Energy-Coupling-Factor (ECF)-Transporter sind Aufnahmesysteme für Vitamine und Übergangsmetallkationen in Prokaryoten. Sie bestehen aus den zwei unverwandten Membranproteinen S und T sowie einem Paar ABC-ATPasen (A). Die S-Einheit vermittelt die Substratspezifität. Die Kombination aus der T- und den A-Einheiten wird als ECF bezeichnet. In dieser Arbeit wurden Fragen zur kontrovers diskutierten Stöchiometrie der Untereinheiten von ECF-Transportern sowie zur zuvor postulierten Substrattransport-Funktion einzelner S-Komponenten auch ohne ECF untersucht. Dazu wurden der ECF-Biotintransporter BioMNY, mehrere natürlicherweise in Organismen ohne ECF existierende biotinspezifische S Einheiten (BioY) sowie zwei Vertreter der metallspezifischen ECF-Systeme genutzt. Die S-Einheit BioY des dreiteiligen Biotinimporters lag in vitro als Monomer und Dimer vor. Oligomeres BioY wurde außerdem in lebenden Bakterienzellen beobachtet. „Pull-down“-Experimente zeigten, dass die T Komponente BioN im BioMNY-Komplex zum Teil als Dimer vorlag. Wachstumsuntersuchungen bestätigten die Transportfunktion von acht solitär vorkommenden BioY. Die in vitro auch für diese BioY-Proteine nachgewiesene Dimerisierung könnte die Transportfunktion von BioY ohne ECF erklären. Die metallspezifischen S Einheiten CbiM/NikM interagieren mit für die Transportfunktion essentiellen, zusätzlichen Transmembranproteinen (N) und zeichnen sich durch eine Topologie mit sieben Transmembranhelices und einem extrem konservierten, weit in das Proteininnere hineinragenden N-Terminus aus. Die Metallbindestelle besteht aus vier Stickstoffatomen von Met1, His2 und His67 und wird durch ein Netz aus Wasserstoffbrückenbindungen stabilisiert. Die Transport¬funktion von CbiMN bzw. Nik(MN) ohne ECF wurde in vivo mittels des nickelabhängigen Enzyms Urease als Indikator für die intrazelluläre Nickelkonzentration verifiziert. Zum gegenwärtigen Zeitpunkt ist die Funktion der für den Transport essentiellen N-Komponente jedoch noch unklar. / Energy-coupling factor (ECF) transporters are uptake systems for vitamins and transition metal cations in prokaryotes. They consist of the two unrelated membrane proteins S and T, and a pair of ABC ATPases (A). The S unit mediates substrate specificity. The combination of the T and the A units is called ECF. In this thesis the controversially discussed stoichiometry of the subunits of ECF transporters and the postulated substrate transport function of solitary S units without ECF were analysed. For this purpose, the biotin-specific ECF transporter BioMNY, several biotin-specific S units (BioY) encoded in organisms lacking any recognizable ECF and two metal-specific ECF transporters were used. The S unit BioY of the tripartite biotin importer existed in vitro as monomer and dimer. Furthermore, oligomeric BioY was observed in living bacterial cells. Oligomerisation of a part of the T unit BioN in the BioMNY complex was shown by “pull-down”- experiments. Growth analyses confirmed the transport function of eight solitary BioY proteins. The dimerisation, also proved for these solitary BioY proteins in vitro, could be an explanation for the transport function of BioY without ECF. The metal-specific S units CbiM/NikM interact with additional and for the transport function essential transmembrane proteins (N). The S units consist of seven transmembrane helices and an extremely conserved N-terminus, which extends deeply into the protein. The metal-binding site consists of four nitrogen atoms from Met1, His2 and His67 and is stabilised by a series of hydrogen bonds. The transport function of CbiMN and Nik(MN) without ECF was verified respectively in vivo using the nickel-depending enzyme urease as an indicator for intracellular nickel concentration, respectively. However, the role of the N component, which is essential for transport activity, is currently under investigation.

In this work two overall subjects were addressed. 1. In recent years high pressure perturbance has become a tool to investigate the folding energy landscape, the volumetric properties and the conformational equilibria of proteins. Conformational states which are not populated at ambient conditions thus become accessible to spectroscopic characterization. In this work a high pressure application was combined with EPR spectroscopy to investigate three spin labeled proteins, BSA from Bos taurus, HisJ from Salmonella enterica serovar Typhimurium and α-synuclein from Homo sapiens. The goal of these studies was to comprehend the influence of pressure on the respective EPR spectra and to identify changes in conformational equilibria and volumetric properties of the investigated proteins. Studies on BSA revealed a negative activation volume for rotational diffusion of the spin labeled site. Moreover, a rotameric equilibrium was derived from the pressure-dependent side chain dynamics and a correlating negative partial molar volume was observed, indicating a shift of the rotameric equilibrium to lesser order. In this regard it was also shown that a chaotropic medium (guanidine hydrochloride) supports the pressure-dependent effect. Spin labeled sites in the substrate binding protein HisJ revealed to be highly influenceable by low pressures between ambient conditions and 200 bar. Pressurization induced oligomerization and precipitation of the protein. Substrate binding revealed differences in pressure-dependence with regard to a decreased precipitation effect but not in relation to oligomerization. The natively unfolded protein α-synuclein plays a key role in Parkinson´s disease and is known for forming β-sheet rich aggregates, so called amyloid fibrils. The experimental data of this work revealed that hydrostatic pressure can induce a non-amyloid aggregation of monomeric α-synuclein which produces an unspecific oligomer. Furthermore, it was shown that α-synuclein amyloid fibrils can be dissolved by hydrostatic pressure. From the pressure dependent conformational equilibrium between the monomer and the fibril form the change of the partial molar volume of the investigated site was determined. 2. The second subject of this work was focused on different import systems, ATP-binding cassette (ABC) transporters and Energy-Coupling-Factor (ECF) transporters, for amino acids, vitamins and metal ions in prokaryotes. Studies on one bacterial ABC and two ECF transporter systems from two different organisms, the histidine ABC-type transporter HisQMP2 from Salmonella enterica serovar Typhimurium, the biotin ECF-type importer BioMNY from Rhodobacter capsulatus and the cobalt-specific ECF-type transporter CbiMNQO from Rhodobacter capsulatus, were performed using DEER and cw EPR spectroscopy. The goal of the studies on HisQMP2 and BioMNY was to shed light on conformations and dynamics connected to their transporter function. Studies on CbiMNQO aimed at the detection of the substrate in the transporter´s substrate binding unit. For HisQMP2 transport cycle dependent conformational changes and interactions with the substrate binding protein HisJ were revealed. Three different distance values between sites H101R1 and H101’R1 in the transporter´s nucleotide binding domains were assigned to the apo-, the ATP-bound and the posthydrolysis state. It was shown that the closed conformation of the nucleotide binding domains is achieved only in the presence of the ligand-bound HisJ which indicates a transmembrane communication of the association of HisJ to the transporter. Furthermore, interspin distances were determined between sites D86R1-A96R1, C197R1-C104R1 and A118R1-G123R1 in the transmembrane domains HisQ and HisM revealing distinguishable conformational states which correlate to the different states of the nucleotide binding sites during the hydrolysis cycle. Measured interspin distances between HisJ and HisM in the HisQMP2 complex showed that interaction only occurred in the closed state of the HisP2 dimer, the nucleotide bound state. Two different, substrate-dependent interactions between site G24R1 in HisJ and site A96R1 in HisQMP2 were observed, revealing that the substrate-free and substrate-bound form of HisJ both associate with HisQMP2. Distance measurements between sites G24R1 and T151R1 in HisJ in the presence and absence of its substrate revealed interspin distance changes that correlate with the proteins open and closed conformation. Investigations on the ECF transporter BioMNY, reconstituted into nanodiscs, revealed a closure and reopening of the nucleotide binding domains between sites H87R1 and H87’R1 using DEER spectroscopy which delivered interspin distance values that correlate with the apo-, the ATP-bound and the posthydrolysis state of the transporter. Further experiments were aimed to shed light on the transporters substrate-translocation mechanism with regard to the so called toppling over mechanism. Unfortunately, the experiments of this work were not able to give a distinct answer with respect to the proposed model because of the transmembrane domains tendency to oligomerize when reconstituted into nanodiscs. In this work we showed that substrate uptake by the substrate binding unit CbiM of the cobalt-specific ECF transporter CbiMNQO depends on the presence of the small transmembrane protein CbiN. Measurements of spin labeled CbiMN in detergent showed oligomerization of CbiM.

ECF (Energy-Coupling Factor)-Transporter sind prokaryotische Aufnahmesysteme für Mikronährstoffe, die eine spezielle Gruppe von Transportern mit ATP-Bindekassette (ABC) darstellen. Sie beinhalten zwei asymmetrische Membranproteine, von denen eins (S) für die spezifische Bindung und Translokation des Substrates und das andere (T) für die Kopplung mit den ATPasen (A1,A2) zuständig ist. Bei ECF-Transportern der Subklasse I bilden diese Komponenten eine Einheit, während bei Vertretern der Subklasse II ein AAT-Modul mit wechselnden S-Einheiten interagiert. In der vorliegenden Arbeit wurde der Transportmechanismus, der eine Drehung der kompletten S-Einheit in der Membran beinhaltet, anhand des Biotintransporters BioMNY erstmals experimentell validiert. Durch Rekonstitution in Lipid-Nanodiscs, chemische Quervernetzung, fluoreszenz- und ESR-spektroskopische Techniken sowie einen Bindungstest mit radioaktivem Biotin wurde gezeigt, dass (i) die ATP-Bindung an die ATPasen zu einer Aufrichtung der S-Einheit (BioY) führt, (ii) diese Bewegung die Substratbeladung ermöglicht und (iii) BioY dabei ununterbrochen mit der T-Einheit (BioN) interagiert. Dies stellt einen Gegensatz zu Systemen der Subklasse II dar, für die ein ATP-abhängiger Austausch von S-Einheiten im Transportzyklus gezeigt worden war. Darüber hinaus wurde ein Escherichia coli-Stamm konstruiert, der durch Blockierung seines hochaffinen Biotintransporters und des -synthesewegs auf Spuren von Biotin nicht wachsen kann. Dieser Stamm ermöglichte einen eindeutigen Nachweis der Transportaktivität einiger solitärer BioY-Proteine. Aufgrund der einheitlichen Topologie von S-Einheiten ist ein Kippen auch für solitäre BioY-Varianten wahrscheinlich. Auch die metallspezifischen S-Einheiten CbiM und NikM besitzen ohne AAT-Modul eine basale Co2+- bzw. Ni2+-Transportaktivität. Ein ESR-spektroskopischer Kobaltnachweises zeigte, dass die aus nur zwei Membranhelices bestehende CbiN-Einheit für die Metallbeladung von CbiM essentiell ist. / ECF (Energy-Coupling Factor) transporters are a subgroup of ABC transporters that mediate uptake of micronutrients into prokaryotic cells. In contrast to canonical ABC importers, ECF transporters comprise two unrelated membrane proteins, one of which is responsible for specific and high affinity substrate binding (S) and the other one constitutes the coupling component (T) between S and the cytosolic ABC-ATPases (A1,A2). Subclass I transporters consist of four dedicated components whereas in subclass II transporters, a central AAT-module may interact with various S units. The biotin specific subclass I ECF transporter BioMNY was used to experimentally verify the hitherto hypothetic transport mechanism, which involves a rotation of the S unit within the membrane. With a series of experiments including reconstitution of BioMNY into lipid nanodiscs, site-specific cross-linking, a substrate binding assay with radioactive biotin and both fluorescence and EPR spectroscopic techniques, the ATP-dependent rotation of BioY (S) as a prerequisite for substrate binding and release was shown for the first time for an ECF transporter. Unlike subclass II transporters, for which an ATP-dependent release of the S unit was proposed, BioY interacts continuously with BioN (T) during the transport cycle. In a second focus of the work, an Escherichia coli reporter strain for biotin transporters was constructed. Due to inactivation of both biotin synthesis and the intrinsic high affinity biotin transporter, this strain was not capable of growing on trace amounts of biotin. With the use of this strain, transport activity of recombinantly produced solitary BioY proteins that naturally lack other ECF components was evidenced. Transport activity in the absence of AAT modules is also a feature of the Co2+ and Ni2+ specific S components CbiM and NikM. An EPR spectroscopic Co2+ detection assay helped underscoring the essential role of the small membrane protein CbiN for Co2+ loading of CbiM.