FMP Publications

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

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References
Theoretical aspects of Magic Angle Spinning - Dynamic Nuclear Polarization
Mentink-Vigier(*), F., Akbey, Ü., Oschkinat, H., Vega(*), S.; Feintuch(*), A.
J Magn Reson, 258:102-120
(2015)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Magic Angle Spinning (MAS) combined with Dynamic Nuclear Polarization (DNP) has been proven in recent years to be a very powerful method for increasing solid-state NMR signals. Since the advent of biradicals such as TOTAPOL to increase the nuclear polarization new classes of radicals, with larger molecular weight and/or different spin properties have been developed. These have led to unprecedented signal gain, with varying results for different experimental parameters, in particular the microwave irradiation strength, the static field, and the spinning frequency. Recently it has been demonstrated that sample spinning imposes DNP enhancement processes that differ from the active DNP mechanism in static samples as upon sample spinning the DNP enhancements are the results of energy level anticrossings occurring periodically during each rotor cycle. In this work we present experimental results with regards to the MAS frequency dependence of the DNP enhancement profiles of four nitroxide-based radicals at two different sets of temperature, 110 and 160K. In fact, different magnitudes of reduction in enhancement are observed with increasing spinning frequency. Our simulation code for calculating MAS-DNP powder enhancements of small model spin systems has been improved to extend our studies of the influence of the interaction and relaxation parameters on powder enhancements. To achieve a better understanding we simulated the spin dynamics of a single three-spin system ea-eb-n during its steady state rotor periods and used the Landau-Zener formula to characterize the influence of the different anti-crossings on the polarizations of the system and their necessary action for reaching steady state conditions together with spin relaxation processes. Based on these model calculations we demonstrate that the maximum steady state nuclear polarization cannot become larger than the maximum polarization difference between the two electrons during the steady state rotor cycle. This study also shows the complexity of the MAS-DNP process and therefore the necessity to rely on numerical simulations for understanding parametric dependencies of the enhancements. Finally an extension of the spin system up to five spins allowed us to probe the first steps of the transfer of polarization from the nuclei coupled to the electrons to further away nuclei, demonstrating a decrease in the spin-diffusion barrier under MAS conditions.

Sensitivity and resolution of proton detected spectra of a deuterated protein at 40 and 60 kHz magic-angle-spinning
Nieuwkoop, A. J., Franks, W. T., Rehbein, K., Diehl, A., Akbey, Ü., Engelke(*), F., Emsley(*), L., Pintacuda(*), G.; Oschkinat, H.
J Biomol NMR, 61:161-171
(2015)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: The use of small rotors capable of very fast magic-angle spinning (MAS) in conjunction with proton dilution by perdeuteration and partial reprotonation at exchangeable sites has enabled the acquisition of resolved, proton detected, solid-state NMR spectra on samples of biological macromolecules. The ability to detect the high-gamma protons, instead of carbons or nitrogens, increases sensitivity. In order to achieve sufficient resolution of the amide proton signals, rotors must be spun at the maximum rate possible given their size and the proton back-exchange percentage tuned. Here we investigate the optimal proton back-exchange ratio for triply labeled SH3 at 40 kHz MAS. We find that spectra acquired on 60 % back-exchanged samples in 1.9 mm rotors have similar resolution at 40 kHz MAS as spectra of 100 % back-exchanged samples in 1.3 mm rotors spinning at 60 kHz MAS, and for (H)NH 2D and (H)CNH 3D spectra, show 10-20 % higher sensitivity. For 100 % back-exchanged samples, the sensitivity in 1.9 mm rotors is superior by a factor of 1.9 in (H)NH and 1.8 in (H)CNH spectra but at lower resolution. For (H)C(C)NH experiments with a carbon-carbon mixing period, this sensitivity gain is lost due to shorter relaxation times and less efficient transfer steps. We present a detailed study on the sensitivity of these types of experiments for both types of rotors, which should enable experimentalists to make an informed decision about which type of rotor is best for specific applications.

Remarkable enhancement of ambient-air electrical conductivity of the perylenediimide pi-stacks isolated in the flexible films of a hydrogen-bonded polymer
Supur(*), M., Yurtsever(*), A.; Akbey, Ü.
Rsc Adv, 5:64240-64246
(2015)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: N,N'-di(2-(trimethylammoniumiodide)ethylene) perylenediimide (TAIPDI), forming extensive pi-stacks through the strong pi-pi interactions of large pi-planes, was isolated in the hydrogen-bonding milieu of polyvinyl alcohol (PVA) from aqueous solutions. The stacking behaviour of TAIPDIs in PVA films was investigated by using UV-vis and magic angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy. It was concluded that the TAIPDI molecules were organized as extensive pi-stacks in the PVA matrix controlled by the interactions with the polymer chains. The resulting films of TAIPDI/PVA were doped with electrons by using a strong reducing agent. The electrical current obtained in the electron-doped TAIPDI/PVA films was 740 times higher under the same bias as compared to that of reduced TAIPDIs cast on a glass substrate without a polymer additive. The significant increase in the conductivity of electron-doped TAIPDI/PVA films reflects the strong effect of the uninterrupted pi-stacking of TAIPDIs extending in the flexible PVA films and the protection of the doped electrons from the oxygen in the air, provided by the H-bonded environment in PVA.

Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation
Nillegoda(*), N. B., Kirstein, J., Szlachcic(*), A., Berynskyy(*), M., Stank(*), A., Stengel(*), F., Arnsburg, K., Gao(*), X., Scior, A., Aebersold(*), R., Guilbride(*), D. L., Wade(*), R. C., Morimoto(*), R. I., Mayer(*), M. P.; Bukau(*), B.
Nature, 524:247-251
(2015)

Tags: Proteostasis in Aging and Disease (Kirstein)

Abstract: Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states. Healthy metazoan cells effectively eliminate intracellular protein aggregates, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control.

The GYF domain protein CD2BP2 is critical for embryogenesis and podocyte function
Albert(*), G. I., Schell(*), C., Kirschner(*), K. M., Schäfer(*), S., Naumann(*), R., Müller(*), A., Kretz(*), O., Kuropka, B., Girbig(*), M., Hübner(*), N., Krause, E., Scholz(*), H., Huber(*), T. B., Knobeloch(*), K. P.; Freund(*), C.
Journal of molecular cell biology, 7:402-414
(2015)

Tags: Mass Spectrometry (Krause, E.)

Abstract: Scaffolding proteins play pivotal roles in the assembly of macromolecular machines such as the spliceosome. The adaptor protein CD2BP2, originally identified as a binding partner of the adhesion molecule CD2, is a pre-spliceosomal assembly factor that utilizes its glycine-tyrosine-phenylalanine (GYF) domain to co-localize with spliceosomal proteins. So far, its function in vertebrates is unknown. Using conditional gene targeting in mice, we show that CD2BP2 is crucial for embryogenesis, leading to growth retardation, defects in vascularization, and premature death at embryonic day 10.5 when absent. Ablation of the protein in bone marrow-derived macrophages indicates that CD2BP2 is involved in the alternative splicing of mRNA transcripts from diverse origins. At the molecular level, we identified the phosphatase PP1 to be recruited to the spliceosome via the N-terminus of CD2BP2. Given the strong expression of CD2BP2 in podocytes of the kidney, we use selective depletion of CD2BP2, in combination with next-generation sequencing, to monitor changes in exon usage of genes critical for podocyte functions, including VEGF and actin regulators. CD2BP2-depleted podocytes display foot process effacement, and cause proteinuria and ultimately lethal kidney failure in mice. Collectively, our study defines CD2BP2 as a non-redundant splicing factor essential for embryonic development and podocyte integrity.

Phenothiazine-derived antipsychotic drugs inhibit dynamin and clathrin-mediated endocytosis
Daniel(*), J. A., Chau(*), N., Abdel-Hamid(*), M. K., Hu(*), L., von Kleist, L., Whiting(*), A., Krishnan(*), S., Maamary(*), P., Joseph(*), S. R., Simpson(*), F., Haucke, V., McCluskey(*), A.; Robinson(*), P. J.
Traffic, 16:635-654
(2015)

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Chlorpromazine is a phenothiazine-derived antipsychotic drug (APD) that inhibits clathrin-mediated endocytosis (CME) in cells by an unknown mechanism. We examined whether its action and that of other APDs might be mediated by the GTPase activity of dynamin. Eight of eight phenothiazine-derived APDs inhibited dynamin I (dynI) in the 2-12 microm range, the most potent being trifluoperazine (IC50 2.6 +/- 0.7 microm). They also inhibited dynamin II (dynII) at similar concentrations. Typical and atypical APDs not based on the phenothiazine scaffold were 8- to 10-fold less potent (haloperidol and clozapine) or were inactive (droperidol, olanzapine and risperidone). Kinetic analysis showed that phenothiazine-derived APDs were lipid competitive, while haloperidol was uncompetitive with lipid. Accordingly, phenothiazine-derived APDs inhibited dynI GTPase activity stimulated by lipids but not by various SH3 domains. All dynamin-active APDs also inhibited transferrin (Tfn) CME in cells at related potencies. Structure-activity relationships (SAR) revealed dynamin inhibition to be conferred by a substituent group containing a terminal tertiary amino group at the N2 position. Chlorpromazine was previously proposed to target AP-2 recruitment in the formation of clathrin-coated vesicles (CCV). However, neither chlorpromazine nor thioridazine affected AP-2 interaction with amphiphysin or clathrin. Super-resolution microscopy revealed that chlorpromazine blocks neither clathrin recruitment by AP-2, nor AP-2 recruitment, showing that CME inhibition occurs downstream of CCV formation. Overall, potent dynamin inhibition is a shared characteristic of phenothiazine-derived APDs, but not other typical or atypical APDs, and the data indicate that dynamin is their likely in-cell target in endocytosis.

Selective inhibitors of the protein tyrosine phosphatase SHP2 block cellular motility and growth of cancer cells in vitro and in vivo
Grosskopf, S., Eckert, C., Arkona(*), C., Radetzki, S., Böhm(*), K., Heinemann(*), U., Wolber(*), G., von Kries, J. P., Birchmeier(*), W.; Rademann(*), J.
Chemmedchem, 10:815-826
(2015)

Tags: Medicinal Chemistry (Rademann), Screening Unit (von Kries)

Abstract: Selective inhibitors of the protein tyrosine phosphatase SHP2 (src homology region 2 domain phosphatase; PTPN11), an enzyme that is deregulated in numerous human tumors, were generated through a combination of chemical synthesis and structure-based rational design. Seventy pyridazolon-4-ylidenehydrazinyl benzenesulfonates were prepared and evaluated in enzyme assays. The binding modes of active inhibitors were simulated in silico using a newly generated crystal structure of SHP2. The most powerful compound, GS-493 (4-(2Z)-2-[1,3-bis(4-nitrophenyl)-5-oxo-1,5-dihydro-4H-pyrazol-4-yliden]hydrazin obenzenesulfonic acid; 25) inhibited SHP2 with an IC50 value of 71+/-15 nM in the enzyme assay and was 29- and 45-fold more active toward SHP2 than against related SHP1 and PTP1B. In cell culture experiments compound 25 was found to block hepatocyte growth factor (HGF)-stimulated epithelial-mesenchymal transition of human pancreatic adenocarcinoma (HPAF) cells, as indicated by a decrease in the minimum neighbor distances of cells. Moreover, 25 inhibited cell colony formation in the non-small-cell lung cancer cell line LXFA 526L in soft agar. Finally, 25 was observed to inhibit tumor growth in a murine xenograft model. Therefore, the novel specific compound 25 strengthens the hypothesis that SHP2 is a relevant protein target for the inhibition of mobility and invasiveness of cancer cells.

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
(2015)

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.

Untangling a Repetitive Amyloid Sequence: Correlating Biofilm-Derived and Segmentally Labeled Curli Fimbriae by Solid-State NMR Spectroscopy
Schubeis(*), T., Yuan(*), P., Ahmed(*), M., Nagaraj, M., van Rossum, B. J.; Ritter(*), C.
Angew Chem Int Ed Engl, 54:14669-14672
(2015)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Curli are functional bacterial amyloids produced by an intricate biogenesis machinery. Insights into their folding and regulation can advance our understanding of amyloidogenesis. However, gaining detailed structural information of amyloids, and their tendency for structural polymorphisms, remains challenging. Herein we compare high-quality solid-state NMR spectra from biofilm-derived and recombinantly produced curli and provide evidence that they adopt a similar, well-defined beta-solenoid arrangement. Curli subunits consist of five sequence repeats, resulting in severe spectral overlap. Using segmental isotope labeling, we obtained the unambiguous sequence-specific resonance assignments and secondary structure of one repeat, and demonstrate that all repeats are most likely structurally equivalent.

Systems Analysis of Protein Fatty Acylation in Herpes Simplex Virus-Infected Cells Using Chemical Proteomics
Serwa(*), R. A., Abaitua(*), F., Krause, E., Tate(*), E. W.; O'Hare(*), P.
Chem Biol, 22:1008-1017
(2015)

Tags: Mass Spectrometry (Krause, E.)

Abstract: Protein fatty acylation regulates diverse aspects of cellular function and organization and plays a key role in host immune responses to infection. Acylation also modulates the function and localization of virus-encoded proteins. Here, we employ chemical proteomics tools, bio-orthogonal probes, and capture reagents to study myristoylation and palmitoylation during infection with herpes simplex virus (HSV). Using in-gel fluorescence imaging and quantitative mass spectrometry, we demonstrate a generalized reduction in myristoylation of host proteins, whereas palmitoylation of host proteins, including regulators of interferon and tetraspanin family proteins, was selectively repressed. Furthermore, we found that a significant fraction of the viral proteome undergoes palmitoylation; we identified a number of virus membrane glycoproteins, structural proteins, and kinases. Taken together, our results provide broad oversight of protein acylation during HSV infection, a roadmap for similar analysis in other systems, and a resource with which to pursue specific analysis of systems and functions.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
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