FMP Publications

Our publications are recorded in a searchable database since 2010, updates will be added regularly.

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References
Addressing protein-protein interactions with small molecules: a Pro-Pro dipeptide mimic with a PPII helix conformation as a module for the synthesis of PRD-binding ligands
Zaminer(*), J., Brockmann, C., Huy(*), P., Opitz, R., Reuter(*), C., Beyermann, M., Freund, C., Müller, M., Oschkinat, H., Kühne, R.; Schmalz(*), H. G.
Angew Chem Int Ed Engl, 49:7111-7115
(2010)

Tags: Computational Chemistry/Drug Design (Kühne), Protein Structure (Oschkinat), Peptide Synthesis (Beyermann)

Azides Derived from Colchicine and their Use in Library Synthesis: a Practical Entry to New Bioactive Derivatives of an Old Natural Drug
Nicolaus(*), N., Zapke, J., Riesterer(*), P., Neudörfl, J. M., Prokop(*), A., Oschkinat, H.; Schmalz(*), H. G.
Chemmedchem, 5:661-665
(2010)

Tags: Protein Structure (Oschkinat)

A novel subtype of AP-1-binding motif within the palmitoylated trans-Golgi network/endosomal accessory protein Gadkin/gamma-BAR
Maritzen(*), T., Schmidt(*), M. R., Kukhtina(*), V., Higman, V. A., Strauss, H., Volkmer(*), R., Oschkinat, H., Dotti(*), C. G.; Haucke, V.
J Biol Chem, 285:4074-4086
(2010)

Tags: Molecular Pharmacology and Cell Biology (Haucke), Protein Structure (Oschkinat)

Abstract: Membrane traffic between the trans-Golgi network (TGN) and endosomes is mediated in part by the assembly of clathrin-AP-1 adaptor complex-coated vesicles. This process involves multiple accessory proteins that directly bind to the ear domain of AP-1gamma via degenerate peptide motifs that conform to the consensus sequence diameterG(P/D/E)(diameter/L/M) (with diameter being a large hydrophobic amino acid). Recently, gamma-BAR (hereafter referred to as Gadkin for reasons explained below) has been identified as a novel AP-1 recruitment factor involved in AP-1-dependent endosomal trafficking of lysosomal enzymes. How precisely Gadkin interacts with membranes and with AP-1gamma has remained unclear. Here we show that Gadkin is an S-palmitoylated peripheral membrane protein that lacks stable tertiary structure. S-Palmitoylation is required for the recruitment of Gadkin to TGN/endosomal membranes but not for binding to AP-1. Furthermore, we identify a novel subtype of AP-1-binding motif within Gadkin that specifically associates with the gamma1-adaptin ear domain. Mutational inactivation of this novel type of motif, either alone or in combination with three more conventional AP-1gamma binding peptides, causes Gadkin to mislocalize to the plasma membrane and interferes with its ability to render AP-1 brefeldin A-resistant, indicating its physiological importance. Our studies thus unravel the molecular basis for Gadkin-mediated AP-1 recruitment to TGN/endosomal membranes and identify a novel subtype of the AP-1-binding motif.

A MAS NMR Study of the Bacterial ABC Transporter ArtMP
Lange, V., Becker-Baldus, J., Kunert, B., van Rossum, B. J., Casagrande(*), F., Engel(*), A., Roske(*), Y., Scheffel(*), F. M., Schneider(*), E.; Oschkinat, H.
Chembiochem, 11:547-555
(2010)

Tags: Protein Structure (Oschkinat)

Abstract: ATP-binding cassette (ABC) transport systems facilitate the translocation of substances, like amino acids, across cell membranes energised by ATP hydrolysis. This work describes first structural studies on the ABC transporter ArtMP from Geobacillus stearothermophilus in native lipid environment by magic-angle spinning NMR spectroscopy. The 2D crystals of ArtMP and 3D crystals of isolated ArtP were prepared in different nucleotide-bound or -unbound states. From selectively C-13,N-15-labelled ArtP, several sequence-specific assignments were obtained, most of which could be transferred to spectra of ArtMP. Residues Tyr133 and Pro134 protrude directly into the ATP-binding pocket at the interface of the ArtP subunits, and hence, are sensitive monitors for structural changes during nucleotide binding and hydrolysis. Distinct sets of NMR shifts were obtained for ArtP with different phosphorylation states of the ligand. Indications were found for an asymmetric or inhomogeneous state of the ArtP dimer bound with triphosphorylated nucleotides. With this investigation, a model system was established for screening all functional states occurring in one ABC transporter in native lipid environment.

Intermolecular protein-RNA interactions revealed by 2D 31P-15N magic angle spinning solid-state NMR spectroscopy
Jehle(*), S., Falb(*), M., Kirkpatrick(*), J. P., Oschkinat, H., van Rossum, B. J., Althoff(*), G.; Carlomagno(*), T.
J Am Chem Soc, 132:3842-3846
(2010)

Tags: Protein Structure (Oschkinat)

Abstract: The structural investigation of large RNP complexes by X-ray crystallography can be a difficult task due to the flexibility of the RNA and of the protein-RNA interfaces, which may hinder crystallization. In these cases, NMR spectroscopy is an attractive alternative to crystallography, although the large size of typical RNP complexes may limit the applicability of solution NMR. Solid-state NMR spectroscopy, however, is not subject to any intrinsic limitations with respect to the size of the object under investigation, with restrictions imposed solely by the sensitivity of the instrumentation. In addition, it does not require large, well-ordered crystals and can therefore be applied to flexible, partially disordered complexes. Here we show for the first time that solid-state NMR spectroscopy can be used to probe intermolecular interactions at the protein-RNA interface in RNP complexes. Distances between the (15)N nuclei of the protein backbone and the (31)P nuclei of the RNA backbone can be measured in TEDOR experiments and used as restraints in structure calculations. The distance measurement is accurate, as proven for the test case of the L7Ae-box C/D RNA complex, for which a crystal structure is available. The results presented here reveal the as yet unexplored potential of solid-state NMR spectroscopy in the investigation of large RNP complexes.

Solid-state NMR and SAXS studies provide a structural basis for the activation of alphaB-crystallin oligomers
Jehle, S., Rajagopal(*), P., Bardiaux, B., Markovic, S., Kühne, R., Stout(*), J. R., Higman, V. A., Klevit(*), R. E., van Rossum, B. J.; Oschkinat, H.
Nat Struct Mol Biol, 17:1037-1042
(2010)

Tags: Protein Structure (Oschkinat), Computational Chemistry/ Drug Design (Kühne)

Abstract: The small heat shock protein alphaB-crystallin (alphaB) contributes to cellular protection against stress. For decades, high-resolution structural studies on oligomeric alphaB have been confounded by its polydisperse nature. Here, we present a structural basis of oligomer assembly and activation of the chaperone using solid-state NMR and small-angle X-ray scattering (SAXS). The basic building block is a curved dimer, with an angle of approximately 121 degrees between the planes of the beta-sandwich formed by alpha-crystallin domains. The highly conserved IXI motif covers a substrate binding site at pH 7.5. We observe a pH-dependent modulation of the interaction of the IXI motif with beta4 and beta8, consistent with a pH-dependent regulation of the chaperone function. N-terminal region residues Ser59-Trp60-Phe61 are involved in intermolecular interaction with beta3. Intermolecular restraints from NMR and volumetric restraints from SAXS were combined to calculate a model of a 24-subunit alphaB oligomer with tetrahedral symmetry.

Optimum levels of exchangeable protons in perdeuterated proteins for proton detection in MAS solid-state NMR spectroscopy
Akbey, Ü., Lange, S., Trent Franks, W., Linser, R., Rehbein, K., Diehl, A., van Rossum, B. J., Reif, B.; Oschkinat, H.
J Biomol NMR, 46:67-73
(2010)

Tags: Protein Structure (Oschkinat), Solid-State NMR Spectroscopy (Reif)

Abstract: We present a systematic study of the effect of the level of exchangeable protons on the observed amide proton linewidth obtained in perdeuterated proteins. Decreasing the amount of D(2)O employed in the crystallization buffer from 90 to 0%, we observe a fourfold increase in linewidth for both (1)H and (15)N resonances. At the same time, we find a gradual increase in the signal-to-noise ratio (SNR) for (1)H-(15)N correlations in dipolar coupling based experiments for H(2)O concentrations of up to 40%. Beyond 40%, a significant reduction in SNR is observed. Scalar-coupling based (1)H-(15)N correlation experiments yield a nearly constant SNR for samples prepared with < or =30% H(2)O. Samples in which more H(2)O is employed for crystallization show a significantly reduced NMR intensity. Calculation of the SNR by taking into account the reduction in (1)H T (1) in samples containing more protons (SNR per unit time), yields a maximum SNR for samples crystallized using 30 and 40% H(2)O for scalar and dipolar coupling based experiments, respectively. A sensitivity gain of 3.8 is obtained by increasing the H(2)O concentration from 10 to 40% in the CP based experiment, whereas the linewidth only becomes 1.5 times broader. In general, we find that CP is more favorable compared to INEPT based transfer when the number of possible (1)H,(1)H interactions increases. At low levels of deuteration (> or =60% H(2)O in the crystallization buffer), resonances from rigid residues are broadened beyond detection. All experiments are carried out at MAS frequency of 24 kHz employing perdeuterated samples of the chicken alpha-spectrin SH3 domain.

Dynamic nuclear polarization of deuterated proteins
Akbey, Ü., Franks, W. T., Linden, A., Lange, S., Griffin(*), R. G., van Rossum, B. J.; Oschkinat, H.
Angew Chem Int Ed Engl, 49:7803-7806
(2010)

Tags: Protein Structure (Oschkinat)

Impact of membrane properties on uptake and transcytosis of colloidal nanocarriers across an epithelial cell barrier model
Orthmann(*), A., Zeisig(*), R., Koklic(*), T., Sentjurc(*), M., Wiesner, B., Lemm(*), M.; Fichtner(*), I.
Journal of pharmaceutical sciences, 99:2423-2433
(2010)

Tags: Cellular Imaging (Wiesner)

Abstract: The aim of this study was to investigate the effect of liposomal membrane properties on cellular uptake and transcytosis across a tight Madin-Darby canine kidney (MDCK) cell barrier in vitro. More than 25 small vesicles were prepared by lipid film hydration/extrusion to generate small unilamellar vesicles. The fluorescence marker calcein was encapsulated to mimic hydrophilic drug transport. Marker uptake by MDCK cells seems to be mediated by different mechanisms for the liposomes used. It was mainly depending on membrane fluidity and vesicle charge. Liposomes L2 with a positive charge (325 +/- 3 pmol/well) and vesicles L3 containing the helper lipid dioleylphosphatidylethanolamine (DOPE) in their membrane (216 +/- 42 pmol/well) were taken up to the most. Selected liposomes were tested for their transcytotic transport across a MDCK monolayer. Liposomes L4 containing equimolar DOPE and octadecyl-1,1-dimethylpiperidin-1-ium-4-yl phosphate (OPP) were the most efficient vesicles for transcellular transport resulting in 808 +/- 30 pmol calcein/cm(2) in the basal medium (28.1% of total liposomal marker added). Transcytosis was positively correlated with membrane fluidity in the outer part of the bilayer, as electron paramagnetic resonance measurements revealed. We expect that an increase in membrane fluidity of vesicles should also improve the restricted transport of hydrophilic drugs across the blood-brain barrier.

Distinct Neuropathologic Phenotypes After Disrupting the Chloride Transport Proteins ClC-6 or ClC-7/Ostm1
Pressey(*), S. N. R., O'Donnell(*), K. J., Stauber, T., Fuhrmann, J. C., Tyynela(*), J., Jentsch, T. J.; Cooper(*), J. D.
J Neuropath Exp Neur, 69:1228-1246
(2010)

Tags: Physiology and Pathology of Ion Transport (Jentsch

Abstract: The proteins ClC-6 and ClC-7 are expressed in the endosomallysosomal system. Because Clcn6-deficient mice display some features of neuronal ceroid lipofuscinosis (NCL), CLCN6 may be a candidate gene for novel forms of NCL. Using landmarks of disease progression from NCL mouse models as a guide, we examined neuropathologic alterations in the central nervous system of Clcn6(-/-), Clcn7(-/-), and gl mice. gl mice bear a mutation in Ostm1, the beta-subunit critical for Clcn7 function. Severely affected Clcn7(-/-) and gl mice have remarkably similar neuropathologic phenotypes, with pronounced reactive changes and neuron loss in the thalamocortical system, similar to findings in early-onset forms of NCL. In contrast, Clcn6(-/-) mice display slowly progressive, milder neuropathologic features with very little thalamic involvement or microglial activation. These findings detail for the first time the markedly different neuropathologic consequences of mutations in these two CLC genes. Clcn7(-/-) and gl mice bear a close resemblance to the progressive neuropathologic phenotypes of early onset forms of NCL, whereas the distinct phenotype of Clcn6-deficient mice suggests that this gene could be a candidate for a later-onset form of mild neurologic dysfunction with some NCL-like features.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
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13125 Berlin, Germany
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info(at)fmp-berlin.de

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