Potassium ions are the most abundant cation and potassium transport is essential in maintaining cellular homeostasis through the regulation of cell turgor and cytoplasmic pH. It allows bacteria to grow and survive, therefore, the potassium pool needs to be strictly controlled, which is mainly performed by transport systems that contain a KTN domain. The potassium efflux system, Kef, is such a KTN-bearing system and it is widespread among Gram negative bacteria. The system provides protection against harmful electrophiles through cytoplasmic acidification. Kef is a glutathione-regulated protein: it is inhibited by glutathione (GSH), but it becomes activated by binding glutathione-S-conjugates (GSX), that are formed in the presence of electrophiles. GSH or GSX are bound in the same pocket that is located in a cytosolic regulatory domain which controls the K+ flux. Previous studies already showed that bacterial growth is inhibited when the gating of Kef is manipulated, which makes Kef a potential target for developing novel antibacterial drugs. Structure-Function studies have already lead to a better understanding of the regulation of potassium efflux activity, but no quantitative analyses had been performed until now. A simplified model Kef system (SdKef) is presented and a novel assay was developed that provided new insights into the structural components necessary for the gating of Kef. This assay makes the search for modulators of Kef, and therefore potential novel antibacterial drugs, more easily accessible. Another objective was to identify the nucleotide(s) bound and to determine its role in controlling the Kef system. This nucleotide was identified as AMP which is essential for stability of the Kef system.

No description available.

The phosphine crown ether, 16-(4'diphenylphosphinophenyl)-1,4,7,10,13-pentaoxa-16azacyclooctadecane (III), was synthesized using a reaction scheme beginning with n-phenyldiethanolamine and the dichloride of tetraethylene glycol, with an overall yield of 4%. Platinum and Palladium complexes of the ligand, of the form MC12L2, were synthesized as well. 13C NMR and picrate extraction data indicate III and IV (the crown-5 analog) both moderately bind sodium (14%, 15%) and potassium ions (17%, 28%). Compound V (a crown-5, triphenylphosphine-based ligand) will bind both sodium and potassium ions as well (18%, 6%). When IV is complexed to nickel carbonyl (Ni(CO)3), the addition of sodium and potassium ions cause the Al carbonyl stretching frequency to increase slightly (0.3 cm-1, 0.2 cm 1). For comparison, the addition of a proton causes the A1 carbonyl stretching frequency to increase 5.2 cm-1. However, the shift in the A1 carbonyl stretching frequency upon the addition of sodium or potassium ions indicates that ion binding by the crown ether is communicated to the phosphorus and finally to the carbonyl groups.Ball State UniversityMuncie, IN 47306 / Department of Chemistry

No description available.

Solid-state NMR (ssNMR) has matured into a versatile method to provide structural information, probe protein dynamics and detect small molecule binding and -protein interaction of a variety of biomolecular assemblies including amyloid fibrils, viral particles and membrane proteins. Membrane proteins embedded in liposomes are natural targets for ssNMR as their native states are solids. Magic angle spinning (MAS) ssNMR studies using moderate spinning frequencies provide detailed structural information and probe subtle conformational change. Development of fast magic angle spinning ssNMR enables proton-detection which increases sensitivity and facilitates protein dynamics measurements. In this dissertation, we applied moderate and fast MAS ssNMR to study potassium ion channel and protein dynamics Chapter 1 will introduce concepts and theory of solid-state NMR pulse sequences and experiments. Chapter 2 will discuss the application and perspectives of solid-state NMR to membrane protein systems. In Chapter 3, we test an allostery mechanism for inactivation using a KcsA mutant (H25R/E118A) that exhibits an open pH gate across a broad range of pH values. We present solid-state NMR measurements of this open mutant at neutral pH to probe the affinity for potassium at the selectivity filter. This result strongly supports our assertion that the open pH gate allosterically affects the potassium binding affinity of the selectivity filter. In this mutant the protonation state of a glutamate residue (E120) in the pH sensor is sensitive to potassium binding, suggesting that this mutant also has flexibility in the activation gate and is subject to transmembrane allostery. In Chapter 4, I optimize protein expression, purification and reconstitution into native environment protocols of a bacterial potassium transporter, KtrB. In chapter 5, methods and experimental details of setting up 60 and 40 kHz fast MAS ssNMR are discussed. With fast MAS ssNMR setup, multidimensional NMR experiments with higher sensitivity could be collected on a perdeuterated sample with less sample mass required. In Chapter 6, we employ fast MAS ssNMR to measure bulk and residue site-specific 15N and carbonyl 13C relaxation of microcrystalline ubiquitin. Carbonyl R1ρ relaxation profiles provide additional information on protein backbone dynamics.

Kaliumionen (K⁺) sind die am häufigsten vorkommenden anorganischen Kationen in Pflanzen. Gemessen am Trockengewicht kann ihr Anteil bis zu 10% ausmachen. Kaliumionen übernehmen wichtige Funktionen in verschiedenen Prozessen in der Pflanze. So sind sie z.B. essentiell für das Wachstum und für den Stoffwechsel. Viele wichtige Enzyme arbeiten optimal bei einer K⁺ Konzentration im Bereich von 100 mM. Aus diesem Grund halten Pflanzenzellen in ihren Kompartimenten, die am Stoffwechsel beteiligt sind, eine kontrollierte Kaliumkonzentration von etwa 100 mM aufrecht.<br><br> Die Aufnahme von Kaliumionen aus dem Erdreich und deren Transport innerhalb der Pflanze und innerhalb einer Pflanzenzelle wird durch verschiedene Kaliumtransportproteine ermöglicht. Die Aufrechterhaltung einer stabilen K⁺ Konzentration ist jedoch nur möglich, wenn die Aktivität dieser Transportproteine einer strikten Kontrolle unterliegt. Die Prozesse, die die Transportproteine regulieren, sind bis heute nur ansatzweise verstanden. Detailliertere Kenntnisse auf diesem Gebiet sind aber von zentraler Bedeutung für das Verständnis der Integration der Transportproteine in das komplexe System des pflanzlichen Organismus. <br><br> In dieser Habilitationsschrift werden eigene Publikationen zusammenfassend dargestellt, in denen die Untersuchungen verschiedener Regulationsmechanismen pflanzlicher Kaliumkanäle beschrieben werden. Diese Untersuchungen umfassen ein Spektrum aus verschiedenen proteinbiochemischen, biophysikalischen und pflanzenphysiologischen Analysen. Um die Regulationsmechanismen grundlegend zu verstehen, werden zum einen ihre strukturellen und molekularen Besonderheiten untersucht. Zum anderen werden die biophysikalischen und reaktionskinetischen Zusammenhänge der Regulationsmechanismen analysiert. Die gewonnenen Erkenntnisse erlauben eine neue, detailliertere Interpretation der physiologischen Rolle der Kaliumtransportproteine in der Pflanze. / Potassium ions (K⁺) are the most abundant anorganic cations in plants. They can constitute up to 10% of the plant dry weight. Potassium ions play important roles in different processes in the plant. For example, they are essential for growth and for metabolism. Many important enzymes work optimally at a K⁺ concentration within the range of about 100 mM. Therefore, plant cells maintain a controlled potassium concentration of approximately 100 mM in their compartments, which are involved in metabolism. <br><br> The uptake of potassium ions from the soil and their transport within the plant and within a plant cell is accomplished by different potassium transporter proteins. However, the maintenance of a stable K⁺ concentration is only possible if the activity of these transporter proteins is subject to strict control. Up today the processes regulating the transporter proteins are only rudimentarily understood. More detailed knowledge in this area is, however, of central importance for the understanding of the integration of the transporter proteins into the complex system of the plant organism. <br><br> This Habilitation-thesis summarizes own publications, in which the investigations of different regulation mechanisms of plant potassium channels are described. These investigations cover a spectrum of different protein-biochemical, biophysical and plant-physiological analyses. In order to understand the regulation mechanisms, on the one hand their structural and molecular characteristics are examined. On the other hand the biophysical and reaction-kinetic properties of the regulation mechanisms are analyzed. The obtained insights allow a new, more detailed view on the physiological role of potassium transporter proteins in the plant.

A procedure was developed for dealing with two problems that have impeded the use of quantal parameters in studies of transmitter release. The first, involving temporal and spatial biasing in the estimates for the number of functional release sites (n̄) and probability of release (p̄), was addressed by reducing temporal variance experimentally and calculating the bias produced by spatial variance in p (var(s)p). The second, involving inaccuracies in the use of nerve-evoked endplate potentials (EPPs), was circumvented by using only miniature EPPs (MEPPs). Intracellular recordings were made from isolated frog cutaneous pectoris, after decapitation and pithing of the animals, and the concentration of K+ ([K+]) was raised to 10 mM to increase the level of transmitter release. The number of quanta released (m̄) by the EPP was replaced by the number of MEPPs in a fixed time interval (bin), and 500 sequential bins used for each quantal estimate. With the use of 50-ms bins, estimates for var(s)p were consistently negative. This was due to too large a bin (and introduction of undetected temporal variance) because the use of smaller bins (5 ms) produced positive estimates of var(s)p. Increases in m, n, and p but not var(s)p were found in response to increases in [K+] or [Ca2+]/[Co2+]. La3+ (20 μM) produced increases in m and n, which peaked after 20 min and declined toward zero. There were also large increases in p and var(s)p, which peaked and declined only to initial control values. The increase in var(s)p was presumed to reflect La3+-induced release of Ca2+ from intracellular organelles. The results suggest that this approach may be used to obtain unbiased estimates of n̄ and p̄ and that the estimates of var(s)p may be useful for studying Ca2+ release from intraterminal organelles.