Structure determination of membrane proteins by solution nuclear magnetic resonance spectroscopy requires that they can be incorporated into detergent micelles in order to ensure that they are maintained in a folded, water-soluble state. However, some detergents appear to disrupt structurally important interactions between transmembrane (TM) helices, while others produce micelle-protein complexes that may be too large for solution NMR. As a result there are a limited number of detergents suitable for solution NMR studies, making the search for better solution conditions one of the remaining challenges for structure elucidation of membrane proteins by this technique. This thesis presents the development and application of methods to address this problem by characterizing detergents for suitability for membrane protein solution NMR. Since the overall size of the membrane protein-detergent complex is one of the major factors that affects the quality of NMR spectra, modified versions of pulsed field gradient (PFG) NMR pulse programs for translational diffusion measurement were set up to estimate the size of these complexes. These methods were applied to a model membrane protein-detergent system allowing an approximate complex size and composition to be determined. 15N relaxation parameters were also measured for this complex and provided evidence that extensive motion of the protein within the micelle can occur to improve the quality of the resulting NMR spectrum. In addition to these experiments that probe complex molecular weight, a model system was also established to help identify detergent characteristics that can promote helix-helix interactions. This system is based on the properties of the single transmembrane segment from the M13 bacteriophage major coat protein (MCPTM) which forms homodimers in both lipid membranes and detergent micelles. Using solution NMR in combination with paramagnetic probes, oligomerization of this peptide under a range of detergent conditions was investigated. The results show that both denaturing and non-denaturing detergents are capable of preserving the TM-helix homodimer interaction. In accord with previous studies on this dimer, results from this study indicate that this peptide exists as a parallel dimeric structure at low concentrations of detergents. Together the methods described in this thesis have the potential to be used in the characterization of a wider range of detergent-protein complexes to assist in the development of new solvents that can ultimately advance the utility of solution NMR of membrane proteins.

Silver nanoparticles can be readily generated in micellar environments by ketyl radicals formed from the photoreduction of benzophenone in the presence of a suitable hydrogen donor. The yield of these ketyl radicals can be increased by extending the lifetime of the triplet radical pair through Zeeman splitting of the triplet sublevels in an externally applied magnetic field. This provides control over the rate of photogeneration of nanoparticles under very mild conditions. The rate of photogeneration can be monitored by the distinctive surface plasmon resonance absorption around 420 nm. In this work, micelles of sodium dodecyl sulphate (SDS) were employed, and 1,4-cyclohexadiene (1,4-CHD), an excellent hydrogen donor, was used to promote the generation of ketyl radicals. When benzophenone and a silver salt are added to this system and it is irradiated in the presence of a magnetic field, the rate of appearance of the plasmon band is enhanced. In addition to serving as a hydrogen donor, 1,4-CHD also has a stabilising influence on the nanoparticles, adsorbing onto the surface and preventing aggregation. 1,4-CHD added to a solution of nanoparticles synthesised without the diene present will even break up existing aggregates.

The lack of photobleaching, minimal sample heating, and high acquisition rates associated with coherent anti-Stokes Raman scattering (CARS) microscopy make it an attractive approach for the chemically specific in vivo imaging of dynamical processes. However, imaging capabilities are currently confined to classes of macromolecules as opposed to specific molecular targets. The use of cyano and deuterium functional group labels, which possess Raman modes in a spectral region devoid of endogenous cellular resonances, has the potential to surmount this limitation, enabling imaging with chemical and molecular specificity using CARS microscopy. Herein, cyano and deuterium vibrational modes have been incorporated into Raman and CARS contrast agents capable of mediating biomolecular modification. Application of this approach will be demonstrated using hepatitis C virus (HCV) RNA and two model protein systems with the end goal of investigating dynamical aspects of HCV molecular virology in real-time in vivo using CARS microscopy. The addition of exogenous CARS labels to a biomolecule can have serious structural and functional consequences that may lead to the expression of a phenotype dictated by the effects of the modification rather than the system under investigation. Therefore, the structural and functional consequences of introducing CARS labels needed to be investigated before applying cyano and deuterium modified HCV RNA and proteins to in vivo analysis using CARS microscopy.

Structure determination of membrane proteins by solution nuclear magnetic resonance spectroscopy require that they can be incorporated into detergent micelles in order to ensure that they are maintained in a folded, water-soluble state. However, detergent molecules produce micelle-protein complexes that pose unique challenges for solution NMR studies. To improve the spectroscopic properties of these complexes, it is possible to use specific isotope labeling strategies developed for the study of large water-soluble proteins by solution NMR. Specifically, the use of a highly deuterated sample that retains protons only in the Val, Leu and Ile (delta1) methyl groups has the potential to be used to determine structures of polytopic helical membrane proteins. However, while the quality of structure that can be obtained from this approach has been examined for water-soluble proteins, its utility for this class of membrane proteins has not been systematically investigated. This thesis evaluates the utility of this method for structure determination of membrane proteins by exploring structure calculation strategies from simulated NMR data sets from membrane proteins of known structure. Here I present results for a set of membrane proteins that consist of pairs or bundles of hydrophobic alpha-helices. I analyzed the impact of intra- and inter-helical NOEs on structure quality for samples specifically labeled with protons only at the methyl and amide positions In addition, I also explored the role of dihedral angles and residual dipolar couplings in an effort to find general trends to improve the accuracy of structures for polytopic a-helical membrane proteins. Based on the results of these calculations it appears that obtaining a uniform distribution of inter-helical NOES and accurately identifying transmembrane helices for dihedral angle restraints are the most important factors determining the accuracy of these simulated membrane protein structures. These results should help to guide future structure determinations for polytopic alpha-helical membrane proteins of unknown structure.

This thesis focuses on various carborane compounds and their uses for targeted cellular imaging. Cellular imaging agents have been developed for the field of medical diagnosis and treatment for some time. Many different processes have been traditionally applied in these fields, including fluorescence tagging, but these are lacking in differentiation from cellular background signals. Herein, carborane compounds have been developed for use as Raman reporters with signature absorption for BH vibrations, inside of the cell silence vibrational range. Carboranes have also been studied for the application of Boron Neutron Capture Therapy, a binary radiation therapy technique. Coupling these two ideas has lead to the formation of specific cellular targeting agents utilizing the unique BH vibrations for imaging, as well as the possible application of BNCT to the malignant tissue. This process has been developed with the aid of silver nanoparticles, which have been shown to enhance the Raman signal up to a factor of 1014 with Surface Enhanced Raman Scattering (SERS) techniques. Functionalized carborane compounds have been developed in order to study BH vibrations, and carborane-containing functionalized silver nanoparticles have been applied to target anti-EGFR antibodies for malignant tissue detection. Resulting SERS images confirmed selective tissue targeting with nanoparticle aggregate hot spots. Overlaying scanning electron microscope images with SERS BH vibrational intensity maps provided additional information on concentrations of cell surface receptors and identifying intercellular structures. Elaboration of the carborane Raman reporter resulted in two other carboranes with key functional groups, which could increase solubility, and have additional Raman handles for identification.

A porous disk device is described that was used for the introduction of sample solutions into a flow system and for on-line extractions. A 0.5$\mu$m 316L stainless steel disk with a 6.35mm diameter and 1.6mm thickness was placed in a Teflon piece of the same thickness and then sandwiched between two blocks of Lucite. Flow channels were made on either side of the disk by cutting oval-shaped holes in Teflon spacers inserted between the disk and the blocks. The disk's void volume was determined with the device placed in a computer-controlled flow manifold. Samples of As(III) were injected, collected in a titration cell and titrated with electrogenerated Ce(IV). The generated titration curves were used to determine the microequivalents of Ce(IV) at the endpoint which were then used with the titration reaction stoichiometry to determine the volume of As(III) injected. The y-intercept of a plot of As(III) volume injected vs. injection times adjusted for flow rates was the void volume of the porous disk. Three determinations using this experiment yielded an average disk volume of 20.60 $\pm$ 0.32$\mu$L. Reproducible injections of a nickel sulfate solution were demonstrated. The device was adapted to perform on-line extractions of caffeine from aqueous solutions into methylene chloride. The aqueous solutions were flowed past the porous disk so that the caffeine was extracted into the CH$\sb2$Cl$\sb2$ contained in the disk. A dispersion of 6.6 was determined for the injection of the plug of solution in the disk into the flow stream and delivery to the detector. A calibration curve of the steady state absorbance vs. concentration of caffeine solutions in $\rm CH\sb2Cl\sb2$ yielded a molar absorptivity coefficient of 6785 M$\sp{-1}$cm$\sp{-1}$. Extraction efficiencies from 0.53 to 2.1%, were obtained for a series of aqueous caffeine solutions ranging in concentration from 0.10002mM to 10.002mM and using a sample volume of 393$\mu$L. The effect of increasing the time that the aqueous caffeine solution was exposed to the $\rm CH\sb2Cl\sb2$ was investigated. The extraction efficiencies increased for hold times up to 180s, after which a decrease in extraction efficiencies was observed. Finally, the feasibility of analyzing real samples containing caffeine was demonstrated through the on-line analysis of a cola sample.

This work includes the investigation and determination of several classes of disinfection by-products from various naturally occurring model compounds using several oxidative and instrumental techniques. Analytical methodology was developed for the determination and identification of nitrosamine species by gas chromatography with time-of-flight mass spectral detection. In addition, this method was successfully evaluated against existing techniques, and applied to real, raw water effluent samples. Identification and determination of various iodinated disinfection byproduct species was accomplished for the further elucidation of potential health risks associated with the municipal disinfected drinking water supply. Finally, products from the oxidation of a model ketoacid (pyruvic acid) by hypochlorous acid were determined to gain a greater understanding of the possible byproducts produced from a treatment facility that utilizes ozonation as primary disinfectant and hypochlorous acid as a secondary oxidizer.

Human serum Transferrin (hTf) is a ∼80 kDa protein, whose function is iron sequestration and transport. The two lobes of the protein (commonly referred to as N- and C-lobes) have a very high degree of structural identity and provide two distinct binding sites for a ferric ion. In addition to iron, serum transferrin also binds a variety of other metals and is believed to provide a route for the in vivo delivery of such metals to cells. In the present study ESI MS is used to investigate interactions between human serum transferrin and two non-ferrous metals (indium and bismuth), conformational changes upon metal binding, as well as characterize human serum transferrin N-lobe (hTf/2N) global dynamics and functionally important local dynamic events. The In-hTf complex was directly detected by ESI MS; the Bi-hTf complex in solution was established by monitoring the evolution of charge state distributions of transferrin ions upon acid-induced protein unfolding in the presence and in the absence of the metal in solution. The large size of Bi3+ ion is likely to prevent formation of a closed conformation (canonical structure of the holo-protein), resulting in a non-native metal coordination which causes anomalous instability of the transferrin-bismuth complex in the gas phase. The apo-hTf/2N and Fe-hTf/2N were used in hydrogen/deuterium exchange (HDX) measurement for characterizing protein dynamics. In this measurement, back-exchange was corrected for every transferrin N-lobe peptic fragment individually. The results showed that iron binding induce more compact conformation and significant decrease of HDX kinetics around hinge regions and Lys206 which is one amino acid from the dilysine-trigger. However, the changes around iron binding sites are not as significant. Our hypothesis is that instead of having frozen states, transferrin has certain frequency of conformational hopping. A new method of rapid detection and identification of disulfide-linked peptides in complex proteolytic mixtures was developed utilizing the tendency of collision-activated peptide ions to lose preferentially side chains of select amino acids in the negative ion mode.

Mass spectrometry has enjoyed enormous popularity over the years for studying biological systems. The theme of this dissertation was to develop and use mass spectrometry based tools to solve five biologically oriented problems associated with protein architecture and extend the utility of these tools to study protein polymer conjugation. The first problem involved elucidating the false negatives of how proteins with few basic residues, forms highly charged ions in electrospray ionization mass spectrometry (ESI MS). This study showed that the unfolding of polypeptide chains in solution leads to the emergence of highly charged protein ions in ESI MS mass spectra, even if the polypeptide chains lack a sufficient number of basic sites. In the second problem, a new technique was developed that can monitor small-scale conformational transitions that triggers protein activity and inactivity using porcine pepsin as a model protein. This work allowed us to revise a commonly accepted scenario of pepsin inactivation and denaturation. The physiological relevance of an enzyme-substrate complex was probed in our third problem. We observed by ESI MS that pepsin forms a facile complex with a substrate protein, N-lobe transferrin under mildly acidic pH. The observed complex could either be a true enzyme-substrate complex or may likely results from an electrostatically driven association. Our investigation suggested that the enzyme binds nonspecifically to substrate proteins under mild acidic pH conditions. The fourth problem dealt with the investigation of conformational heterogeneity of natively unstructured proteins using a combination of spectroscopic techniques and ESI MS as tools. It was observed that four different conformations of alpha-synuclein coexist in equilibrium. One of these conformations appeared to be tightly folded. Conclusions regarding the nature of these states were made by correlating the abundance evolution of the conformers as a function of pH with earlier spectroscopic measurements. The final problem was aimed at monitoring conformational transitions in polypeptide and polymer segments of PEGylated proteins using PEGylated ubiquitin as a model system. This studies suggested that for a PEGylated protein, polypeptides maintain their folded conformation to a greater extent whiles the polymer segments are bound freely to the protein.

Cellular Retinoic acid binding protein I (CRABP I), a member of intracellular lipid binding proteins (iLBPs), binds physiologically to a mostly hydrophobic ligand, all-trans Retinoic Acid (RA). The binding site is inside an internal cavity, inaccessible in a static apo-protein conformation. Binding of RA to CRABP I does not result in significant changes of the protein tertiary structure, suggesting significance of dynamics in the ligand recognition and binding processes. One of the proposed scenarios for the protein-ligand binding process invokes the notion of a flexible portal region adjacent to the binding site, while another model suggests that the requisite dynamic events are induced by dimerization of the apo-protein in solution. In this work RA binding to CRABP I is studied in dilute solutions (low micro-molar range), where no dimer and/or oligomer formation occurs. Modulation of backbone dynamics within various segments of the protein by its ligand is assessed using a combination of hydrogen exchange, electrospray ionization mass spectrometry and collision-induced dissociation of protein ions in the gas phase. Consistent with the portal model of ligand entry, several protein segments are flexible in the absence of the ligand. At the same time, the two segments containing arginine residues forming a salt bridge with RA form the least flexible region in the apo-form of the protein. Although the presence of RA in solution reduces flexibility of all protein segments, the largest effect is observed within four strands that form one of the two β-sheets enveloping a cavity, which houses the ligand-binding site. These results are consistent with a model in which ligand binding occurs through a partially unstructured state of the protein with unobstructed access to the ligand-binding site. This intermediate (whose core is formed by the two stable arginine-containing strands) corresponds to a relatively low-energy local minimum on the apo-protein energy surface and is frequently sampled under native conditions. Intermediate states were visualized under various mildly denaturing conditions and characterized by HDX/CAD ESI MS. The similar amide backbone protection pattern suggests that the intermediate states bear significant resemblance with the activated intermediate state.