Chitosan films are an excellent media for binding metal ions due to the electrostatic nature of the chitosan molecules. Addition of cross-linking or plasticizing agents alters texture of the films, but their effect on metal-binding capacity has not been fully characterized. The objective of this research was to determine effects of plasticizers and cross-linkers on physical and metal-binding properties of chitosan films and coatings prepared by casting and by spincoating. Chitosan films were prepared using 1% w/w chitosan in 1% acetic acid with or without (control) additives. Plasticizing agents were tetraethylene glycol (TEG) and glycerol while citric acid, ethylenediamine tetraacetic acid (EDTA), and tetraethylene glycol diacrylate (TEGDA) were used as cross-linkers. The additives were applied in concentrations of 0.10%, 0.25%, and 0.50% w/w of film-forming solution. The films were prepared by casting and by spincoating. Films were cast at ambient conditions for tests within one week (fresh films) and eight weeks (aged) after casting. The cast films were evaluated for thickness, residual moisture (by the Karl Fischer method), Cr(VI) binding capacity, puncture strength, and puncture deformation while the chitosan coatings were tested for thickness, Cr(VI) binding capacity, solubility in aqueous solution, and surface morphology (using atomic force microscopy). Cast films with cross-linkers showed an increase in resistance to puncture while plasticized films become more elastomeric. Control films bound 97.2% Cr(VI) ions from solution (0.56 mg Cr(VI)/g film), and addition of plasticizers did not affect chromium binding, tying up to 96.7% Cr(VI) ions from solution (0.56 mg Cr(VI)/g film). Films containing cross-linkers yielded binding capabilities ranging from 42.3% to 94.3% bound Cr(VI) ions (0.26-0.52 mg Cr(VI)/g film). Ultrathin coatings also possess the ability to bind Cr(VI) from solution, though only a maximum of 7.4% of Cr(VI) ions could be bound from solution, the thin films had the ability to bind up to 224 mg Cr(VI)/g ultrathin film. These coatings use less chitosan, but they display greater binding per mass. Overall, plasticizers do not alter, while cross-linkers may reduce, the binding capacity of chitosan films, but physical properties of the films can be controlled by inclusion of additives.

abstract: A novel small metal-binding protein (SmbP), with only 93 residues and no similarity to other known proteins, has been isolated from the periplasm of Nitrosomonas europaea. It is characterized by its high percentage (17%) of histidines, a motif of ten repeats of seven residues, a four α-helix bundle structure, and a high binding affinity to about six equivalents of Cu2+. The goal of this study is to investigate the Cu2+ binding sites in SmbP and to understand how Cu2+ stabilizes the protein. Preliminary folding experiments indicated that Cu2+ greatly stabilizes SmbP. In this study, protein folding data from circular dichroism (CD) spectroscopy was used to elucidate the role of Cu2+ in stabilizing SmbP structure against unfolding induced by decreased pH, increased temperature, and chemical denaturants. The significant stabilization effects of Cu2+ were demonstrated by the observation that Cu2+-SmbP remained fully folded under extreme environmental conditions, such as acidic pH, 96 °C, and 8 M urea. Also, it was shown that Cu2+ is able to induce the refolding of unfolded SmbP in acidic solutions. These findings imply that the coordination of Cu2+ to histidine residues is responsible for the stabilization effects. The crystal structure of SmbP without Cu2+ has been determined. However, attempts to crystallize Cu2+-SmbP have not been successful. In this study, multidimensional NMR experiments were conducted in order to gain additional information regarding the Cu2+-SmbP structure, in particular its metal binding sites. Unambiguous resonance assignments were successfully made. Cα secondary chemical shifts confirmed that SmbP has a four α-helical structure. A Cu2+-protein titration experiment monitored by NMR indicated a top-to-bottom, sequential metal binding pattern for SmbP. In addition, several bioinformatics tools were used to complement the experimental approach and identity of the ligands in Cu2+-binding sites in SmbP is proposed. / Dissertation/Thesis / Ph.D. Chemistry 2010

No description available.

This dissertation aims at analyzing complex problems arising in the context of dynamical networks, proteomics, and disease prevention. First, a new graph-based method for proving global stability of synchronization in directed dynamical networks is developed. This method utilizes stability and graph theories to clarify the interplay between individual oscillator dynamics and network topology. Secondly, a graph-theoretical algorithm is proposed to predict Ca2+-binding site in proteins. The new algorithm enables us to identify previously-unknown Ca2+-binding sites, and deepens our understanding towards disease-related Ca2+-binding proteins at a molecular level. Finally, an optimization model and algorithm to solve a disease prevention problem are described at the population level. The new resource allocation model is designed to assist clinical managers to make decisions on identifying at-risk population groups, as well as selecting a screening and treatment strategy for chlamydia and gonorrhea patients under a fixed budget. The resource allocation model and algorithm can have a significant impact on real treatment strategy issues.

The biosynthesis of [NiFe]-hydrogenase and urease in Helicobacter pylori requires several accessory proteins for proper assembly of the nickel-containing active sites. Critical to the maturation of both enzymes in H. pylori is the GTPase HypB. In this work, the metal-binding properties of H. pylori HypB (HpHypB) were investigated and a link between metal binding and the other biochemical properties of HpHypB was established. HpHypB binds stoichiometric nickel or zinc with nanomolar affinities, in partially overlapping sites located between two major GTPase motifs. Upon metal binding, the GTP hydrolysis activity and oligomeric properties of the protein are modulated. Furthermore, the stoichiometry and affinity of the nickel is altered when HpHypB is bound to nucleotide, a change not observed for zinc. Mutagenesis of the metal ligands suggest that a conserved cysteine is responsible for transducing the metal-bound state to altered GTPase activity and a conserved histidine is a required nickel ligand only in the nucleotide-bound state. Together, these results suggest that the metal-binding and GTP hydrolysis properties of HpHypB are intimately linked and may comprise a mechanism through which the [NiFe]-hydrogenase and urease maturation pathways can discriminate between Ni(II) and Zn(II). Characterization of the Escherichia coli GTPase YjiA, a member of the same GTPase family as HpHypB, demonstrated that YjiA can bind Ni(II), Zn(II), or Co(II) at a site in a similar location as in HpHypB. Metal binding also regulates the GTPase activity and oligomerization of YjiA. This finding suggests that metal-responsive GTPase activity may be a trait of this family of GTPases. Together, this work describes a unique link between the metal-binding and biochemical properties of the G3E GTPases and provides insight into the role of HpHypB in [NiFe]-hydrogenase and urease maturation.

The function of the metalloenzymes is mainly determined by four structural features: the metal core, the metal binding motif, the second sphere residues in the active site and the electronic statistics. Cysteamine dioxygenase (ADO) and cysteine dioxygenase (CDO) are the only known enzymes that oxidize free thiol containing molecules in mammals by inserting of a dioxygen molecue. Both ADO and CDO are known as non-heme iron dependent enzymes with 3-His metal binding motif. However, the mechanistic understanding of both enzymes is obscure. The understanding of the mechanistic features of the two thiol dioxygenases is approached through spectroscopic and metal substitution in this dissertation. Another focus of the dissertation is the understanding of the function of a second sphere residue His228 in a 3-His-1-carboxyl zinc binding decarboxylase α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD). ACMSD catalyzes the decarboxylation through a hydrolase-like mechanism that is initialized by the deprotonation of metal bounded water molecule. Our study reveled that the second sphere residue His228 is responsible for the water deprotonation through hydrogen bonding. The spectroscopic and crystallographic data showed the H228Y mutation binds ferric iron instead of native zinc metal and the active site water is replaced by the Tyr228 residue ligation. Thus, we concluded that, H228Y not only plays a role of stabilizing and deprotonating the active site water but also is an essential residue on metal selectivity.

The biosynthesis of [NiFe]-hydrogenase and urease in Helicobacter pylori requires several accessory proteins for proper assembly of the nickel-containing active sites. Critical to the maturation of both enzymes in H. pylori is the GTPase HypB. In this work, the metal-binding properties of H. pylori HypB (HpHypB) were investigated and a link between metal binding and the other biochemical properties of HpHypB was established. HpHypB binds stoichiometric nickel or zinc with nanomolar affinities, in partially overlapping sites located between two major GTPase motifs. Upon metal binding, the GTP hydrolysis activity and oligomeric properties of the protein are modulated. Furthermore, the stoichiometry and affinity of the nickel is altered when HpHypB is bound to nucleotide, a change not observed for zinc. Mutagenesis of the metal ligands suggest that a conserved cysteine is responsible for transducing the metal-bound state to altered GTPase activity and a conserved histidine is a required nickel ligand only in the nucleotide-bound state. Together, these results suggest that the metal-binding and GTP hydrolysis properties of HpHypB are intimately linked and may comprise a mechanism through which the [NiFe]-hydrogenase and urease maturation pathways can discriminate between Ni(II) and Zn(II). Characterization of the Escherichia coli GTPase YjiA, a member of the same GTPase family as HpHypB, demonstrated that YjiA can bind Ni(II), Zn(II), or Co(II) at a site in a similar location as in HpHypB. Metal binding also regulates the GTPase activity and oligomerization of YjiA. This finding suggests that metal-responsive GTPase activity may be a trait of this family of GTPases. Together, this work describes a unique link between the metal-binding and biochemical properties of the G3E GTPases and provides insight into the role of HpHypB in [NiFe]-hydrogenase and urease maturation.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease causing degeneration of upper and lower motor neurons. Most ALS cases are sporadic; only 6% are associated with mutations in Cu, Zn superoxide dismutase (SOD1). It is believed, however, that sporadic and familiar forms of ALS share a common mechanism, where SOD1 plays an important role: SOD1 knockout mice do not develop ALS, whereas the overexpression of human SOD1 in mice produces ALS-like symptoms. Increasing evidence suggest that the SOD1 structure gains cytotoxic properties, but detailed description of the toxic species is missing. This thesis work is focused on understanding how structural and dynamic properties of SOD1 change along its folding free-energy landscape and indicates the structural hot-spots from where the cytotoxic species may originate. Thus, binding of the zinc controls folding, stability and turnover of SOD1: (i) miscoordination of Zn2+ by the Cu-ligands speeds up folding of the SOD1 core structure, however, it stabilizes SOD1 in a state where both active-site loops IV and VII are unfolded, (ii) coordination of Zn2+ in the Zn-site, induces the folding of loop VII and stabilizes the native and  functional fold of both active-site loops and (iii) the tremendous stability gain due to Zn-site metallation corresponds to a folded state’s lifetime of  &gt; 100 years, thus the cellular lifetime of SOD1 is likely controlled by Zn2+ release, which again is coupled to opening of active-site loops. Hence the active-site loops IV and VII stand out as critical and floppy parts of the SOD1 structure. Moreover, a number of ALS-associated mutations, benign to apo-SOD1 stability, are shown here to affect integrity of active-site loops in holo-SOD1, which, in turn, increases population of SOD1 species with these loops disorganized. Finally, the close relation between SOD1 and Zn2+ can also act in the reverse direction: a perturbed folding free-energy landscape of SOD1 can disturb Zn2+ homeostasis. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

Proteins perform an incredible array of functions facilitated by a diverse set of biochemical properties. Changing these properties is an essential molecular mechanism of evolutionary change, with major questions in protein evolution surrounding this topic. How do new functional biochemical features evolve? How do proteins change following gene duplication events? I used the S100 protein family as a model to probe these aspects of protein evolution. The S100s are signaling proteins that play a diverse range of biological roles binding Calcium ions, transition metal ions, and other proteins. Calcium drives a conformational change allowing S100s to bind to diverse peptide regions of target proteins. I used a phylogenetic approach to understand the evolution of these diverse biochemical features. Chapter I comprises an introduction to the disseration. Chapter II is a co-authored literature review assessing available evidence for global trends in protein evolution. Chapter III describes mapping of transition metal binding onto a maximum likelihood S100 phylogeny. Transition metal binding sites and metal-driven structural changes are a conserved, ancestral features of the S100s. However, they are highly labile at the amino acid level. Chapter IV further characterizes the biophysics of metal binding in the S100A5 lineage, revealing that the oft–cited Ca2+/Cu2+ antagonism of S100A5 is likely due to an experimental artifact of previous studies. Chapter V uses the S100 family to investigate the evolution of binding specificity. Binding specificity for a small set of peptides in the duplicate S100A5 and S100A6 clades. Ancestral sequence reconstruction reveals a pattern of clade-level conservation and apparent subfunctionalization along both lineages. In chapter VI, peptide phage display, deep-sequencing, and machine-learning are combined to quantitatively reconstruct the evolution of specificity in S100A5 and S100A6. S100A5 has subfunctionalized from the ancestor, while S100A6 specificity has shifted. The importance of unbiased approaches to measure specificity are discussed. This work highlights the lability of conserved functions at the biochemical level, and measures changes in specificity following gene duplication. Chapter VII summarizes the results of the dissertation, considers the implications of these results, and discusses limitations and future directions. This dissertation includes both previously published/unpublished and co- authored material.

Zinc is an essential metal ion for the activity of multiple enzymes and transcription factors. Among many other transporting proteins human serum albumin (HSA) is the main carrier of Zn(II) in the blood plasma. HSA displays multiple ligand binding sites with extraordinary binding capacity for a wide range of ions and molecules including fatty acids. Hence, HSA controls the availability and distribution of those molecules throughout the body. Previous studies have established that the existence of one zinc site with high affinity (MBS-A) that is modulated by the presence of fatty acids. Therefore, the fatty acid concentration in the blood influences zinc distribution which may result in a significant effect on both normal physiological processes and a range of diseases. Based on the current knowledge of HSA's structure and its coordination chemistry with zinc ion, here, we attempted to investigate zinc interactions and coordination with HSA and the effect of different fatty acids on the protein structure, stability and on Zn(II) binding. By NMR titration, we examine the Zn(II) binding to HSA and the spectra show distinct movements of some resonances showing a conformational change has occurred as a result of Zn(II) binding. Isothermal calorimetry titrations study was performed to evaluate zinc binding affinity to HSA in the absence and presence of fatty acids. Free HSA results indicates the existence of one high affinity site and multiple low affinity sites. Upon the binding of fatty acids to HSA, three distinct behaviors of Zn(II) affinity was observed ranging from no effect to moderate to significant depending on the FAs. By the use of circular dichroism, we investigate secondary and tertiary structure of HSA in the presence and absence of FAs and Zn(II). We found albumin is predominately α-helical and the overall conformation of the protein remains unchanged even after interacting with FAs and Zn(II) with some exception. The structural stability of HSA was evaluated by obtaining the denaturation temperature in the presence and absence of fatty acid and we found the thermal denaturation of HSA increases with the increase of amount of fatty acids.