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The carnitine transporter CaiT from Escherichia coli belongs to the betaine, choline, and carnitine transporter family of secondary transporters. It acts as an L-carnitine/gamma-butyrobetaine exchanger and is predicted to span the membrane 12 times. Unlike the other members of this transporter family, it does not require an ion gradient and does not respond to osmotic stress (Jung, H., Buchholz, M., Clausen, J., Nietschke, M., Revermann, A., Schmid, R., and Jung, K. (2002) J. Biol. Chem. 277, 39251-39258). The structure and oligomeric state of the protein was examined in detergent and in lipid bilayers. Blue native gel electrophoresis indicated that CaiT was a trimer in detergent solution. This result was further supported by gel filtration and cross-linking studies. Electron microscopy and single particle analysis of the protein showed a triangular structure of three masses or two parallel elongated densities. Reconstitution of CaiT into lipid bilayers yielded two-dimensional crystals that indicated that CaiT was a trimer in the membrane, similar to its homologue BetP. The implications of the trimeric structure on the function of CaiT are discussed.
My graduate thesis is on the "Structural studies of membrane transport proteins". Transporters are membrane proteins that have multiple membrane-spanning a-helices. They are dynamic and diverse proteins, undergoing a large conformational change and transporting wide range of susbtrates. Based on their energy source they can be classified into primary and secondary transport systems. Primary transport systems are driven by the use of chemical (ATP) or light energy, while secondary transporters utilize ion gradients to transport substrates. I began my PhD dissertation on secondary transporters by two-dimensional crystallization and electron crystallographic analysis and recently my focus also has shifted towards 3D crystallization. The following projects constitute my PhD thesis: 1) 2D crystallization of MjNhaP1 and pH induced structural change: MjNhaP1, a Na+/H+ antiporter that is regulated by pH has been implicated in homeostasis of H+ and Na+ in Methanococcus jannaschii, a hyperthermophilic archaeon that grows optimally at 85°C. MjNhaP1 was cloned and expressed in E. coli. Two-dimensional crystals were obtained from purified protein at pH4. Electron cryo-microscopy yielded an 8Å projection map. The map of MjNhaP1 shows elongated densities in the centre of the dimer and a cluster of density peaks on either side of the dimer core, indicative of a bundle of 4-6 membrane-spanning helices. The effect of pH on the structure of MjNhaP1was studied in situ in 2D crystals revealing a major change in density within the helix bundle relative to the dimer interface. This change occurred at pH6 and above. The two conformations at low and high pH most likely represent the closed and open states of the antiporter, respectively. This is the first instance where a conformational change associated with the regulation of a secondary transporter appears to map structurally. Reconstruction of 3D map and high-resolution structure by x-ray crystallography would be necessary to understand the mechanism of ion transport and regulation by pH. 2) 2D crystallization of Proline transporter: Proline transporter (PutP) from E.coli belongs the sodium-solute symporter family that includes disease related sodium dependent glucose and iodide transporter in humans. Sodium and proline are co-transported with a stoichiometry of 1:1. Purified PutP was reconstituted to yield 2D crystals that were hexagonal in nature. The 2D crystals had tendency to stack indicating their willingness to form 3D crystals. A projection map of PutP from negatively stained crystals showed trimeric arrangement of protein. Other members of the SSF family have been shown to be monomers. My analysis of oligomeric state of PutP in detergent by blue native gel indicates a monomer in detergent solution. It is likely that PutP can function as a monomer but at higher concentration and in lipid bilayer it tends to form trimer. 3) Oligomeric state and crystallization of carnitine transporter from E.coli: E.coli carnitine transporter (CaiT) belongs to the BCCT (Betaine, Carnitine and Choline) superfamily that transports molecules with quaternary amine groups. CaiT is predicted to span the membrane 12 times and acts as a L-carnitine/g-butyrobetaine exchanger. Unlike other members in this transporter family, it does not require an ion gradient and does not respond to osmotic stress. Over-expression of the protein yielded ~2mg of protein/L of culture. The structure and oligomeric state of the protein were analyzed in detergent and lipid bilayers. Blue native gel electrophoresis indicated that CaiT was a trimer in detergent solution. Gel filtration and cross-linking studies further support this. Reconstitution of CaiT into lipid bilayers resulted in 2D crystals. Analysis of negatively stained 2D crystals confirmed that CaiT is a trimer in the membrane. Initial 3D crystallization trials have been successful and currently, the crystals diffract to 6Å and are being improved. 4) Monomeric porin OmpG: OmpG is a bacterial outer membrane b-barrel protein. It is monomeric and its size (33kDa) places it as a prime candidate for a structural solution, using the recently developed method of solid state NMR (work in collaboration with Prof.Hartmut Oskinat, FMP, Berlin). A long-term aim would be to study porins as templates for designing nanopores, for DNA sequencing and identification. I have expressed OmpG in inclusion bodies and refolded at an efficiency of >90% into a functional form using detergent. OmpG was then crystallized by 2D crystallization yielding an 8Å projection map whose structure was similar to native protein. In addition, these crystals were used for structure determination by solid state NMR. An initial spectrum of heavy isotopically labeled OmpG has allowed identification of specific amino acid residues including threonine and proline. Additionally, I obtained 3D crystals in detergent that diffract to 5.5Å and are being improved.
Riboswitches are a novel class of genetic control elements that function through the direct interaction of small metabolite molecules with structured RNA elements. The ligand is bound with high specificity and affinity to its RNA target and induces conformational changes of the RNA's secondary and tertiary structure upon binding. To elucidate the molecular basis of the remarkable ligand selectivity and affinity of one of these riboswitches, extensive all-atom molecular dynamics simulations in explicit solvent ({approx}1 µs total simulation length) of the aptamer domain of the guanine sensing riboswitch are performed. The conformational dynamics is studied when the system is bound to its cognate ligand guanine as well as bound to the non-cognate ligand adenine and in its free form. The simulations indicate that residue U51 in the aptamer domain functions as a general docking platform for purine bases, whereas the interactions between C74 and the ligand are crucial for ligand selectivity. These findings either suggest a two-step ligand recognition process, including a general purine binding step and a subsequent selection of the cognate ligand, or hint at different initial interactions of cognate and noncognate ligands with residues of the ligand binding pocket. To explore possible pathways of complex dissociation, various nonequilibrium simulations are performed which account for the first steps of ligand unbinding. The results delineate the minimal set of conformational changes needed for ligand release, suggest two possible pathways for the dissociation reaction, and underline the importance of long-range tertiary contacts for locking the ligand in the complex.
Intoxication of class II chloroplasts of spinach with Cu(II) leads to inhibition of millisecond luminescence. The degree of inhibition depends on Cu (II) -concentration. The investgation of the pH dpendence of the inhibition curve of luminescence revealed that (1) there is an inhibition site of copper on the donor side of photosystem II, (2) copper (II) does not act as an uncoupler of photophosphorylation, (3) a protonation equilibrium is involved in the inhibition mechanism, and (4) copper (II) binds to a dissociated residue of a membrane protein.
ncubation of class II chloroplasts of spinach with copper in the light at pH = 8 in concentrations that inhibit oxygen evolution results in the formation of a copper (II) protein complex with the photosynthetic membrane. The EPR spectra indicate that the four nearest ligands to Cu(II) consist of three oxygen atoms and one nitrogen atom. The copper (II) protein appears to be pre dominantly associated with photosystem II. The formation of this protein as measured by the EPR signal amplitude of its room temperature spectrum correlates with the inhibition of oxygen evolution and of electron transport within photosystem I. This result indicates that the inhibition of photosynthetic electron transport by copper may be due to the formation of a copper (II) chelate with a membrane protein.
Spectrophotometric investigation of the kinetics of the spontaneous reduction of the central metal ion in K2[Mn (IV)-2-α-hydroxyethyl-isochlorine e4] acetate in aqueous alkaline solution in the absence of any reducing agent reveals that it is a pseudo-first order reaction which is specifically hydroxide ion catalyzed. The pKα-value of the acid-base equilibrium has been estimated to be 14.4.
Electron transfer to the central metal ion is the rate limiting step. The measurements of its temperature dependence yields an activation enthalpy of ∆H‡ = 12 kcal/mol and an entropy of activation ∆S‡ = - 30 e.u. thus indicating that the electron transfer step is a bimolecular reaction. The most likely reactant is water. The reduction reaction does not take place with appreciable reaction rates at physiological pH. Thus, when bound to a suitable ligand of the chlorin type, Mn (IV)-compounds are sufficiently stable with respect to autoxidation to play some role in biological redox reactions as postulated recently for the photoreactivation process of the water splitting system in photosynthesis.
The hypothesis of GLIKMAN and ZABRODA (Biochemistry [USSR] 84,, 239 [1969]) that the primary electron donor during photoreduction of manganese(III) in Mn(III)-hydroxychlorin compounds in oxygen free aqueous alkaline solutions is the axially bound OH- ion was tested with Mn(III)-2-a-hydroxyethyl-isochlorin e4. It has been shown that
1) the primary generation of OH radicals upon irradiation of the complex is highly improbable,
2) light is not essential for the reduction reaction,
3) the kinetics of photoreduction of the Mn(III)-compound in 2 N NaOH clearly is not compatible with OH radical formation.
The effect of NH4Cl on the kinetics of the back reaction of photosystem II as derived from luminescence measurements was investigated in dark adapted Chlorella in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) at different temperatures. The kinetics of the back reaction which, under these conditions, leads to the reduction of the S2 state by the primary electron acceptor Q- of photosystem II was observed to be considerably slowed down in the presence of NH4Cl.
Analysis of the kinetic results in the light of the theory of the back reaction developed by Mar and Roy (J. Theor. Biol. 48, 257-281 (1974)) revealed two opposite effects of NH4Cl to be present simultaneously:
1) The enthalpy of activation of the back reaction was lowered (catalyzing effect of NH4Cl)
2) The frequenca factor which indicates the number of collisions of the reacting molecules in the membrane per second is largely decreased (inhibitory effect of NH4Cl).
This reduction of the mobility of the recombining species of the back reaction is the predominant effect of NH4Cl. It is suggested that this effect is due to a change of the conformational state of the membrane induced by dissolution of relative large amounts of NH? within the lipid phase of the thylakoid membrane. This hypothesis is supported by the observation that the value of the exciton yield of the back reaction changes upon addition of NH4Cl.
Photosystem II
It is shown that the kinetics of the back reaction of photosystem II in the seconds time range as derived from the luminescence decay curve in the presence of DCMU is controlled by the internal pH of the thylakoids. Modifications of the conformational state of the photosynthetic membrane while leaving the internal pH unchanged, however, reaction.
The electron paramagnetic resonance of copper (II)-tetrammine nitrate in solution of methanol and water has been investigated. The data obtained from the spectra at room temperature and 97 °K together with the optical transition energies determined from single crystal polarized absorption spectra at 77 °K by other authors were used to calculate the LCAO-MO bonding parameters. The bonding orbital of the ammonia molecule cannot be described by the concept of sp2 hybridization which was exclusively used in the theory. Therefore a calculation of the overlap integral S(n) for α bonding and of the superhyperfine splitting was carried out in terms of an arbitrary hybridization parametern. For ammonia, n was taken from the Duncan-Pople hybrid wave function for the lone pair orbital. The o bonding and the out-of-plane π bonding appear to have a moderate degree of covalency (α = Ϭ = 0.91; α’= 0.49). The covalent in-plane n bonding is somewhat stronger (β = 0.87) but is by no means so strongly covalent as is observed in compounds with ligands which do not exclusively coordinate through the lone pair electrons.
At low temperature nine ligand nuclear superhyperfine structure lines corresponding to the interaction of four magnetically equivalent nitrogen nuclei have been observed. The value of α' derived from the superhyperfine splitting is in excellent agreement with that obtained from the copper nucleus hyperfine structure.
Ziel der vorliegenden Dissertation war es, die Dynamik des Retinalchromophors in archaealen, bakteriellen sowie eukaryotischen Retinalproteinen zeitaufgelöst zu untersuchen und so Informationen über die unterschiedlichen lichtgesteuerten zyklischen Reaktionen zu erhalten. Für das bakterielle Proteorhodopsin (PR) wurde die Primärdynamik im sichtbaren Spektralbereich unter D2O-Bedingungen bei unterschiedlichen pD-Werten untersucht. Es zeigte sich, dass das isomerisierte K-Photoprodukt mit zwei Zeitkonstanten im Bereich von 1 ps und 20 ps gebildet wird. Der Vergleich mit Messungen in H2O erlaubte es den kinetischen Isotopeneffekt für die Deaktivierung des S1-Zustandes zu berechnen. Die Ergebnisse weisen dabei auf unterschiedliche Wasserstoffbrückenmuster unter sauren und alkalischen Bedingungen hin. Um diesem Resultat weiter nachzugehen, wurde die D97N-Mutante untersucht, bei der der primäre Protonenakzeptors ungeladen vorliegt. Die gefundene Primärdynamik von PR D97N läuft nur unwesentlich langsamer ab als die des Wildtyp-Proteins bei pD 6,4. Um weitergehende Einsichten in die Primärdynamik von PR zu erlangen, wurden am Wildtyp-Protein sowie der D97N-Mutante transiente Absorptionsmessungen im Bereich der C=C- und C=N-Schwingung des Retinals durchgeführt. Es stellte sich heraus, dass die Quantenausbeute der K-Bildung unabhängig vom pD-Wert ist. In einem weiteren Schritt wurde der Einfluss des hochkonservierten His-75 auf die Isomerisierungsdynamik untersucht. Hierfür wurden die Mutanten H75N und H75M verwendet. Die Kurzzeitmessungen lassen keinen ausgeprägten Einfluss auf die Isomerisierungsdynamik erkennen. Auch der nachfolgende Teil des Photozyklus war im Blickpunkt dieser Arbeit. Die Tieftemperaturstudien im sichtbaren Spektralbereich erlaubten das in kinetischen Messungen nicht beobachtete M-Intermediat des sauren Photozyklus nachzuweisen. Um strukturelle Einblicke in den Photozyklus zu erlangen und die am Pumpvorgang beteiligten Aminosäuren zu identifizieren, wurden nachfolgend Tieftemperaturuntersuchungen im infraroten Spektralbereich durchgeführt. Die Implementierung eines Faserspektrometers in den Strahlengang des FTIR-Aufbaus erlaubte hierbei die simultane Aufnahme der lichtinduzierten Änderungen der Bandenposition im sichtbaren Spektralbereich und der Änderungen der Proteinstruktur sowie der Seitenketten. Für den M-Zustand bei pH 5,1 konnte gezeigt werden, dass auch hier eine Aspartat- oder Glutamat-Seitenkette als Protonenakzeptor fungiert. Weiterhin konnte dargelegt werden, dass der Photozyklus von PR nicht nur vom pKa-Wert des Protonen-akzeptors Asp-97 abhängt, sondern von einem Zusammenspiel mehrerer pH-abhängiger Gleichgewichte, da schon kleinste Änderungen des pH-Werts im Bereich des pKa großen Einfluss auf die beobachteten Differenzspektren sowie die Dynamik haben. Auch für das in jüngster Vergangenheit zur optogenetischen Kontrolle neuronaler Netze eingesetzte eukaryotische Retinalprotein Channelrhodopsin-2 (ChR-2) wurden umfangreiche Photozyklusstudien durchgeführt. Mit Hilfe von transienter Absorptionsspektroskopie im Sichtbaren sowie der Fluoreszenz-aufkonvertierung konnte gezeigt werden, dass der angeregte Zustand monoexponentiell mit 0,4 ps zerfällt. Die Reaktion setzt sich mit einem Kühlprozess und kleineren Änderungen der Linienbreite des K-Photoprodukts fort. Durch die schnelle Deaktivierung des angeregten Zustands war es zudem möglich die direkten Auswirkungen der Retinalisomerisierung auf die Proteinumgebung zu beobachten. Die Vielzahl ausgeprägter Differenzbanden zeigte hierbei, dass neben der schnellen Isomerisierung auch der Energietransfer der im Retinal gespeicherten Überschussenergie an das Protein sehr effizient ist. Über Blitzlichtphotolyseexperimente konnte die Langzeitdynamik des ChR-2-Photozyklus erstmals mit einer sub-µs-Zeitauflösung charakterisiert werden. Neben der für Retinalproteine typischen Abfolge von blau- und rot-verschobenen Intermediaten, ist der Photozyklus mit einer Dauer von etwa 5 s signifikant langsamer als der gemeinhin schon langsame Zyklus der sensorischen Retinalproteine. Um die Aktivierungs-barrieren des ChR-2-Photozyklus zu untersuchen, wurden weiterhin temperaturabhängige Messungen durchgeführt. Diese ergaben, dass der Photozyklus durch entropische Faktoren bestimmt wird. In einem letzten Ansatzpunkt wurde die Imidazol-Abhängigkeit der Langzeitdynamik des ChR-2-Photozyklus untersucht. Es zeigte sich, dass die Dynamik um die De- und Reprotonierung stark von diesem externen Donor beeinflusst wird. Es wurde jedoch nicht nur eine Beschleunigung der Reprotonierungsreaktion beobachtet, sondern auch der molekulare Mechanismus scheint sich nach Zugabe von Imidazol geändert zu haben. Diese Effekte können am ehesten durch eine Verstärkung des Histidin-Donor-Effekts durch das strukturell verwandte Imidazol erklärt werden. Genau dieser Einfluss externer Donor-Moleküle stand ebenfalls in einer Kurzzeit-Studie archaealer Retinalproteine im Fokus. Vorausgegangene Studien konnten zeigen, dass die Zugabe von Azid-Anionen die Isomerisierungsdynamik sowie den nachfolgenden spektral stillen Übergang der Protonenakzeptor-mutante von SRII D75N beeinflusst. Die vorliegende Arbeit stellte heraus, dass dieser Effekt ein einzigartiges Merkmal dieser Mutante ist. Abschließend wurde überdies die Bedeutung des in der Zelle in 2:2-Stöchiometrie beobachteten Transducerkomplexes auf die Primärreaktion von SRII untersucht. Es zeigte sich, dass dieser keinen Einfluss auf die Isomerisierungsdynamik aufweist, was eine wichtige Information bezüglich der Signalweitergabe sensorischer Retinalproteine ist.
Enhanced labeling density and whole-cell 3D dSTORM imaging by repetitive labeling of target proteins
(2018)
With continuing advances in the resolving power of super-resolution microscopy, the inefficient labeling of proteins with suitable fluorophores becomes a limiting factor. For example, the low labeling density achieved with antibodies or small molecule tags limits attempts to reveal local protein nano-architecture of cellular compartments. On the other hand, high laser intensities cause photobleaching within and nearby an imaged region, thereby further reducing labeling density and impairing multi-plane whole-cell 3D super-resolution imaging. Here, we show that both labeling density and photobleaching can be addressed by repetitive application of trisNTA-fluorophore conjugates reversibly binding to a histidine-tagged protein by a novel approach called single-epitope repetitive imaging (SERI). For single-plane super-resolution microscopy, we demonstrate that, after multiple rounds of labeling and imaging, the signal density is increased. Using the same approach of repetitive imaging, washing and re-labeling, we demonstrate whole-cell 3D super-resolution imaging compensated for photobleaching above or below the imaging plane. This proof-of-principle study demonstrates that repetitive labeling of histidine-tagged proteins provides a versatile solution to break the "labeling barrier" and to bypass photobleaching in multi-plane, whole-cell 3D experiments.
The discovery of antibiotics represented a key milestone in the history of medicine. However, with the rise of these life-saving drugs came the awareness that bacteria deploy defense mechanisms to resist these antibiotics, and they are good at it. Today, we appear at a crossroads between discovery of new potent drugs and omni-resistant superbugs. Moreover, the misuse of antibiotics in different industries has increased the rate of resistance development by providing permanent selective pressure and, subsequently, enrichment of multidrug resistant pathogens. As a result, antimicrobial resistance has now become an urgent threat to public health worldwide (http:// www.who.int/drugresistance/documents/surveillancereport/en/). The development of multidrug resistance (MDR) in an increasing number of pathogens, including Pseudomonas, Acinetobacter, Klebsiella, Salmonella, Burkholderia, and other Gram-negative bacteria is a serious issue. Membrane efflux pump complexes of the Resistance-Nodulation-Division (RND) superfamily play a key role in the development of MDR in these bacteria. These pumps, together with other transporters, contribute to intrinsic and acquired resistance of bacteria toward most, if not all, of the compounds available in our antimicrobial arsenal. Given the enormous drug polyspecificity of MDR efflux pumps, studies on their mechanism of action are extremely challenging, and this has negatively impacted both on the development of new antibiotics that are able to evade these efflux pumps and on the design of pump inhibitors. The collection of articles in this eBook, published as a Research Topic in Frontiers in Microbiology, section of Antimicrobials, Resistance, and Chemotherapy, aims to update the reader about the latest advances on the structure and function of RND efflux transporters, their roles in the overall multidrug resistance phenotype of Gram-negative pathogens, and on the strategies to inhibit their activities. ...
The structure of the title compound, (C15H15N2O4)[AgI2], consists of an organic 4-[3-(isonicotinoyloxy)propoxycarbonyl]pyridinium cation which has a gauche–gauche (O/C/C/C—O/C/C/C or GG’) conformation and lies on a twofold rotation axis, which passes through the central C atom of the aliphatic chain, and an inorganic [AgI2]− anion. In the complex anion, the Ag+ cation is bound to two I− anions in a linear geometry. The anion was modelled assuming disorder around a crystallographic inversion centre near the location of the Ag+ cation. The crystal packing is stabilized by a strong intermolecular N—H[cdots, three dots, centered]N hydrogen bond, which links the cations into zigzag chains with graph-set notation C(16) running along the face diagonal of the ac plane. The N-bound H atom is disordered over two equally occupied symmetry-equivalent sites, so that the molecule has a pyridinium ring at one end and a pyridine ring at the other.
In the title compound, [Ag(BF4)(C14H12N2O4)]n, the coordination of the Ag+ ion is trigonal–bipyramidal with the N atoms of two ethane-1,2-diyl bis(pyridine-3-carboxylate) ligands in the apical positions and three F atoms belonging to different tetrafluoridoborate anions in the equatorial plane. The material consists of infinite chains of [Ag(C14H12N2O4)] units running along [001], held together by BF4 − bridging anions.
Molecules of the title compound (alternative name: butane-1,4-diyl dinicotinate), C16H16N2O4, lie on a inversion centre, located at the mid-point of the central C—C bond of the aliphatic chain, giving one half-molecule per asymmetric unit. The butane chain adopts an all-trans conformation. The dihedral angle between the mean plane of the butane-3-carboxylate group [for the non-H atoms, maximum deviation = 0.0871 (15) Å] and the pyridine ring is 10.83 (7)°. In the crystal, molecules lie in planes parallel to (122). The structure features weak π–π interactions with a centroid–centroid distance of 3.9281 (11) Å.
Im Rahmen dieser Arbeit ist es gelungen, die über die 1,´-Position phenylenverbrückten Bis(silolyl)verbindungen 120, 123, 125, 156, 158, 160, 135, 137 und 139, sowie die entsprechenden Phenylsilole 126, 163 und 141 (siehe Schema 4.1) zu synthetisieren und NMR-spektroskopisch, massenspektroskopisch und zum Teil auch durch Kristallstrukturen zu charakterisieren. Weiterhin ist es gelungen eine über die 3,3´-Position verknüpfte phenylenverbrückte Bis(silolyl)verbindung 175, sowie auch die Vorstufe zu einer 2,2´-verknüpften Bis(silolyl)verbindung 177 zu synthetisieren und zweifelsfrei zu charakterisieren.
Antibody library technology represents a powerful tool for the discovery and design of antibodies with high affinity and specificity for their targets. To extend the technique to the expression and selection of antibody libraries in an eukaryotic environment, we provide here a proof of concept that retroviruses can be engineered for the display and selection of variable single-chain fragment (scFv) libraries. A retroviral library displaying the repertoire obtained after a single round of selection of a human synthetic scFv phage display library on laminin was generated. For selection, antigen-bound virus was efficiently recovered by an overlay with cells permissive for infection. This approach allowed more than 10(3)-fold enrichment of antigen binders in a single selection cycle. After three selection cycles, several scFvs were recovered showing similar laminin-binding activities but improved expression levels in mammalian cells as compared with a laminin-specific scFv selected by the conventional phage display approach. Thus, translational problems that occur when phage-selected antibodies have to be transferred onto mammalian expression systems to exert their therapeutic potential can be avoided by the use of retroviral display libraries.
Methanogenic archaea share one ion gradient forming reaction in their energy metabolism catalyzed by the membrane-spanning multisubunit complex N5-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH or simply Mtr). In this reaction the methyl group transfer from methyl-tetrahydromethanopterin to coenzyme M mediated by cobalamin is coupled with the vectorial translocation of Na+ across the cytoplasmic membrane. No detailed structural and mechanistic data are reported about this process. In the present work we describe a procedure to provide a highly pure and homogenous Mtr complex on the basis of a selective removal of the only soluble subunit MtrH with the membrane perturbing agent dimethyl maleic anhydride and a subsequent two-step chromatographic purification. A molecular mass determination of the Mtr complex by laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) and size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) resulted in a (MtrABCDEFG)3 heterotrimeric complex of ca. 430 kDa with both techniques. Taking into account that the membrane protein complex contains various firmly bound small molecules, predominantly detergent molecules, the stoichiometry of the subunits is most likely 1:1. A schematic model for the subunit arrangement within the MtrABCDEFG protomer was deduced from the mass of Mtr subcomplexes obtained by harsh IR-laser LILBID-MS.
Some physical and chemical properties of the cancerostat cyclophosphamide (generic name: ENDOXAN) and its basic constituents H3PO4 and nor-N-mustard have been calculated with the help of a modified CNDO/S-method. The spectroscopic data of the H3PO4 , which is the starting-point for a corresponding calculation of cyclophosphamide, has been studied by taking account of the 3 d electron of the phosphorus. Nor-N-mustard is a very reactive compound, characterized by the ability to split off chloride ions and to act as an alkylating agent. The binding of the nor-N-mustard to the cyclic phosphate ester (cyclophosphamide) modifies the chemical reactivity of the mustard group in an essential way, and the 3d electron of the phosphorus plays an important role with respect to the excitability of the C -Cl bonds. Cyclophosphamide must be metabolized in a suitable way to develop the same alkylating activity as the nor-N-mustard. The computation of the excited states of cyclophosphamide revealed a similar term scheme as it was found by Clar in the case of the carcinogenic polycyclic hydrocarbons.