Franz Müller

Publications (3)7.37 Total impact

• Article: 13C Isotopologue editing of FMN bound to phototropin domains.

  Wolfgang Eisenreich, Monika Joshi, Boris Illarionov, Gerald Richter, Werner Römisch-Margl, Franz Müller, Adelbert Bacher, Markus Fischer
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  ABSTRACT: The plant blue light receptor phototropin comprises a protein kinase domain and two FMN-binding LOV domains (LOV1 and LOV2). Blue light irradiation of recombinant LOV domains is conducive to the addition of a cysteinyl thiolate group to carbon 4a of the FMN chromophore, and spontaneous cleavage of that photoadduct completes the photocycle of the receptor. The present study is based on (13)C NMR signal modulation observed after reconstitution of LOV domains of different origins with random libraries of (13)C-labeled FMN isotopologues. Using this approach, all (13)C signals of FMN bound to LOV1 and LOV2 domains of Avena sativa and to the LOV2 domain of the fern, Adiantum capillus-veneris, could be unequivocally assigned under dark and under blue light irradiation conditions. (13)C Chemical shifts of FMN are shown to be differently modulated by complexation with the LOV domains under study, indicating slight differences in the binding interactions of FMN and the apoproteins.
  FEBS Journal 12/2007; 274(22):5876-90. · 3.79 Impact Factor
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  Available from: Adelbert Bacher

  Article: 13C-, 15N- and 31P-NMR studies of oxidized and reduced low molecular mass thioredoxin reductase and some mutant proteins.

  Wolfgang Eisenreich, Kristina Kemter, Adelbert Bacher, Scott B Mulrooney, Charles H Williams, Franz Müller
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  ABSTRACT: Thioredoxin reductase (TrxR) from Escherichia coli, the mutant proteins E159Y and C138S, and the mutant protein C138S treated with phenylmercuric acetate were reconstituted with [U-(13)C(17),U-(15)N(4)]FAD and analysed, in their oxidized and reduced states, by (13)C-, (15)N- and (31)P-NMR spectroscopy. The enzymes studied showed very similar (31)P-NMR spectra in the oxidized state, consisting of two peaks at -9.8 and -11.5 p.p.m. In the reduced state, the two peaks merge into one apparent peak (at -9.8 p.p.m.). The data are compared with published (31)P-NMR data of enzymes closely related to TrxR. (13)C and (15)N-NMR chemical shifts of TrxR and the mutant proteins in the oxidized state provided information about the electronic structure of the protein-bound cofactor and its interactions with the apoproteins. Strong hydrogen bonds exist between protein-bound flavin and the apoproteins at C(2)O, C(4)O, N(1) and N(5). The N(10) atoms in the enzymes are slightly out of the molecular plane of the flavin. Of the ribityl carbon atoms C(10alpha,gamma,delta) are the most affected upon binding to the apoprotein and the large downfield shift of the C(10gamma) atom indicates strong hydrogen bonding with the apoprotein. The hydrogen bonding pattern observed is in excellent agreement with X-ray data, except for the N(1) and the N(3) atoms where a reversed situation was observed. Some chemical shifts observed in C138S deviate considerably from those of the other enzymes. From this it is concluded that C138S is in the FO conformation and the others are in the FR conformation, supporting published data. In the reduced state, strong hydrogen bonding interactions are observed between C(2)O and C(4)O and the apoprotein. As revealed by the (15)N chemical shifts and the N(5)H coupling constant the N(5) and the N(10) atom are highly sp(3) hybridized. The calculation of the endocyclic angles for the N(5) and the N(10) atoms shows the angles to be approximately 109 degrees, in perfect agreement with X-ray data showing that the flavin assumes a bent conformation along the N(10)/N(5) axis of the flavin. In contrast, the N(1) is highly sp(2) hybridized and is protonated, i.e. in the neutral state. Upon reduction of the enzymes, the (13)C chemical shifts of some atoms of the ribityl side chain undergo considerable changes also indicating conformational rearrangements of the side-chain interactions with the apoproteins. The chemical shifts between native TrxR and C138S are now rather similar and differ from those of the two other mutant proteins. This strongly indicates that the former enzymes are in the FO conformation and the other two are in the FR conformation. The data are discussed briefly in the context of published NMR data obtained with a variety of flavoproteins.
  European Journal of Biochemistry 05/2004; 271(8):1437-52. · 3.58 Impact Factor
• Article: 31P‐NMR spectroscopy of human and Paracoccus denitrificans electron transfer flavoproteins, and 13C‐ and 15N‐NMR spectroscopy of human electron transfer flavoprotein in the oxidised and reduced states

  Kurt J. Griffin, Gregory D. Degala, Wolfgang Eisenreich, Franz Müller, Adelbert Bacher, Frank E. Frerman
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  ABSTRACT: Human and Paracoccus denitrificans wild-type electron transfer flavoproteins have been investigated by 31P-NMR in the oxidised and reduced states. The 31P chemical shifts of the diphosphate moiety of the protein-bound FAD were similar in the proteins and were independent of the redox state. The chemical shifts were remarkably similar to those of ferredoxin−NADP+ reductase and, to a lesser degree, with those of NADPH−cytochrome P-450 reductase.The wild-type human electron transfer apoprotein was reconstituted with [2,4a-13C2]FAD, [4,10a-13C2]FAD, or [U-15N4]FAD. The reconstituted proteins were studied by 13C- and 15N-NMR techniques in the oxidised and reduced states. The chemical shifts were compared with those of free flavin in aqueous solution or in chloroform, and those of flavoproteins published in the literature.In the oxidised state, strong hydrogen bonds exist between residues of the apoprotein and C(2)O and N(5) of FAD. The N(1) atom is also hydrogen bonded and, as shown by X-ray data, involves the C′(4)-OH group of FAD. The sp2 hybridisation of N(10) is small compared to other flavoproteins.In the reduced state, there are strong hydrogen bonds involving C(2)O and N(5) of FAD. The N(1) atom is ionised as observed also in other flavoproteins when investigated by NMR. The intramolecular hydrogen bond between the C′(4)-OH group and the N(1) atom of FAD is maintained in the reduced state, suggesting an involvement in the stabilisation of a certain configuration of the diphosphate group of protein-bound FAD in both redox states. The N(10) atom in the reduced protein is highly sp3 hybridised in comparison to those of other flavoproteins.
  European Journal of Biochemistry. 06/1998; 255(1):125 - 132.