Development of protein-catalyzed capture (PCC) agents with application to the specific targeting of the E17K Point mutation of Akt1

This thesis describes the expansion and improvement of the iterative in situ click chemistry OBOC peptide library screening technology. Previous work provided a proof-of-concept demonstration that this technique was advantageous for the production of protein-catalyzed capture (PCC) agents that could be used as drop-in replacements for antibodies in a variety of applications. Chapter 2 describes the technology development that was undertaken to optimize this screening process and make it readily available for a wide variety of targets. This optimization is what has allowed for the explosive growth of the PCC agent project over the past few years.

These technology improvements were applied to the discovery of PCC agents specific for single amino acid point mutations in proteins, which have many applications in cancer detection and treatment. Chapter 3 describes the use of a general all-chemical epitope-targeting strategy that can focus PCC agent development directly to a site of interest on a protein surface. This technique utilizes a chemically-synthesized chunk of the protein, called an epitope, substituted with a click handle in combination with the OBOC in situ click chemistry libraries in order to focus ligand development at a site of interest. Specifically, Chapter 3 discusses the use of this technique in developing a PCC agent specific for the E17K mutation of Akt1. Chapter 4 details the expansion of this ligand into a mutation-specific inhibitor, with applications in therapeutics.

Lynx1 and the β2V287L mutation affect the stoichiometry of the α4β2 nicotinic acetylcholine receptor

GPI-anchored neurotoxin-like receptor binding proteins, such as lynx modulators, are topologically positioned to exert pharmacological effects by binding to the extracellular portion of nAChRs. These actions are generally thought to proceed when both lynx and the nAChRs are on the plasma membrane. Here, we demonstrate that lynx1 also exerts effects on α4β2 nAChRs within the endoplasmic reticulum. Lynx affects assembly of nascent α4 and β2 subunits, and alters the stoichiometry of the population that reaches the plasma membrane. Additionally, these data suggest that lynx1 alters nAChR stoichiometry primarily through this intracellular interaction, rather than via effects on plasma membrane nAChRs. To our knowledge, these data represent the first test of the hypothesis that a lynx family member, or indeed any GPI-anchored protein, could act within the cell to alter assembly of multi-subunit protein.

Primary cosmic-ray positrons and negatrons in 1968 at energies between 11 and 204 MeV

The cosmic-ray positron and negatron spectra between 11 and 204 MeV have been measured in a series of 3 high-altitude balloon flights launched from Fort Churchill, Manitoba, on July 16, July 21, and July 29, 1968. The detector system consisted of a magnetic spectrometer utilizing a 1000-gauss permanent magnet, scintillation counters, and a lucite Čerenkov counter.

Launches were timed so that the ascent through the 100 g/cm² level of residual atmosphere occurred after the evening geomagnetic cutoff transition. Data gathered during ascent are used to correct for the contribution of atmospheric secondary electrons to the flux measured at float altitude. All flights floated near 2.4 g/cm².

A pronounced morning intensity increase was observed in each flight. We present daytime positron and negatron data which support the interpretation of the diurnal flux variation as a change in the local geomagnetic cutoff. A large diurnal variation was observed in the count rate of positrons and negatrons with magnetic rigidities less than 11 MV and is evidence that the nighttime cutoff was well below this value.

Using nighttime data we derive extraterrestrial positron and negatron spectra. The positron-to-total-electron ratio which we measure indicates that the interstellar secondary, or collision, source contributes ≾50 percent of the electron flux within this energy interval. By comparing our measured positron spectrum with the positron spectrum calculated for the collision source we derive the absolute solar modulation for positrons in 1968. Assuming negligible energy loss during modulation, we derive the total interstellar electron spectrum as well as the spectrum of directly accelerated, or primary, electrons. We examine the effect of adiabatic deceleration and find that many of the conclusions regarding the interstellar electron spectrum are not significantly altered for an assumed energy loss of up to 50 percent of the original energy.

Photochemical electron transfer at fixed distance : a synthetic model of the photosynthetic primary process

A series of meso-phenyloctamethylporphyrins covalently bonded at the 4'phenyl position to quinones via rigid bicyclo[2.2.2]octane spacers were synthesized for the study of the dependence of electron transfer reaction rate on solvent, distance, temperature, and energy gap. A general and convergent synthesis was developed based on the condensation of ac-biladienes with masked quinonespacer-benzaldehydes. From picosecond fluorescence spectroscopy emission lifetimes were measured in seven solvents of varying polarity. Rate constants were determined to vary from 5.0x10⁹sec^-1 in N,N-dimethylformamide to 1.15x10¹⁰ Sec^-1 in benzene, and were observed to rise at most by about a factor of three with decreasing solvent polarity. Experiments at low temperature in 2-MTHF glass (77K) revealed fast, nearly temperature-independent electron transfer characterized by non-exponential fluorescence decays, in contrast to monophasic behavior in fluid solution at 298K. This example evidently represents the first photosynthetic model system not based on proteins to display nearly temperature-independent electron transfer at high temperatures (nuclear tunneling). Low temperatures appear to freeze out the rotational motion of the chromophores, and the observed nonexponential fluorescence decays may be explained as a result of electron transfer from an ensemble of rotational conformations. The nonexponentiality demonstrates the sensitivity of the electron transfer rate to the precise magnitude of the electronic matrix element, which supports the expectation that electron transfer is nonadiabatic in this system. The addition of a second bicyclooctane moiety (15 Å vs. 18 Å edge-to-edge between porphyrin and quinone) reduces the transfer rate by at least a factor of 500-1500. Porphyrinquinones with variously substituted quinones allowed an examination of the dependence of the electron transfer rate constant κ_ET on reaction driving force. The classical trend of increasing rate versus increasing exothermicity occurs from 0.7 eV≤ |ΔG^0'(R)| ≤ 1.0 eV until a maximum is reached (κ_ET = 3 x 10⁸ sec^-1 rising to 1.15 x 10¹⁰ sec^-1 in acetonitrile). The rate remains insensitive to ΔG⁰ for ~ 300 mV from 1.0 eV≤ |ΔG^0’(R)| ≤ 1.3 eV, and then slightly decreases in the most exothermic case studied (cyanoquinone, κ_ET = 5 x 10⁹ sec^-1).