plot shows a slightly increased spread of results. Paired T-test shows that Vitros 5.1 FS and QuikRead CRP does not produces the same results. This study shows that QuikRead CRP is a user-friendly instrument that fits well in near patient testing. QuikRead CRP and Vitros 5.1 FS did not give the same results of P-CRP. This does not affect the results in regards to distinguish a viral infection (10-50 mg / L) from a bacterial infection (> 100 mg / L).

The Major Intrinsic Proteins are represented in all forms of life; plants, animals, bacteria and recently archaebacteria have all been shown to express at least one member of this superfamily of integral membrane proteins. We have overexpressed the E. coli aquaglyceroporin, glycerol facilitator (GlpF), to use as a model for studying membrane protein structure, folding and stability. Understanding membrane protein folding, stability, and dynamics is required for a molecular explanation of membrane protein function and for the development of interventions for the hundreds of membrane protein folding diseases. X-ray analysis of GlpF crystals shows that the protein exits as a tetramer in the crystallized state [1]. However, preparations of stable aqueous detergent solutions of GlpF in its native oligomeric state have been difficult to make; the protein readily unfolds and forms non-specific aggregates in many detergents. Here, I report the study of the structure and stability of the glycerol facilitator in several detergent solutions by blue native and sodium dodecyl sulphate polyacrylamide gel electrophoresis, circular dichroism, and fluorescence. For the first time, stable protein tetramers were prepared in two different detergent solutions (dodecyl maltoside (DDM) and lyso-myristoyl phosphatidylcholine (LMPC)) at neutral pH. Thermal unfolding experiments show that the protein is slightly more stable in LMPC than in DDM and that the thermal stability of the helical core at 95oC is slightly greater in the former detergent. In addition, tertiary structure unfolds before quaternary and secondary structures in LMPC whereas unfolding is more cooperative in DDM. The high stability of the protein is also evident from the unfolding half-life of 8 days in 8 M urea suggesting that hydrophobic interactions contribute to the stability. The GlpF tetramers are less resistant to acidic conditions; LMPC-solubilized GlpF shows loss of tertiary and quaternary structure by pH 6, while in DDM the tertiary structure is lost by pH 5, however the tetramer remains mostly intact at pH 4. The implications of thermal and chemical stress on the stability of the detergent-solubilized protein and its in vivo folding are discussed. / October 2008

Amyotrophic lateral sclerosis (ALS) is a debilitating, incurable, neurodegenerative disease characterized by degradation of motor neurons leading to paralysis and ultimately death in ~3-5 years. Approximately 10% of ALS cases have a dominant inheritance pattern, termed familial ALS (fALS). Mutations in the gene encoding the dimeric superoxide scavenger Cu, Zn superoxide dismutase (SOD), were found to be associated with ~20% of fALS cases. Over 110 predominantly missense SOD mutations lead to fALS by an unknown mechanism; however, it is thought that mutant SOD acquires a toxic gain of function. Mice as well as human post mortem studies have identified mutant SOD-rich aggregates in affected neurons, leading to the hypothesis that mutations in SOD increase the tendency of the protein to form toxic aggregates. SOD has a complex maturation process whereby the protein is synthesized in an apo or demetalated state, followed by formation of an intramolecular disulfide bond and binding of Zn2+ and Cu2+. Each of these post-translational modifications increases the stability of the protein. SOD has been shown to aggregate more readily from destabilized immature states, including the apo state both with and without the disulfide bond, highlighting the importance of these states. Thermal unfolding monitored by differential scanning calorimetry (DSC) and chemical denaturation monitored by optical spectroscopy were used to elucidate the folding mechanism and stability of both the apo SOD disulfide-intact and disulfide-reduced states. Chemically and structurally diverse fALS-associated mutants were investigated to gain insights into why mutant SODs may be more prone to misfold and ultimately aggregate. The mutations were introduced into a pseudo wild-type (PWT) background lacking free cysteines, resulting in highly reversible unfolding amenable to accurate thermodynamic analysis. Similarly to what was previously described for fully metallated (holo) SODs, chemical denaturation of the apo disulfide-intact SODs is well described by a 3-state dimer mechanism with native dimer, monomeric intermediate and unfolded monomer populated at equilibrium. Although removal of metals has a relatively small effect on the stability of the dimer interface, the stability of the monomer intermediate is dramatically reduced. Thermal unfolding of some disulfide-intact apo SOD mutants as well as PWT is well described by a 2-state dimer mechanism, while others unfold via a 3-state mechanism similar to chemical denaturation. All but one of the studied disulfide-intact apo mutations are destabilizing as evidenced by reductions in ΔG of unfolding. Additionally, several mutants show an increased tendency to aggregate in thermal unfolding studies through increased ratios of van’t Hoff to calorimetric enthalpy (HvH/ Hcal ). The effects of the mutations on dimer interface stability in the apo disulfide-intact form were further investigated by isothermal titration calorimetry (ITC) which provided a quantitative measure of the dissociation constant of the dimer (Kd). ITC results revealed that disulfide-intact apo SOD mutants generally have increased Kd values and hence favor dimer dissociation to the less stable monomer which has been proposed to be a precursor to toxic aggregate formation. Reduction of the disulfide bond in apo SOD leads to marked destabilization of the dimer interface, and both thermal unfolding and chemical denaturation of PWT and mutants are well described by a 2-state monomer unfolding mechanism. Most mutations destabilize the disulfide-reduced apo SOD to such an extent that the population of unfolded monomer under physiological conditions exceeds 50%. The disulfide-reduced apo mutants show increased tendency to aggregate relative to PWT in DSC experiments through increased HvH /Hcal, low or negative change in heat capacity of unfolding and/or decreased unfolding reversibility. Further evidence of enhanced aggregation tendency of disulfide-reduced apo mutants was derived from analytical ultracentrifugation sedimentation equilibrium experiments that revealed the presence of weakly associated aggregates. Overall, the results presented here provide novel insights into SOD maturation and the possible impact of stability on aggregation.

Lipoxygenases from legumes

Amyotrophic lateral sclerosis (ALS) is a debilitating, incurable, neurodegenerative disease characterized by degradation of motor neurons leading to paralysis and ultimately death in ~3-5 years. Approximately 10% of ALS cases have a dominant inheritance pattern, termed familial ALS (fALS). Mutations in the gene encoding the dimeric superoxide scavenger Cu, Zn superoxide dismutase (SOD), were found to be associated with ~20% of fALS cases. Over 110 predominantly missense SOD mutations lead to fALS by an unknown mechanism; however, it is thought that mutant SOD acquires a toxic gain of function. Mice as well as human post mortem studies have identified mutant SOD-rich aggregates in affected neurons, leading to the hypothesis that mutations in SOD increase the tendency of the protein to form toxic aggregates. SOD has a complex maturation process whereby the protein is synthesized in an apo or demetalated state, followed by formation of an intramolecular disulfide bond and binding of Zn2+ and Cu2+. Each of these post-translational modifications increases the stability of the protein. SOD has been shown to aggregate more readily from destabilized immature states, including the apo state both with and without the disulfide bond, highlighting the importance of these states. Thermal unfolding monitored by differential scanning calorimetry (DSC) and chemical denaturation monitored by optical spectroscopy were used to elucidate the folding mechanism and stability of both the apo SOD disulfide-intact and disulfide-reduced states. Chemically and structurally diverse fALS-associated mutants were investigated to gain insights into why mutant SODs may be more prone to misfold and ultimately aggregate. The mutations were introduced into a pseudo wild-type (PWT) background lacking free cysteines, resulting in highly reversible unfolding amenable to accurate thermodynamic analysis. Similarly to what was previously described for fully metallated (holo) SODs, chemical denaturation of the apo disulfide-intact SODs is well described by a 3-state dimer mechanism with native dimer, monomeric intermediate and unfolded monomer populated at equilibrium. Although removal of metals has a relatively small effect on the stability of the dimer interface, the stability of the monomer intermediate is dramatically reduced. Thermal unfolding of some disulfide-intact apo SOD mutants as well as PWT is well described by a 2-state dimer mechanism, while others unfold via a 3-state mechanism similar to chemical denaturation. All but one of the studied disulfide-intact apo mutations are destabilizing as evidenced by reductions in ΔG of unfolding. Additionally, several mutants show an increased tendency to aggregate in thermal unfolding studies through increased ratios of van’t Hoff to calorimetric enthalpy (HvH/ Hcal ). The effects of the mutations on dimer interface stability in the apo disulfide-intact form were further investigated by isothermal titration calorimetry (ITC) which provided a quantitative measure of the dissociation constant of the dimer (Kd). ITC results revealed that disulfide-intact apo SOD mutants generally have increased Kd values and hence favor dimer dissociation to the less stable monomer which has been proposed to be a precursor to toxic aggregate formation. Reduction of the disulfide bond in apo SOD leads to marked destabilization of the dimer interface, and both thermal unfolding and chemical denaturation of PWT and mutants are well described by a 2-state monomer unfolding mechanism. Most mutations destabilize the disulfide-reduced apo SOD to such an extent that the population of unfolded monomer under physiological conditions exceeds 50%. The disulfide-reduced apo mutants show increased tendency to aggregate relative to PWT in DSC experiments through increased HvH /Hcal, low or negative change in heat capacity of unfolding and/or decreased unfolding reversibility. Further evidence of enhanced aggregation tendency of disulfide-reduced apo mutants was derived from analytical ultracentrifugation sedimentation equilibrium experiments that revealed the presence of weakly associated aggregates. Overall, the results presented here provide novel insights into SOD maturation and the possible impact of stability on aggregation.

This thesis describes some experiments developed to probe the fundamental aspects of the interfacial behaviour of proteins. The contents of this thesis can be broadly divided into two parts. In the first part, we studied how the size of the nanoparticles and other variables such as pH and bulk protein concentration affect the structure of the adsorbed protein layers. We also probed how these factors can influence the binding activity of adsorbed proteins. Study on the adsorption of IgG, Protein A and streptavidin on gold nanoparticles reveals that not all proteins are similarly affected by the size of the adsorbing surface. We found that though the optical properties of all the proteins vary with the size of the nanoparticle, their functionalities are not similarly affected by nanoparticle curvature. Protein A and streptavidin retain their binding capacity to IgG and biotin, respectively, irrespective of the size of the gold nanoparticle that they are attached to. On the other hand, a reduction/ loss in binding of adsorbed IgG to Protein A molecules is observed. The reduction in biological activity further depends on the radius of curvature of the adsorbing surface. The second part of the thesis describes how nanoparticles can used as a probe to study the complex interfacial behaviour of proteins. We have utilized the extreme sensitivity of localized surface plasmon resonance (LSPR) of gold nanoparticles to local refractive index to determine the optical properties of BSA adsorbed on various polymer surfaces. The dielectric properties of the adsorbed protein depend on the nature of the substrate. Further, we have developed a model to determine the refractive index profile of adsorbed protein as a function of the distance from the substrate.

Disulfide bonds play crucial roles to predict the three-dimensional structure and the function of a protein. This thesis develops two algorithms to predict the disulfide bonding state of each cysteine in a protein sequence. These methods are based on the multi-stage framework and the multi-classifier of the support vector machine (SVM). The first algorithm achieves 94.0% accuracy of cysteine state prediction for dataset PDB4136, but in some datasets the results are not as good as our expectation. Thus the second algorithm is designed to improve the predicting ability for the proteins which have oxidized and reduced cysteines simultaneously. In addition, a new training strategy is also developed to increase the prediction accuracy. It appends the probabilities which are obtained from the SVM to the existing features and then starts a new training procedure repeatedly to get better performance. The experiments are performed on the datasets derived from well-known databases, such as Protein Data Bank and SWISS-PROT. It gets 94.3% accuracy for predicting disulfide bonding state on dataset PDB4136, which gets improvement 3.6% compared with the previously best result 90.7%.

For understanding the function of a protein, the protein structure plays an important role. The prediction of protein structure from its primary sequence has significant assistance in bioinformatics. Generally, the real protein structures can be reconstructed by some costly techniques, but predicting the protein structures helps us guess the functional expression of a protein in advance. In this thesis, we develop three terms as the materials of the fitness function that can be successfully used in protein backbone structure prediction. In the result of this thesis, it shows that over 80% of good values calculated from our fitness function, which are generated by the genetic programming, are better than the average in the CASP8.