Single molecule studies of enzymes have revealed that nominally identical individual enzyme molecules are functionally heterogeneous. Different individual molecules exhibit different catalytic rates under identical conditions, and individual enzyme molecules show fluctuating rates over broad timescales. The structural basis and the biological sources for such heterogeneity remains poorly understood. Herein, studies are presented of the β-galactosidase from Escherichia coli, using capillary electrophoresis with laser-induced fluorescence (CE-LIF), to investigate the sources of catalytic heterogeneity at the single molecule level. Limited proteolysis as a possible source for single molecule heterogeneity, and for the changes in activity of a population of individual molecules over time, was investigated by inducing enzyme expression in two E.coli strains in the presence of a broad spectrum of protease inhibitors. The effect of protease inhibitors was found to be limited. β-Galactosidase was expressed from a lacZ linear template from two different E. coli strains using an in vitro protein expression system to determine if in vitro synthesized enzyme was identical to its in vivo counterpart. In vitro synthesized enzyme was found to be less active than in vivo sources. The differences were attributed to deficient N-terminal methionine removal and the higher rates of translation error associated with in vitro protein synthesis. Single molecule separations revealed that individual molecules of β-galactosidase were electrophoretically distinct, and that the electrophoretic heterogeneity was independent of source of enzyme, method of measurement, or of capillary coating. Electrophoretic modeling indicated that slight variation of hydrodynamic radius is the most likely source of electrophoretic mobility heterogeneity. The extent of single molecule catalytic variation was reduced in a mutant with a hyperaccurate translation phenotype implying that translation error is a source of the heterogeneity. Streptomycin-induced translation error reduced average activity, but did not lead to an increase in catalytic heterogeneity. No relationship between translation error and electrophoretic heterogeneity was observed. A novel CE-LIF assay was developed for the continuous monitoring of the catalytic activity and electrophoretic mobility of individual β-galactosidase molecules. Thermally-induced catalytic fluctuations were observed suggesting that individual enzyme molecules were capable of conformational fluctuations that supported different catalytic rates.

Data is presented for a range of experiments that have been performed using a selected ion flow tube (SIFT) instrument operated at room temperature (~ 298K) with carrier gas pressures typically in the range of 0.3 – 0.6 Torr. The majority of the experiments discussed are performed on a Voice100 instrument that has not been described in detail previously. The Voice100 is a novel instrument that has been designed particularly for quantitative trace gas analysis using the SIFT-MS technique. A mixture of helium and argon carrier gases are employed in the Voice100 flow tube. By mixing carrier gases, the flow dynamics and diffusion characteristics of a flow tube are altered when compared to classic single carrier gas models. Therefore firstly, optimal flow conditions for the operation of a Voice100 are characterised. The diffusion of an ion in a mixture of carrier gases is then characterised using theoretical models and experimental techniques. This research requires that a new parameter Mp be defined regarding the mass discrimination of an ion in the non-field-free region near the downstream ion sampling orifice. Furthermore, a new method is described for the simultaneous measurement of rate coefficients for the reactions of H₃O⁺.(H₂O)n (n = 1, 2, 3) ions with analytes. Rate coefficients and branching ratios for the reactions of SIFT-MS precursor ions with specific analytes related to four individual applications are presented. For each application, the kinetic parameters are determined so as to facilitate the quantitative detection of the analytes relevant to that application. The GeoVOC application involves the measurement of hydrocarbon concentrations in the headspace of soil and water across a range of humidities. Alkyl esters are investigated to allow for the quantitative detection of each compound in fruits and vegetables. Chemical warfare agents, their surrogates and precursor compounds are studied which allows for the quantitative or semi-quantitative detection of a range of highly toxic compounds. Finally, 17 compounds classified by the US-EPA as hazardous air pollutants are studied that enables SIFT-MS instruments to replicate sections of the TO-14A and TO-15 methods.

In dieser Dissertation werden die Bindungseigenschaften von Rezeptor-Ligand-Komplexen mit Hilfe von Optischen Pinzetten untersucht. Aufgrund ihrer außerordentlichen Orts- (2nm) und Kraftauflösung (0,2pN) ist es möglich, diese spezifischen Interaktionen anhand einzelner Bindungsereignisse zu charakterisieren. Als Modellsysteme dienen die Wechselwirkungen zwischen den phosphorylierungsspezifischen, monoklonalen Antikörpern HPT-101 und HPT-104 und dem Morbus Alzheimer relevanten Tau-Peptid. Dieses pathogen veränderte Peptid wird krankheitsspezifisch an den Aminosäuren Threonin231 und Serin235 phosphoryliert, sodass die Detektion dieses Phosphorylierungsmusters mit Hilfe von monoklonalen Antikörpern eine mögliche Früherkennung der Alzheimer-Krankheit darstellt. Eine notwendige Voraussetzung dafür ist jedoch die exakte Kenntnis der Bindungsstellen des Liganden am Rezeptor. Ziel des ersten Teils dieser Arbeit ist es, das Epitop des monoklonalen Antikörpers HPT-101 zu bestimmen. Dazu werden mögliche bindungsrelevante Aminosäuren durch ein Alanin ausgetauscht (Alanin-Scan) und so insgesamt sieben neue Tau-Isoformen aus dem ursprünglichen doppelt-phosphorylierten Peptid Tau[pThr231/pSer235] hergestellt. Die jeweiligen Interaktionen zwischen den modifizierten Peptiden und dem Antikörper werden mit der dynamischen Kraftspektroskopie untersucht und mit Hilfe eines literaturbekannten Modells analysiert. Die sich daraus ergebenden Bindungsparameter (Lebensdauer der Bindung, charakteristische Bindungslänge, freie Aktivierungsenergie und Affinitätskonstante) werden zusammen mit den relativen Bindungshäufigkeiten erstmals genutzt, um Kriterien für essentielle, sekundäre und nicht-essentielle Aminosäuren im Tau-Peptid zu definieren. Bemerkenswerterweise existieren für insgesamt drei dieser Parameter (Bindungslebensdauer, Bindungslänge und Affinitätskonstante) scharfe Klassengrenzen, mit denen eine objektive Einteilung des Epitops von Antikörper HPT-101 möglich ist. Die erhaltenen Ergebnisse sind in überzeugender Weise im Einklang mit ELISA-Messungen zu diesem Antikörper-Peptid-Komplexen, sie liefern jedoch einen tieferen Einblick in die Natur einer spezifischen Bindung, da den kraftspektroskopischen Messungen auch die Bindungskinetik zugänglich ist. Das zweite Projekt der vorliegenden Dissertation etabliert eine Methodik, um die Datenvarianz in der Bestimmung der relativen Bindungshäufigkeit zu reduzieren. Anhand einer Kombination aus Fluoreszenz- und kraftspektroskopischen Messungen werden die Wechselwirkungen zwischen dem monoklonalen Antikörper HPT-104 und dem fluoreszenzmarkierten Peptid Tau[Fl-pThr231] untersucht. Es wird gezeigt, dass durch Vorsortieren der Peptid-beschichteten Kolloide, entsprechend ihrer Oberflächenbeladung, die Datenvarianz in den Bindungshäufigkeitsmessungen signifikant reduziert wird.

The syntheses, structures, and magnetic properties of a series of di/trivalent hydridotris(3,5-dimethylpyrazol-1-yl)borate (Tp*) cyanomanganates were investigated. Treatment of manganese(III)acetylacetonate with KTp* followed by tetra(ethyl)- ammonium cyanide affords [NEt4][(Tp*)MnII(acac)(CN)] (1). Attempts to oxidize 1 with iodine affords {(Tp*)MnII(κ2O-acac-CN)}n (7); a minor complex {[NEt4][(Tp*)MnII(κ2O-acac-3-CN)]2(µ-CN) (8) was also isolated. The manganese(II) complex [NEt4][(Tp*)MnII(κ2O-acac-3-CN)(κ1N -3-NC-acac)] (2) was obtained via treatment of Mn(3-acacCN)3 with KTp* and [NEt4]CN. [NEt4]2[MnII(CN)4] (3) was prepared via treatment of Mn(OTf)2 with excess [NEt4]CN. [NEt4][(Tp*)MnIII(CN)3] (4), is prepared via treatment of 4 with Mn(3-acacCN)3, KTp* and excess [NEt4]CN. [PPN][(Tp*)MnIII(CN)3] (5) is obtained via treatment of [PPN]3[MnII(CN)6] with (Tp*)SnBu2Cl. Combination of 4 with [MnII(bipy)2(OH2)2][OTf]2 afforded a tetranuclear rectangular cluster {MnIII 2MnII 2} (9). At low temperature, {MnIII2NiII2} (10) was prepared via treatment of 4 and [Ni(II)(bipy)2(H2O)2][OTf]2. Treatment of 4 with [CoII(bipy)2(OH2)2][OTf]2 at low temperature failed to give the desired {MnIII2CoII2} complex. Magnetic measurements indicate that 1, 2, and 7 contain high-spin isotropic MnII with no long-range magnetic order observed for 7 (T > 2 K); 4 contains low-spin MnIII that likely adopt an isotropic 3A2 spin ground state. Surprisingly 9 and 10 do not exhibit slow relaxation of the magnetization (for T > 1.8 K) despite the presence of significant molecular anisotropy.

This thesis describes the development of sensitive and high-resolution fluorescence spectroscopic and microscopic techniques and their application to probe biomolecules and their interactions in solution, lipid membrane model systems and in cells. Paper I-IV are largely focused on methodological developments. In paper I, a new fluorescence method based on fluorescence correlation spectroscopy (FCS) for detecting single particles was realized, requiring no fluorescent labeling of the particles. The method can yield information both about the diffusion properties of the particles as well as about their volumes. In paper II, a modified fluorescence cross correlation spectroscopy procedure with well characterized instrumental calibration was developed and applied to study cis interactions between an inhibitory receptor and its Major Histocompatibility Complex class I ligand molecule, both within the same cellular membranes. The quantitative analysis brought new insights into the Nature killer cell’s self-regulating of tolerance and aggressiveness for immune responses. Paper III describes a multi-color STED (STimulated Emission Depletion) microscopy procedure, capable of imaging four different targets in the same cells at 40nm optical resolution, which was developed and successfully demonstrated on platelets. In paper IV, a modified co-localization algorithm for fluorescence images analysis was proposed, which is essentially insensitive to resolutions and molecule densities. Further, the performance of this algorithm and of using STED microscopy for co-localization analysis was evaluated using both simulated and experimentally acquired images. Papers V-VII have their main emphasis on the application side. In paper V, transient state imaging was demonstrated on live cells to image intracellular oxygen concentration and successfully differentiated different breast cancer cell lines and the different metabolic pathways they adopted to under different culturing conditions. Paper VI describes a FCS-based study of proton exchange at biological membranes, the size-dependence of the membrane proton collecting antenna effect as well as effects of external buffer solutions on the proton exchange, in a nanodisc lipid membrane model system. These findings provide insights for understanding proton transport at and across membranes of live cells, which has a central biological relevance. In paper VII, STED imaging and co-localization analysis was applied to analyze cell adhesion related protein interactions, which are believed to have an important modulating role for the proliferation, differentiation, survival and motility of the cells. The outcome of efforts taken to develop means for early cancer diagnosis are also presented. It is based on single cells extracted by fine needle aspiration and the use of multi-parameter fluorescence detection and STED imaging to detect protein interactions in the clinical samples. Taken together, detailed studies at a molecular level are critical to understand complex systems such as living organisms. It is the hope that the methodologies developed and applied in this thesis can contribute not only to the development of fundamental science, but also that they can be of benefit to mankind in the field of biomedicine, especially with an ultimate goal of developing novel techniques for cancer diagnosis. / <p>QC 20140609</p>

Affibody molecules are promising candidates for targeted radionuclide-based imaging and therapy applications. Optimisation of targeting properties would permit the in vivo visualization of cancer-specific surface receptors with high contrast. In therapy, this may increase the ratio of radioactivity uptake between tumour and normal tissues.  This thesis work is based on 5 original research articles (papers I-V) and focuses on optimisation of targeting properties of anti-HER2 affibody molecules by optimising the labelling chemistry. Paper I and II report the comparative evaluation of the anti-HER2 ZHER2:2395 affibody molecule site specifically labelled with 111In (suitable for SPECT imaging) and 68Ga (suitable for PET imaging) using the thiol reactive derivatives of DOTA and NODAGA as chelators. The incorporation of different macrocyclic chelators and labelling with different radionuclides modified the biodistribution properties of affibody molecules. This indicates that the labelling strategy may have a profound effect on the targeting properties of radiotracers and must be carefully optimized. Paper III reports the study of the mechanism of renal reabsorption of anti-HER2 ZHER2:2395 affibody molecule. An unknown receptor (not HER2) is suspected to be responsible for the high reabsorption of ZHER2:2395 molecules in the kidneys. Paper IV reports the optimization and development of in vivo targeting properties of 188Re-labelled anti-HER2 affibody molecules. By using an array of peptide based chelators, it was found that substitution of one amino acid by another or changing its position can have a dramatic effect on the biodistribution properties of 188Re-labelled affibody molecules. This permitted the selection of –GGGC chelator whichdemonstrated the lowest retention of radioactivity in kidneys compared to other variants and showed excellent tumour targeting properties. Paper V reports the preclinical evaluation of 188Re-ZHER2:V2 as a potential candidate for targeted radionuclide therapy of HER2-expressing tumours. In vivo experiments in mice along with dosimetry assessment in both murine and human models revealed that future human radiotherapy studies using 188Re-ZHER2:V2 may be feasible. It would be reasonable to believe that the results of optimisation of anti-HER2 affibody molecules summarized in this thesis can be of importance for the development of other scaffold protein-based targeting agents.

In recent years, semiconductor quantum dots (QDs) have arisen as a new class of fluorescent probes that possess unique optical and electronic properties well-suited for single-molecule imaging of dynamic live cell processes. Nonetheless, the large size of conventional QD-ligand constructs has precluded their widespread use in single-molecule studies, especially on cell interiors. A typical QD-ligand construct can range upwards of 35 nm in diameter, well exceeding the size threshold for cytosolic diffusion and posing steric hindrance to binding cell receptors. The objective of this research is to develop tagging strategies that allow QD-ligand conjugates to specifically bind their target proteins while maintaining a small overall construct size. To achieve this objective, we utilize the HaloTag protein (HTP) available from Promega Corporation, which reacts readily with a HaloTag ligand (HTL) to form a covalent bond. When HaloTag ligands are conjugated to size-minimized multidentate polymer coated QDs, compact QD-ligand constructs less than 15 nm in diameter can be produced. These quantum dot-HaloTag ligand (QD-HTL) conjugates can then be used to covalently bind and track cellular receptors genetically fused to the HaloTag protein. In this study, size-minimized quantum dot-HaloTag ligand conjugates are synthesized and evaluated for their ability to bind specifically to purified and cellular HTP. The effect of QD-HTL surface modifications on different types of specific and nonspecific cellular binding are systematically investigated. Finally, these QD-HTL conjugates are utilized for single-molecule imaging of dynamic live cell processes. Our results show that size-minimized QD-HTLs exhibit great promise as novel imaging probes for live cell imaging, allowing researchers to visualize cellular protein dynamics in remarkable detail.

We applied single-molecule fluorescence (SMF) methods to probe the properties of individual fluorescent probes, and to characterize the proteins of interest to which these probes were attached. One remarkable advantage of SMF spectroscopy is the ability to investigate heterogeneous subpopulations of the ensemble, which are buried in ensemble averaging in other measurements. Other advantages include the ability to probe the entire dynamic sequences of a single molecule transitioning between different conformational states. For the purpose of having an extended observation of single molecules, while maintaining the native nanoscale surroundings, we developed an improved vesicle preparation method for encapsulating scarce biological samples. SMF investigations revealed that molecules trapped in vesicles exhibit nearly ideal single-emitter behavior, which therefore recommends the vesicle encapsulation for reproducible and reliable SMF studies. Hyperactive Signal-Transducer-and-Activator-of-Transcription 3 (STAT3) protein contributes significantly to human cancers, such as leukemia and lymphoma. We have proposed a novel therapeutic strategy by designing a cholesterol-based protein membrane anchor (PMA), to tether STAT3 to the cell membrane and thus inhibit unwanted transcription at the cell nucleus. We designed in vitro proof-of-concept experiments by encapsulating STAT3 and PMAs in phospholipid vesicles. The efficiency and the stability of STAT3 anchoring in the lipid membrane were interrogated via quantitative fluorescence imaging and multiparameter SMF spectroscopy. Our in vitro data paved the way for the in vivo demonstration of STAT3 inhibition in live cells, thus demonstrating that PMA-induced protein localization is a conceptually viable therapeutic strategy. The recent discovery of intrinsically disordered proteins (IDPs) highlights important exceptions to the traditional structure-function paradigm. SMF methods are very suited for probing the properties of such highly heterogeneous systems. We studied in detail the effects of electrostatics on the conformational disorder of an IDP protein, Sic1 from yeast, and found that the electrostatic repulsion is a major factor controlling the dimensions of Sic1. Based on our data we also conclude that a rod-like shape seems a better candidate than a random Gaussian chain to describe and predict the behavior of Sic1.

We applied single-molecule fluorescence (SMF) methods to probe the properties of individual fluorescent probes, and to characterize the proteins of interest to which these probes were attached. One remarkable advantage of SMF spectroscopy is the ability to investigate heterogeneous subpopulations of the ensemble, which are buried in ensemble averaging in other measurements. Other advantages include the ability to probe the entire dynamic sequences of a single molecule transitioning between different conformational states. For the purpose of having an extended observation of single molecules, while maintaining the native nanoscale surroundings, we developed an improved vesicle preparation method for encapsulating scarce biological samples. SMF investigations revealed that molecules trapped in vesicles exhibit nearly ideal single-emitter behavior, which therefore recommends the vesicle encapsulation for reproducible and reliable SMF studies. Hyperactive Signal-Transducer-and-Activator-of-Transcription 3 (STAT3) protein contributes significantly to human cancers, such as leukemia and lymphoma. We have proposed a novel therapeutic strategy by designing a cholesterol-based protein membrane anchor (PMA), to tether STAT3 to the cell membrane and thus inhibit unwanted transcription at the cell nucleus. We designed in vitro proof-of-concept experiments by encapsulating STAT3 and PMAs in phospholipid vesicles. The efficiency and the stability of STAT3 anchoring in the lipid membrane were interrogated via quantitative fluorescence imaging and multiparameter SMF spectroscopy. Our in vitro data paved the way for the in vivo demonstration of STAT3 inhibition in live cells, thus demonstrating that PMA-induced protein localization is a conceptually viable therapeutic strategy. The recent discovery of intrinsically disordered proteins (IDPs) highlights important exceptions to the traditional structure-function paradigm. SMF methods are very suited for probing the properties of such highly heterogeneous systems. We studied in detail the effects of electrostatics on the conformational disorder of an IDP protein, Sic1 from yeast, and found that the electrostatic repulsion is a major factor controlling the dimensions of Sic1. Based on our data we also conclude that a rod-like shape seems a better candidate than a random Gaussian chain to describe and predict the behavior of Sic1.

There is a need for new antiviral drugs. Especially for the treatment of adenovirus infections, since no approved anti-adenoviral drugs are available. Adenovirus infections in healthy persons are most often associated with respiratory disease, diarrhea and infections of the eye. These infections can be severe, but are most often self-limiting. However, in immunocompromised patients, adenovirus infections are associated with morbidity and high mortality rates. These patients are mainly stem cell or bone marrow transplantation recipients, however solid organ transplantation recipients or AIDS patients may be at risk as well. In addition, children are at higher risk to develop disseminated disease. Due to the need for effective anti-adenoviral drugs, we have developed a cell based screening assay, using a replication-competent GFP expressing adenovirus vector based on adenovirus type 11 (RCAd11GFP). This assay facilitates the screening of chemical libraries for antiviral activity. Using this assay, we have screened 9800 small molecules for anti-adenoviral activity with low toxicity. One compound, designated Benzavir-1, was identified with activity against representative types of all adenovirus species. In addition, Benzavir-1 was more potent than cidofovir, which is the antiviral drug used for treatment of adenovirus disease. By structure-activity relationships analysis (SAR), the potency of Benzavir-1 was improved. Hence, the improved compound is designated Benzavir-2. To assess the antiviral specificity, the activity of Benzavir-1 and -2 on both types of herpes simplex virus (HSV) was evaluated. Benzavir-2 displayed better efficacy than Benzavir-1 and had an activity comparable to acyclovir, which is the original antiviral drug used for therapy of herpes virus infections. In addition, Benzavir-2 was active against acyclovir-resistant clinical isolates of both HSV types. To expand our search for compounds with antiviral activity, we turned to the natural products. An ethyl acetate extract library was established, with extracts derived from actinobacteria isolated from sediments of the Arctic Sea. Using our screening assay, several extracts with anti-adenoviral activity and low toxicity were identified. By activity-guided fractionation of the extracts, the active compounds could be isolated. However, several compounds had previously been characterized with antiviral activity. Nonetheless, one compound had uncharacterized antiviral activity and this compound was identified as a butenolide. Additional butenolide analogues were found and we proposed a biosynthetic pathway for the production of these compounds. The antiviral activity was characterized and substantial differences in their toxic potential were observed. One of the most potent butenolide analogues had minimal toxicity and is an attractive starting point for further optimization of the anti-adenoviral activity. This thesis describes the discovery of novel antiviral compounds that targets adenovirus and HSV infections, with the emphasis on adenovirus infections. The discoveries in this thesis may lead to the development of new antiviral drugs for clinical use.