Membrane proteins contribute about 30% of the proteome and about 60% of the drugs in the market target membrane proteins. But only about 1.5% of structures in Protein Data Bank are membrane proteins which reflect the challenges in membrane protein studies. In this dissertation work M2 full-length protein from Influenza A, a drug target to treat Influenza, is studied in lipid bilayer environment. ssNMR, the best technique to date to study membrane proteins at atomic resolution, has been utilized and intermonomer distance measurement is carried out throughout the study. Differently isotopically labeled protein mixtures were utilized for experiments to obtain unambiguous measurements, even though this method significantly reduces the sensitivity. Influence of amantadine, an antiviral drug, towards distinctive residues at different locations of the channel, such as N-terminus channel pore opening, channel interior and C-terminus of the channel has been characterized. M2 is known to be sensitive to its environment and here the influence of factors such as drugs, pH and membrane constituents such as cholesterol is studied. Distance information obtained for M2 full-length is compared with some of the existing M2 structures of truncated constructs and the importance of studying M2 protein as much as its native-like environment is emphasized. Among multiple drug resistant mutations of M2, S31N mutation contribute almost 100% of drug resistant strains observed in flu infected humans. Therefore, studies involving S31N mutation is an essential part of M2 proton channel studies. Hence, multiple experiments has been performed to obtain structural insights of this oligomeric protein and to compare them with that of M2 wild-type protein, in addition to study of its effect to environmental conditions such as pH. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester 2015. / July 01, 2015. / Amantadine binding, Cholesterol binding of M2 protein, Influenza A M2 protein, Inter-monomer distance measurements, S31N mutant of M2 protein, Solid-state NMR bspectroscopy / Includes bibliographical references. / Timothy A. Cross, Professor Directing Dissertation; Debra A. Fadool, University Representative; William T. Cooper, Committee Member; Alan G. Marshall, Committee Member.

The work in this dissertation presents methods for study of glucose stimulated insulin secretion as well as other hormones involved in glucose homeostasis. A microfluidic system was developed to investigate the entrainment of insulin secretion from islets of Langerhans to oscillatory glucose levels. A gravity-driven perfusion system was integrated with a microfluidic system to deliver sinusoidal glucose waveforms to the islet chamber. Insulin levels in the perfusate were measured using an online competitive electrophoretic immunoassay with a sampling period of 10 s. The insulin immunoassay had a detection limit of 3 nM with RSDs of calibration points ranging from 2–8%. At 11 mM glucose, insulin secretion from single islets was oscillatory with a period ranging from 3–6 min. Application of a small amplitude sinusoidal wave of glucose with a period of 5 or 10 min, shifted the period of the insulin oscillations to this forcing period. Exposing groups of 6–10 islets to a sinusoidal glucose wave synchronized their behavior, producing a coherent pulsatile insulin response from the population. These results demonstrate the feasibility of the developed system for the study of oscillatory insulin secretion. A dual detection microscopy system was developed to simultaneously image intracellular messengers and monitor insulin secretion from islets of Langerhans. Glucose stimulated insulin secretion plays a critical role in glucose homeostasis, but the underlying mechanism is still unclear. To develop an automated system to simultaneously study the correlation between intracellular events and insulin secretion, fluorescence imaging and laser induced fluorescence detection for insulin immunoassay were integrated into a single microscopy system. Intracellular calcium ([Ca2+]i) was used as a representative secondary messenger and studied with insulin secretion from islets during exposure to constant and oscillatory glucose levels. Both of [Ca2+]i and insulin were oscillatory during constant glucose levels and entrained to a small amplitude sinusoidal wave of glucose (0.5 – 2 mM). [Ca2+]i and insulin oscillations were temporally but not necessarily quantitatively correlated. Oscillatory glucose waveforms amplified insulin oscillation amplitude without further increasing [Ca2+]i, which could be related to amplifying pathway. The developed system was also applied to study the effect of glucokinase activator 22 on [Ca2+]i and insulin secretion. These results indicated the robustness of the developed method, which can be potentially expanded to include other intracellular messengers to study the mechanism of glucose stimulated insulin secretion. A method was developed that allowed simultaneous monitoring of the acute secretory dynamics of insulin and islet amyloid polypeptide (IAPP) from islets of Langerhans using a microfluidic system with two-color detection. A flow-switching feature enabled changes in the perfusion media within 5 s, allowing rapid exchange of constant glucose concentrations delivered to groups of islets. The perfusate was continuously sampled by electroosmotic flow and mixed online with Cy5-labeled insulin, fluorescein isothiocyanate (FITC)-labeled IAPP, anti-insulin, and anti-IAPP antibodies in an 8.15 cm mixing channel maintained at 37 °C. The immunoassay mixture was injected for 0.3 s onto a 1.5 cm separation channel at 11.75 s intervals and immunoassay reagents detected using 488 and 635 nm lasers with two independent photomultiplier tubes for detection of the FITC and Cy5 signal. RSD of the bound-to-free immunoassay ratios ranged from 2 to 7% with LODs of 20 nM for insulin and 1 nM for IAPP. Simultaneous secretion profiles of the two peptides were monitored from groups of 4−10 islets during multiple step changes in glucose concentration. Insulin and IAPP were secreted in an approximately 10:1 ratio and displayed similar responses to step changes from 3 to 11 or 20 mM glucose. The ability to monitor the secretory dynamics of multiple peptides from islets of Langerhans in a highly automated fashion is expected to be a useful tool for investigating hormonal regulation of glucose homeostasis. The ability to detect picomolar concentrations of glucagon and amylin using fluorescently labeled mirror image aptamers, so-called Spiegelmers, is demonstrated. Using Spiegelmers as affinity probes, noncompetitive capillary electrophoresis affinity assays of glucagon and murine amylin were developed and optimized. The detection limit for glucagon was 6 pM and for amylin was 40 pM. Glucagon-like peptide-1 and -2 did not interfere with the glucagon assay, while the amylin assay showed cross-reactivity to calcitonin gene related peptide. The developed assays were combined with a competitive immunoassay for insulin to measure glucagon, amylin, and insulin secretion from batches of islets after incubation with different glucose concentrations. The development of these assays is an important step towards incorporation into an online measurement system for monitoring dynamic secretion from single islets. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the Doctor of Philosophy. / Spring Semester 2016. / April 4, 2016. / affinity assay, capillary electrophoresis, hormone, islet of Langerhans, microfluidics / Includes bibliographical references. / Michael G. Roper, Professor Directing Dissertation; P. Bryant Chase, University Representative; John G. Dorsey, Committee Member; Kenneth L. Knappenberger, Jr., Committee Member.

Histone variants and post-translational modifications (PTMs) are known to play central roles in genome regulation and maintenance. Quantitavely characterization of the variants and the associated PTMs is the first step to understand their biological functions. However, many variants and the PTM combinations are inaccessible by antibody-based methods or bottom-up tandem mass spectrometry. For many, the only tractable approach is with intact protein top-down tandem mass spectrometry. In this dissertation, a method of identification of histone H4 proteoforms is developed, revealing the challenge of top-down proteomics. Then a method of proteoform identification and relative quantitation of histones H2A and H2B by FT-ICR MS and MS/MS is developed, yielding quantitative identification of all detected H2A and H2B isobaric and isomeric proteoforms with a label-free approach in HeLa. The method is further extended to 1) normal and HIV-infected U937 cells; 2) the Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) stem cells sorted into early G1, late G1, S, and G2/M phases; 3) MDA-MB-231 breast cancer cells synchronized to S and M phases. The top-down MS/MS approach provides a path forward for more extensive elucidation of the biological roles of many previously unstudied histone variants and PTMs. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester 2016. / June 27, 2016. / Includes bibliographical references. / Alan G. Marshall, Professor Directing Dissertation; David M. Gilbert, University Representative; Michael G. Roper, Committee Member; Timothy M. Logan, Committee Member.

High resolution mass spectrometry, especially Fourier transform ion cyclotron resonance mass spectrometry (FT ICR MS) is a widely practiced technique of choice in proteomics and lipidomics due to its high sensitivity, reproducibility and wide dynamic range. FT-ICR MS enables quick assignments of hundreds of peptides and lipids with extreme complexity. Chapter 1 introduces the fundamental of FT ICR phenomena for mass measurement and basic theories of LC-MS based hydrogen deuterium exchange (HDX) technique for high order structure studies. Chapter 1 also introduces the application of mass spectrometry in lipidomics including lipid classification and MS analysis. Chapter 2 described the characterization of the binding interfaces in R2TP complex by hydrogen/deuterium exchange mass spectrometry. The two closely related AAA+ family ATPase Rvb1 and Rvb2 form a tight functional complex with two Hsp90 interactors: Pih1p and Tah1p. The R2TP complex involves in multiple biological processes including apoptosis, PIKK signaling, and RNA polymerase II assembly, and snoRNP biogenesis. The current lack of structural information on R2TP complex prevents a mechanistic understanding of many biological processes. By use of solution-phase HDX MS, we probed the contact surfaces on Pih1p-Tah1p upon Rvb1/2p binding. The present results demonstrate that Pih1p-Tah1p interacts with Rvb1/2p through N-terminal and IDR2 regions of Pih1p. Significantly, HDX also detected a rearrangement of residues 38–60 of Pih1p and 1–44 of Tah1p upon formation of the R2TP complex. Chapter 3 depicts the study of conformations of activated, disease-associated glucokinase variants by a comparative hydrogen/deuterium exchange mass spectrometry. Human glucokinase (GCK) acts as the body’s primary glucose sensor and plays a critical role in glucose homeostatic maintenance. Previous biochemical and biophysical studies suggest the existence of two activated variants. HDX results demonstrate that a disordered active site, which is folds upon binding of glucose, is protected from exchange in α helix variant. Additionally, α helix variant displays an increased level of exchange near enzyme’s hinge region. In contrast, β hairpin variant does not show substantial difference in global or local exchange relative to that of wild type GCK. The work elucidates the structural and dynamics origins of GCK’s unique kinetic cooperativity. Chapter 4 investigated the structure of an antibody with ‘Knob-into-hole’ mutations by HDX MS. Bispecific antibodies (BsAbs) have flourished in the biopharmaceutical industry for targeting two distinct antigens simultaneously. ‘Knob-into-hole’ approach is a way to manufacture bispecific antibodies. The applicability and advantage of ‘Knob-into-hole’ engineered bispecific antibody is vast. However, concerns about the conformational change and immunogenicity risks posed by the new approach has have been raised. To better understand the conformations and dynamics impacted by the ‘knob’ and ‘hole’ mutations, HDX MS is used to characterize peptide-level conformational changes of a ‘Knob-into-hole’ engineered antibody. The study shows that there is no significant structural alternation induced by ‘Knob-into-hole’ framework. In Chapter 5, the applicability of resolving HDX-derived isotopic fine structure by ultrahigh resolving power FT ICR mass spectrometry was discussed. In an HDX experiment, labeling protein with deuterium causes the deuterium incorporation, resulting in distributions of various combinations of 13C1H and 12C2H (Δm = 2.9 mDa). The isotopic fine structure typically cannot be used to evaluate deuteration level due to the difficulty of .resolving fine structures for all proteolytic peptides spanning wide mass range from HDX experiments. The introduction of hexapolar cell triples the observed resolving power on 14.5 tesla FT-ICR mass spectrometer, thus we successfully extend the capability of resolving isotopic fine structure to most of identified peptides. Additionally, a new method of analysis of isotopic fine structure-resolved HDX data was proposed to determine degrees of deuterium incorporation. Another research area I have worked on is characterization of polar lipids by LC coupled with FT-ICR mass spectrometry. Algae lipids contain long-chain saturated and polyunsaturated fatty acids. The lipids may be transesterified to generate biodiesel fuel. In Chapter 6, I compared polar lipid compositions for two microalgae, Nannochloropsis oculata and Haematococcus pluvialis, that are prospective lipid-rich feedstock candidates for an emerging biodiesel industry. Online nano liquid chromatography coupled with negative electrospray ionization 14.5 T Fourier transform ion cyclotron resonance mass spectrometry ((−) ESI FT-ICR MS) with newly modified ion optics provides ultrahigh mass accuracy and resolving power to identify hundreds of unique elemental compositions. Assignments are confirmed by isotopic fine structure for a polar lipid extract. Collision-induced-dissociation (CID) MS/MS provides additional structural information. H. pluvialis exhibits more highly polyunsaturated lipids than does N. oculata. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester 2018. / June 13, 2018. / FT ICR Mass Spectrometry, Hydrogen Deuterium Exchange, Isotopic Fine Structure, Lipidomics, Liquid Chromatography, Protein High Order Structure / Includes bibliographical references. / Alan G. Marshall, Professor Directing Dissertation; Hengli Tang, University Representative; John G. Dorsey, Committee Member; Brian G. Miller, Committee Member.

toc / Chemistry / Master / Master of Science

Malonaldehyde, a very reactive member of the homologous series of dialdehydes, is associated with the autoxidative deterioration of lipids. Its measurement, in autoxidized lipid systems is an expression of the extent of oxidation, in lipids. Malonaldehyde lends itself well to such determinations because of the sensitivity and specificity of its quantification in complex lipid systems. Complete knowledge of the formation of malonaldehyde in autoxidized lipids. is lacking. Such knowledge would undoubtedly promote a better understanding of lipid autoxidation mechanisms. In this investigation, a method for detecting malonaldehyde through the use of its reaction with p-aminobenzoic acid was developed. This was adapted for use in measuring malonaldehyde in lipids and in tissue samples. The reaction between p-aminobenzoic acid and malonaldehyde was partially characterized, and a mechanism for the formation of the reaction product was postulated. The quantification of malonaldehyde in lipid systems by the use of p-aminobenzoic acid involves the use of a mild reducing agent such as stannous chloride to prevent interference from hyd roperoxides present in the system. The p-aminobenzoic acid reaction is highly specific for malonaldehyde and proceeds smoothly and rapidly at room temperature in a non-aqueous chloroform-methanol solvent system to yield a highly colored compound having a maximum absorbance at 406 mμ. and with a molar absorptivity of 73,500. The absorbance value may be converted directly to parts-per-million malonaldehyde through the use of a conversion factor in a simple equation. The limits of detectability of themethod are on the order of one ppm malonaldehyde. The measurement of malonaldehyde in the lipid fraction of tissue samples involves the prior extraction of the lipid with a non-aqueous chloroform-methanol solvent, by an extraction method which was developed for this purpose. The reaction of malonaldehyde with p-aminobenzoic acid involves the condensation of two molecules of p-aminobenzoic acid with one molecule of malonaldehyde. The reaction exhibits a rate maximum at a hydrogen ion concentrations of about 0.1 molar, and also exhibits rate dependencies upon the concentrations of both malonaldehyde and p-aminobenzoic acid. This strongly suggests that the reaction proceeds according to an S [subscript N] 2 mechanism. A postulated mechanism involves nucleophilic 1,4-addition of the amino nitrogen of p-aminobenzoic acid to the enol of malonaldehyde followed by-loss of water to form the enamine. The reaction with a second molecule of p-aminobenzoic acid involves nucleophilic substitution of the amino nitrogen at the carbonyl function of malonaldehyde followed by loss of water to form an imine linkage. / Graduation date: 1968

The rate of oxidation of copper metal to copper (I) by copper (II) in the presence of organic ligands has been determined for a variety of ligands using a flow injection analysis system to sample the reaction. The results obtained using the FIA sampling method compare favorably with those obtained in eariler work when allyl alcohol was used as the ligand. The mechanism proposed in that earlier work has been shown to be a general mechanism for the oxidation of copper metal in the presence of complexing ligands. It was found that some ligands increase the rate of the reaction beyond the ability of conventional FIA to sample many data points before the reaction reaches equilibrium. A method has been developed that allows for more rapid sampling of the reaction mixture by the FIA system, resulting in overlapping FIA peaks. These overlapping peaks can then be deconvolved by an iterative curve fitting/digital subtraction technique in which each peak in the FIA output is fitted to a model function and subtracted from the output, thus removing its effect on the peak height of subsequent peaks. The iodination of acetylacetone in acidic solution was also studied by the fast injection FIA technique. Iodine was generated coulometrically in the solution containing acetylacetone and hydrochloric acid. By this method, the keto-enol equilibrium constant of acetylacetone was determined over a range of pH's. They ranged from 2.3 x 10⁻⁵ at a pH of 2.63 to 1.8 x 10⁻⁴ at a pH of 4.03. It was also found that the reaction was first order with respect to the acetylacetone concentration.

The optimization of the competitive binding and sandwich immunoassays was investigated in order to examine some commonly encountered experimental aberrations. Experimental and theoretical results were integrated to provide a better understanding of the causes of the "hook" or "prozone" effect. The "hook" generates ambiguous results for the test sample in an immunoassay. The competitive binding assay manifests a low dose "hook" effect while the sandwich immunoassay demonstrates a high dose "hook" effect. Human growth hormone (hGH) having no repeating epitopes was examined as a model for the "hook" effect in the competitive binding assay. Three model analytes, hGH, the dimeric form of hGH (D-hGH, having a discrete number of repeating epitopes) and ferritin (multiple epitopes) with differing immunological properties were employed. To elucidate the low dose "hook" effects in the competitive binding assay the interaction of two different monoclonal antibodies with hGH was investigated. The individual monoclonal antibodies show normal behavior in a competitive binding assay, but mixtures of antibodies demonstrate a "hook" attributed to formation of multicomponent complexes in solution. Size exclusion chromatography was employed identify higher molecular weight complexes. The experimental data were supported by theoretical models assuming a circular tetrameric complex formation. The one-step sandwich immunoassay suffers from the "hook" effect irrespective of the analyte characteristics. Model analytes, hGH, D-hGH, and ferritin offer new insights into general guidelines for assay procedures allowing the analyst to quickly optimize assay conditions without a priori knowledge of the immunological characteristics of the antibody or the antigen. Experimental and theoretical data show that the high capacity solid-phase antibodies shifted the "hook" to relatively higher analyte concentrations. The effect of the concentration of labeled antibody on assay response was examined theoretically. The cause of the "hook" effect in two-step sandwich immunoassay is attributed to the desorption of the bound analyte most likely due to a conformational change after the labeled antibody interacts with the several epitopes of the analyte, hence the assay for hGH shows no "hook" effect. Two different protocols for D-hGH were implemented. These assays demonstrated a "hook" effect if the labeled antibody was allowed to interact with more than one epitope of the analyte. Multiple epitope interactions with the labeled antibody, as exemplified by ferritin, demonstrate the "hook" effect. The effect of the ferritin concentration, capture antibody and the labeled antibody was examined.

Capillary Zone Electrophoresis (CZE) is an important analytical technique used to analyze the chemical makeup of small samples with volumes on the order of picoliters. However, in the quest for smaller sample volumes, smaller capillaries are used. This research has focussed on analyzing the optical properties of these small capillaries to determine if and how they affect the fluorescence spectrum of the molecules inside the capillary. In the course of this research we have determined that for fluorescent liquids, there is no evidence of angular dependencies in the fluorescent emissions for the total intensity or the linear polarization components of the total intensity. This angular isotropy does not show any noticeable temperature dependence. We have also determined that the fluorescence spectrum from these molecules is not smooth. Even with weak reflections from the capillary inner walls, enough feedback exists to superimpose resonances on the otherwise smooth spectrum. If the reflectivity is high enough, typically when the core index of refraction is higher than the capillary's index, it is possible to cause some materials to lase. This lasing has been shown in a variety of systems by several researchers. However, total internal reflection is not required for resonances, although weak, to be present. Resonances between the cavity dimensions and the incident laser light are also responsible for a nonlinearity in fluorescence intensity normalized to volume. This normalized intensity nonlinearity may also be due partially to photoinduced decay processes that were observed. In any event, some care is required in the selection of capillary size for CZE in order to maximize fluorescence intensity per unit volume from the capillary. The use of small capillaries exposes the experimenter to a wide variety of physical phenomena which make their use and appropriate interpretation of the data somewhat complex, although manageable.

The use of scientifically operated charge-transfer device array detectors are investigated for several spatial and spectroscopic optical imaging applications in chemical analysis and are described in this dissertation. The improved optical detection capabilities of this class of solid state detectors, made up of both charge-injection and charge-coupled device (CCD) detectors, offers a number of significant advantages over previous optical detection technologies utilized for these applications. The results obtained from the investigation of the use of a scientifically operated charge-injection device based echelle spectrograph system for the on-line monitoring of heteropoly acids, after chromatographic separation, for the indirect determination of phosphate, silicate and arsenate are presented. The obtained results represent an improvement over direct aspiration into an emission source for these nonmetals, showing excellent linearity over three orders of magnitude. Results from the evaluation of a scientific CCD detector based technique for the luminescence imaging of latent fingerprint residues on glass and paper, after chemical treatment with fluorescence enhancement agents, is presented. The use of a scientific CCD for fingerprint imaging is demonstrated to be a viable alternative to those methodologies requiring the use of high power laser excitation sources and photographic film imaging. Results of the use of a scientific CCD for the spectroscopic determination of seawater pH are also presented. Discussion is given to general system design, detector characteristics and modes of operation which will result in a ship board instrument capable of making both sensitive fluorometric and precise absorbance measurements. Additional investigations presented include the use of a scientific CCD for the in situ detection of DNA fragments separated in agarose electrophoretic slab gels and aflatoxins separated on thin-layer chromatographic plates. Scientific CCD detection, in contrast to previous optical detection techniques, is demonstrated here to provide for a competitive means of component detection in these media, offering significant advantages in speed, precision and ease of analysis.