FMP Publications

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

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The efficacy of trivalent cyclic hexapeptides to induce lipid clustering in PG/PE membranes correlates with their antimicrobial activity
Finger(*), S., Kerth(*), A., Dathe, M.; Blume(*), A.
Biochim Biophys Acta, 1848:2998-3006

Tags: Peptide-Lipid-Interaction/ Peptide Transport (Dathe)

Abstract: Various models have been proposed for the sequence of events occurring after binding of specific antimicrobial peptides to lipid membranes. The lipid clustering model arose by the finding that antimicrobial peptides can induce a segregation of certain negatively charged lipids in lipid model membranes. Anionic lipid segregation by cationic peptides is initially an effect of charge interaction where the ratio of peptide and lipid charges is thought to be the decisive parameter in the peptide induced lipid demixing. However, the sequence of events following this initial lipid clustering is more complex and can lead to deactivation of membrane proteins involved in cell division or perturbation of lipid reorganization essential for cell division. In this study we used DSC and ITC techniques to investigate the effect of binding different cyclic hexapeptides with varying antimicrobial efficacy, to phosphatidylglycerol (PG)/phosphatidylethanolamine (PE) lipid membranes and their ability to induce lipid segregation in these mixtures. We found that these cyclic hexapeptides consisting of three charged and three aromatic amino acids showed indeed different abilities to induce lipid demixing depending on their amino acid composition and their sequence. The results clearly showed that the cationic amino acids are essential for electrostatic binding but that the three hydrophobic amino acids in the peptides and their position in the sequence also contribute to binding affinity and to the extent of induction of lipid clustering. The efficacy of these different hexapeptides to induce PG clusters in PG/PE membranes was found to be correlated with their antimicrobial activity.

Thermodynamics of protein destabilization in live cells
Danielsson(*), J., Mu(*), X., Lang(*), L., Wang(*), H., Binolfi, A., Theillet, F. X., Bekei, B., Logan(*), D. T., Selenko, P., Wennerstrom(*), H.; Oliveberg(*), M.
Proc Natl Acad Sci U S A, 112:12402-12407

Tags: In-Cell NMR (Selenko)

Abstract: Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a beta-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 degrees C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein's interplay with the functionally optimized "interaction landscape" of the cellular interior.

Copper binding to the N-terminally acetylated, naturally occurring form of alpha-synuclein induces local helical folding
Miotto(*), M. C., Valiente-Gabioud(*), A. A., Rossetti(*), G., Zweckstetter(*), M., Carloni(*), P., Selenko, P., Griesinger(*), C., Binolfi, A.; Fernandez(*), C. O.
J Am Chem Soc, 137:6444-6447

Tags: In-Cell NMR (Selenko)

Abstract: Growing evidence supports a link between brain copper homeostasis, the formation of alpha-synuclein (AS)-copper complexes, and the development of Parkinson disease (PD). Recently it was demonstrated that the physiological form of AS is N-terminally acetylated (AcAS). Here we used NMR spectroscopy to structurally characterize the interaction between Cu(I) and AcAS. We found that the formation of an AcAS-Cu(I) complex at the N-terminal region stabilizes local conformations with alpha-helical secondary structure and restricted motility. Our work provides new evidence into the metallo-biology of PD and opens new lines of research as the formation of AcAS-Cu(I) complex might impact on AcAS membrane binding and aggregation.

Real-time NMR monitoring of biological activities in complex physiological environments
Smith(*), M. J., Marshall(*), C. B., Theillet, F. X., Binolfi, A., Selenko, P.; Ikura(*), M.
Curr Opin Struct Biol, 32:39-47

Tags: In-Cell NMR (Selenko)

Abstract: Biological reactions occur in a highly organized spatiotemporal context and with kinetics that are modulated by multiple environmental factors. To integrate these variables in our experimental investigations of 'native' biological activities, we require quantitative tools for time-resolved in situ analyses in physiologically relevant settings. Here, we outline the use of high-resolution NMR spectroscopy to directly observe biological reactions in complex environments and in real-time. Specifically, we discuss how real-time NMR (RT-NMR) methods have delineated insights into metabolic processes, post-translational protein modifications, activities of cellular GTPases and their regulators, as well as of protein folding events.

Disruption of adaptor protein 2mu (AP-2mu) in cochlear hair cells impairs vesicle reloading of synaptic release sites and hearing
Jung(*), S., Maritzen, T., Wichmann(*), C., Jing(*), Z., Neef(*), A., Revelo(*), N. H., Al-Moyed(*), H., Meese(*), S., Wojcik(*), S. M., Panou(*), I., Bulut(*), H., Schu(*), P., Ficner(*), R., Reisinger(*), E., Rizzoli(*), S. O., Neef(*), J., Strenzke(*), N., Haucke, V.; Moser(*), T.
EMBO J, 34:2686-2702

Tags: Membrane Traffic and Cell Motility (Maritzen), Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Active zones (AZs) of inner hair cells (IHCs) indefatigably release hundreds of vesicles per second, requiring each release site to reload vesicles at tens per second. Here, we report that the endocytic adaptor protein 2mu (AP-2mu) is required for release site replenishment and hearing. We show that hair cell-specific disruption of AP-2mu slows IHC exocytosis immediately after fusion of the readily releasable pool of vesicles, despite normal abundance of membrane-proximal vesicles and intact endocytic membrane retrieval. Sound-driven postsynaptic spiking was reduced in a use-dependent manner, and the altered interspike interval statistics suggested a slowed reloading of release sites. Sustained strong stimulation led to accumulation of endosome-like vacuoles, fewer clathrin-coated endocytic intermediates, and vesicle depletion of the membrane-distal synaptic ribbon in AP-2mu-deficient IHCs, indicating a further role of AP-2mu in clathrin-dependent vesicle reformation on a timescale of many seconds. Finally, we show that AP-2 sorts its IHC-cargo otoferlin. We propose that binding of AP-2 to otoferlin facilitates replenishment of release sites, for example, via speeding AZ clearance of exocytosed material, in addition to a role of AP-2 in synaptic vesicle reformation.

Gas-Phase Rearrangement in Lysine Phosphorylated Peptides During Electron-Transfer Dissociation Tandem Mass Spectrometry
Bertran-Vicente, J., Schümann, M., Hackenberger, C. P.; Krause, E.
Anal Chem, 87:6990-6994

Tags: Mass Spectrometry (Krause, E.), Chemical Biology II (Hackenberger)

Abstract: Tandem mass spectrometry (MS/MS) strategies coupled with collision-induced dissociation (CID) or radical-driven fragmentation techniques such as electron-capture dissociation (ECD) or electron-transfer dissociation (ETD) have been successfully used for comprehensive phosphoproteome analysis. However, the unambiguous characterization of the phosphorylation site remains a significant challenge due to phosphate-related neutral losses and gas-phase rearrangements, which have been observed during CID. In particular, for the analysis of labile N-phosphorylated peptides, ECD and ETD are emerging as a complementary method. In contrast to CID, the phosphorylation site of histidine, arginine, and lysine phosphorylated peptides can be characterized by ETD. Here, we present a study on the application of ETD for analysis of phospholysine (pLys) peptides. We show that, depending on the charge state of the precursor ion as well as the presence of basic amino acid side chains, phosphate transfer reactions during the ETD process can be observed leading to ambiguous fragment ion spectra. Basically, pLys is stable under ETD conditions allowing an unambiguous assignment of the site of phosphorylation, but some factors/parameters have to be considered to avoid gas-phase rearrangement which would lead to false positive results in phosphoproteomic studies.

Direct access to site-specifically phosphorylated-lysine peptides from a solid-support
Bertran-Vicente, J., Schümann, M., Schmieder, P., Krause, E.; Hackenberger, C. P. R.
Organic & Biomolecular Chemistry, 13:6839-6843

Tags: Chemical Biology II (Hackenberger), Mass Spectrometry (Krause, E), Solution NMR (Schmieder)

Abstract: Phosphorylation is a key process for changing the activity and function of proteins. The impact of phospho-serine (pSer), -threonine (pThr) and -tyrosine (pTyr) is certainly understood for some proteins. Recently, peptides and proteins containing N-phosphorylated amino acids such as phosphoarginine (pArg), phosphohistidine (pHis) and phospholysine (pLys) have gained interest because of their different chemical properties and stability profiles. Due to its high intrinsic lability, pLys is the least studied within this latter group. In order to gain insight into the biological role of pLys, chemical and analytical tools, which are compatible with the labile P(vO)-N bond, are highly sought-after. We recently reported an in-solution synthetic approach to incorporate pLys residues in a site-specific manner into peptides by taking advantage of the chemoselectivity of the Staudinger-phosphite reaction. While the in-solution approach allows us to circumvent the critical TFA cleavage, it still requires several transformations and purification steps to finally deliver pLys peptides. Here we report the synthesis of site-specific pLys peptides directly from a solid support by using a base labile resin. This straightforward and highly efficient approach facilitates the synthesis of various site-specific pLys-containing peptides and lays the groundwork for future studies about this elusive protein modification.

Perspectives for sensitivity enhancement in proton-detected solid-state NMR of highly deuterated proteins by preserving water magnetization
Chevelkov, V., Xiang(*), S. Q., Giller(*), K., Becker(*), S., Lange, A.; Reif(*), B.
J. Biomol. NMR, 61:151-160

Tags: Molecular Biophysics (Lange, A.)

Abstract: In this work, we show how the water flip-back approach that is widely employed in solution-state NMR can be adapted to proton-detected MAS solid-state NMR of highly deuterated proteins. The scheme allows to enhance the sensitivity of the experiment by decreasing the recovery time of the proton longitudinal magnetization. The method relies on polarization transfer from non-saturated water to the protein during the inter-scan delay.

Specific 13C labeling of leucine, valine and isoleucine methyl groups for unambiguous detection of long-range restraints in protein solid-state NMR studies
Fasshuber, H. K., Demers, J. P., Chevelkov, V., Giller(*), K., Becker(*), S.; Lange, A.
J Magn Reson, 252:10-19

Tags: Molecular Biophysics (Lange, A.)

Abstract: Here we present an isotopic labeling strategy to easily obtain unambiguous long-range distance restraints in protein solid-state NMR studies. The method is based on the inclusion of two biosynthetic precursors in the bacterial growth medium, alpha-ketoisovalerate and alpha-ketobutyrate, leading to the production of leucine, valine and isoleucine residues that are exclusively (13)C labeled on methyl groups. The resulting spectral simplification facilitates the collection of distance restraints, the verification of carbon chemical shift assignments and the measurement of methyl group dynamics. This approach is demonstrated on the type-three secretion system needle of Shigella flexneri, where 49 methyl-methyl and methyl-nitrogen distance restraints including 10 unambiguous long-range distance restraints could be collected. By combining this labeling scheme with ultra-fast MAS and proton detection, the assignment of methyl proton chemical shifts was achieved.

Structural heterogeneity in microcrystalline ubiquitin studied by solid-state NMR
Fasshuber, H. K., Lakomek(*), N. A., Habenstein(*), B., Loquet(*), A., Shi, C., Giller(*), K., Wolff(*), S., Becker(*), S.; Lange, A.
Protein Sci, 24:592-598

Tags: Molecular Biophysics (Lange, A.)

Abstract: By applying [1-(13) C]- and [2-(13) C]-glucose labeling schemes to the folded globular protein ubiquitin, a strong reduction of spectral crowding and increase in resolution in solid-state NMR (ssNMR) spectra could be achieved. This allowed spectral resonance assignment in a straightforward manner and the collection of a wealth of long-range distance information. A high precision solid-state NMR structure of microcrystalline ubiquitin was calculated with a backbone rmsd of 1.57 to the X-ray structure and 1.32 A to the solution NMR structure. Interestingly, we can resolve structural heterogeneity as the presence of three slightly different conformations. Structural heterogeneity is most significant for the loop region beta1-beta2 but also for beta-strands beta1, beta2, beta3, and beta5 as well as for the loop connecting alpha1 and beta3. This structural polymorphism observed in the solid-state NMR spectra coincides with regions that showed dynamics in solution NMR experiments on different timescales.

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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 
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