FMP Publications

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

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Amyloid beta 42 peptide (Abeta42)-lowering compounds directly bind to Abeta and interfere with amyloid precursor protein (APP) transmembrane dimerization
Richter(*), L., Munter(*), L. M., Ness(*), J., Hildebrand(*), P. W., Dasari, M., Unterreitmeier(*), S., Bulic(*), B., Beyermann, M., Gust(*), R., Reif, B., Weggen(*), S., Langosch(*), D.; Multhaup(*), G.
Proc Natl Acad Sci U S A, 107:14597-14602

Tags: Solid-State NMR Spectroscopy (Reif), Peptide Synthesis (Beyermann)

Abstract: Following ectodomain shedding by beta-secretase, successive proteolytic cleavages within the transmembrane sequence (TMS) of the amyloid precursor protein (APP) catalyzed by gamma-secretase result in the release of amyloid-beta (Abeta) peptides of variable length. Abeta peptides with 42 amino acids appear to be the key pathogenic species in Alzheimer's disease, as they are believed to initiate neuronal degeneration. Sulindac sulfide, which is known as a potent gamma-secretase modulator (GSM), selectively reduces Abeta42 production in favor of shorter Abeta species, such as Abeta38. By studying APP-TMS dimerization we previously showed that an attenuated interaction similarly decreased Abeta42 levels and concomitantly increased Abeta38 levels. However, the precise molecular mechanism by which GSMs modulate Abeta production is still unclear. In this study, using a reporter gene-based dimerization assay, we found that APP-TMS dimers are destabilized by sulindac sulfide and related Abeta42-lowering compounds in a concentration-dependent manner. By surface plasmon resonance analysis and NMR spectroscopy, we show that sulindac sulfide and novel sulindac-derived compounds directly bind to the Abeta sequence. Strikingly, the attenuated APP-TMS interaction by GSMs correlated strongly with Abeta42-lowering activity and binding strength to the Abeta sequence. Molecular docking analyses suggest that certain GSMs bind to the GxxxG dimerization motif in the APP-TMS. We conclude that these GSMs decrease Abeta42 levels by modulating APP-TMS interactions. This effect specifically emphasizes the importance of the dimeric APP-TMS as a promising drug target in Alzheimer's disease.

Narrow carbonyl resonances in proton-diluted proteins facilitate NMR assignments in the solid-state
Linser, R., Fink, U.; Reif, B.
J. Biomol. NMR, 47:1-6

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: HNCO/HNCACO type correlation experiments are an alternative for assignment of backbone resonances in extensively deuterated proteins in the solid-state, given the fact that line widths on the order of 14-17 Hz are achieved in the carbonyl dimension without the need of high power decoupling. The achieved resolution demonstrates that MAS solid-state NMR on extensively deuterated proteins is able to compete with solution-state NMR spectroscopy if proteins are investigated with correlation times tau (c) that exceed 25 ns.

Assignment of dynamic regions in biological solids enabled by spin-state selective NMR experiments
Linser, R., Fink, U.; Reif, B.
J Am Chem Soc, 132:8891-8893

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: Structural investigations are a prerequisite to understand protein function. Intermediate time scale motional processes (ns-micros) are deleterious for NMR of biological solids and obscure the detection of amide moieties in traditional CP based solid-state NMR approaches as well as in regular scalar coupling based experiments. We show that this obstacle can be overcome by using TROSY type techniques in triple resonance experiments, which enable the assignment of resonances in loop regions of a microcrystalline protein. The presented approach provides an exemplified solution for the analysis of secondary structure elements undergoing slow dynamics that might be particularly crucial for understanding protein function.

Microsecond time scale mobility in a solid protein as studied by the 15N R(1rho) site-specific NMR relaxation rates
Krushelnitsky(*), A., Zinkevich(*), T., Reichert(*), D., Chevelkov, V.; Reif, B.
J Am Chem Soc, 132:11850-11853

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: For the first time, we have demonstrated the site-resolved measurement of reliable (i.e., free of interfering effects) (15)N R(1rho) relaxation rates from a solid protein to extract dynamic information on the microsecond time scale. (15)N R(1rho) NMR relaxation rates were measured as a function of the residue number in a (15)N,(2)H-enriched (with 10-20% back-exchanged protons at labile sites) microcrystalline SH3 domain of chicken alpha-spectrin. The experiments were performed at different temperatures and at different spin-lock frequencies, which were realized by on- and off-resonance spin-lock irradiation. The results obtained indicate that the interfering spin-spin contribution to the R(1rho) rate in a perdeuterated protein is negligible even at low spin-lock fields, in contrast to the case for normal protonated samples. Through correlation plots, the R(1rho) rates were compared with previous data for the same protein characterizing different kinds of internal mobility.

Glycogen synthase kinase 3beta interaction protein functions as an A-kinase anchoring protein
Hundsrucker, C., Skroblin, P., Christian, F., Zenn(*), H. M., Popara, V., Joshi, M., Eichhorst, J., Wiesner, B., Herberg(*), F. W., Reif, B., Rosenthal(*), W.; Klussmann, E.
J Biol Chem, 285:5507-5521

Tags: Anchored Signalling (Klussmann), Solid-State NMR Spectroscopy (Reif), Cellular Imaging (Wiesner)

Abstract: A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3beta interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3beta (glycogen synthase kinase 3beta). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3beta by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3beta and thereby provides a mechanism for the integration of PKA and GSK3beta signaling pathways.

Quantification of protein backbone hydrogen-deuterium exchange rates by solid state NMR spectroscopy
del Amo, J. M., Fink, U.; Reif, B.
J Biomol NMR, 48:203-212

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: We present the quantification of backbone amide hydrogen-deuterium exchange rates (HDX) for immobilized proteins. The experiments make use of the deuterium isotope effect on the amide nitrogen chemical shift, as well as on proton dilution by deuteration. We find that backbone amides in the microcrystalline alpha-spectrin SH3 domain exchange rather slowly with the solvent (with exchange rates negligible within the individual (15)N-T (1) timescales). We observed chemical exchange for 6 residues with HDX exchange rates in the range from 0.2 to 5 s(-1). Backbone amide (15)N longitudinal relaxation times that we determined previously are not significantly affected for most residues, yielding no systematic artifacts upon quantification of backbone dynamics (Chevelkov et al. 2008b). Significant exchange was observed for the backbone amides of R21, S36 and K60, as well as for the sidechain amides of N38, N35 and for W41epsilon. These residues could not be fit in our previous motional analysis, demonstrating that amide proton chemical exchange needs to be considered in the analysis of protein dynamics in the solid-state, in case D(2)O is employed as a solvent for sample preparation. Due to the intrinsically long (15)N relaxation times in the solid-state, the approach proposed here can expand the range of accessible HDX rates in the intermediate regime that is not accessible so far with exchange quench and MEXICO type experiments.

Comparison of solid-state dipolar couplings and solution relaxation data provides insight into protein backbone dynamics
Chevelkov, V., Xue(*), Y., Linser, R., Skrynnikov(*), N. R.; Reif, B.
J Am Chem Soc, 132:5015-5017

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: Analyses of solution (15)N relaxation data and solid-state (1)H(N)-(15)N dipolar couplings from a small globular protein, alpha-spectrin SH3 domain, produce a surprisingly similar pattern of order parameters. This result suggests that there is little or no ns-mus dynamics throughout most of the sequence and, in particular, in the structured portion of the backbone. At the same time, evidence of ns-mus motions is found in the flexible loops and termini. These findings, corroborated by the MD simulations of alpha-spectrin SH3 in a hydrated crystalline environment and in solution, are consistent with the picture of protein dynamics that has recently emerged from the solution studies employing residual dipolar couplings.

High Resolution H-1-Detected Solid-State NMR Spectroscopy of Protein Aliphatic Resonances: Access to Tertiary Structure Information
Asami, S., Schmieder, P.; Reif, B.
J. Am. Chem. Soc., 132:15133-15135

Tags: Solid-State NMR Spectroscopy (Reif), Solution NMR (Schmieder)

Abstract: Biological magic angle spinning (MAS) solid-state nuclear magnetic resonance spectroscopy has developed rapidly over the past two decades. For the structure determination of a protein by solid-state NMR, routinely C-13,C-13 distance restraints as well as dihedral restraints are employed. In protonated samples, this is achieved by growing the bacterium on a medium which contains [1,3]-C-13 glycerol or [2]-C-13 glycerol to dilute the C-13 spin system. Labeling schemes, which rely on heteronuclei, are insensitive both for detection and in terms of quantification of distances, since they are relying on low-gamma nuclei. Proton detection can in principle provide a gain in sensitivity by a factor of 8 and 31, compared to the C-13 or N-15 detected version of the experiment. We report here a new labeling scheme, which enables H-1-detection of aliphatic resonances with high resolution in MAS solid-state NMR spectroscopy. We prepared microcrystals of the SH3 domain of chicken a-spectrin with 5% protonation at nonexchangeable sites and obtained line widths on the order of 25 Hz for aliphatic H-1 resonances. We show further that C-13 resolved 3D-H-1,H-1 correlation experiments yield access to long-range proton-proton distances in the protein.

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

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.

Identification of hydroxyl protons, determination of their exchange dynamics, and characterization of hydrogen bonding in a microcrystallin protein
Agarwal, V., Linser, R., Fink, U., Faelber, K.; Reif, B.
J Am Chem Soc, 132:3187-3195

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: Heteronuclear correlation experiments employing perdeuterated proteins enable the observation of all hydroxyl protons in a microcrystalline protein by MAS solid-state NMR. Dipolar-based sequences allow magnetization transfers that are >50 times faster compared to scalar-coupling-based sequences, which significantly facilitates their assignment. Hydroxyl exchange rates were measured using EXSY-type experiments. We find a biexponential decay behavior for those hydroxyl groups that are involved in side chain-side chain C-O-H...O horizontal lineC hydrogen bonds. The quantification of the distances between the hydroxyl proton and the carbon atoms in the hydrogen-bonding donor as well as acceptor group is achieved via a REDOR experiment. In combination with X-ray data and isotropic proton chemical shifts, availability of (1)H,(13)C distance information can aid in the quantitative description of the geometry of these hydrogen bonds. Similarly, correlations between backbone amide proton and carbonyl atoms are observed, which will be useful in the analysis of the registry of beta-strand arrangement in amyloid fibrils.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
Robert-Roessle-Str. 10
13125 Berlin, Germany
+4930 94793 - 100 
+4930 94793 - 109 (Fax)

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