TY - GEN A1 - Best, Robert B. A1 - Zheng, Wenwei A1 - Borgia, Alessandro A1 - Buholzer, Karin A1 - Borgia, Madeleine B. A1 - Hofmann, Hagen A1 - Soranno, Andrea A1 - Nettels, Daniel A1 - Gast, Klaus A1 - Grishaev, Alexander A1 - Schuler, Benjamin T1 - Comment on "Innovative scattering analysis shows that hydrophobic disordered proteins are expanded in water" T2 - Science N2 - Riback et al. (Reports, 13 October 2017, p. 238) used small-angle x-ray scattering (SAXS) experiments to infer a degree of compaction for unfolded proteins in water versus chemical denaturant that is highly consistent with the results from Forster resonance energy transfer (FRET) experiments. There is thus no "contradiction" between the two methods, nor evidence to support their claim that commonly used FRET fluorophores cause protein compaction. Y1 - 2018 U6 - https://doi.org/10.1126/science.aar7101 SN - 0036-8075 SN - 1095-9203 VL - 361 IS - 6405 PB - American Assoc. for the Advancement of Science CY - Washington ER - TY - JOUR A1 - Blasig, Ingolf E. A1 - Winkler, Lars A1 - Lassowski, Birgit A1 - Müller, Sandra L. A1 - Zuleger, Nikolaj A1 - Krause, Eberhard A1 - Krause, Gerd A1 - Gast, Klaus A1 - Kolbe, Michael A1 - Piontek, Jörg T1 - On the self-association potential of transmembrane tight junction proteins N2 - Tight junctions seal intercellular clefts via membrane-related strands, hence, maintaining important organ functions. We investigated the self-association of strand-forming transmembrane tight junction proteins. The regulatory tight junction protein occludin was differently tagged and cotransfected in eucaryotic cells. These occludins colocalized within the plasma membrane of the same cell, coprecipitated and exhibited fluorescence resonance energy transfer. Differently tagged strand-forming claudin-5 also colocalized in the plasma membrane of the same cell and showed fluorescence resonance energy transfer. This demonstrates self-association in intact cells both of occludin and claudin-5 in one plasma membrane. In search of dimerizing regions of occludin, dimerization of its cytosolic C-terminal coiled-coil domain was identified. In claudin-5, the second extracellular loop was detected as a dimer. Since the transmembrane junctional adhesion molecule also is known to dimerize, the assumption that homodimerization of transmembrane tight junction proteins may serve as a common structural feature in tight junction assembly is supported Y1 - 2006 UR - http://www.springerlink.com/content/101193 U6 - https://doi.org/10.1007/s00018-005-5472-x SN - 1420-682X ER - TY - JOUR A1 - Keller, S. A1 - Sauer, I. A1 - Strauss, H. A1 - Gast, Klaus A1 - Dathe, M. A1 - Bienert, Michael C. T1 - Membrane-mimetic nanocarriers formed by a dipalmitoylated cell-penetrating peptide Y1 - 2005 ER - TY - JOUR A1 - Fabian, H. A1 - Gast, Klaus A1 - Filimonov, Vladimir V. A1 - Zamyatkin, D. F. A1 - Rogov, V. V. T1 - Thermal unfolding of two designed monomeric lambda Cro repressor variants N2 - The thermal unfolding of the wild-type lambda Cro repressor and of two designed variants, Cro K56-[DGEVK] and Cro K56-[DGEVK] Q16L, was studied by Fourier transform infrared spectroscopy and dynamic light scattering. The engineered Cro K56-[DGEVK] monomer has five additional amino acids inserted after position 56 of the wild-type sequence, while the K56-[DGEVK] Q16L variant differs only in one position (Gln-16 to Leu substitution) from the Cro K56-[DGEVK] sequence. The temperature dependence of selected protein backbone infrared `marker' bands revealed that Cro K56- [DGEVK] is slightly more stable than the wild-type protein, while the replacement of Gln-16 by Leu increases the thermal transition temperature by similar to 20 degrees C. Moreover, thermal unfolding of the two Cro variants was found to proceed through equilibrium unfolding intermediates and to involve the formation of oligomers. The first thermal transition of Cro K56-[DGEVK] involves the melting of major parts of its native secondary structure and is accompanied by the formation of dinners and non-native beta-sheet structures. These structures unfold during a second transition at higher temperatures, accompanied by the dissociation of the dimers. In contrast to the Cro K56-[DGEVK] protein, the intermediate state of the Cro K56-[DGEVK] Q16L variant is less well defined, and involves the formation of oligomers of different size. (c) 2005 Elsevier B.V. All rights reserved Y1 - 2005 SN - 0924-2031 ER - TY - JOUR A1 - Hanisch, Uwe-Karsten A1 - van Rossum, D. A1 - Gast, Klaus A1 - Misselwitz, Rolf A1 - Goldstein, Gundars A1 - Koistinaho, Jari A1 - Möller, Thomas T1 - The microglia-activating potential of thrombin : is the protease able to induce cyto- and chemokines? Y1 - 2004 ER - TY - JOUR A1 - Piontek, J. A1 - Winkler, Lars A1 - Bal, M. S. A1 - Lassowski, Birgit A1 - Mueller, Sandra L. A1 - Gast, Klaus A1 - Blasig, Ingolf E. T1 - Investigating of homophilic interactions of the tight junction proteins occludin and claudin-5 Y1 - 2004 ER - TY - JOUR A1 - Modler, Andreas Johannes A1 - Fabian, H. A1 - Sokolowski, F. A1 - Lutsch, G. A1 - Gast, Klaus A1 - Damaschun, Gregor T1 - Polymerization of proteins into amyloid protofibrils shares common critical oligomeric states but differs in the mechanisms of their formation N2 - Amyloid protofibril formation of phosphoglycerate kinase (PGK) and Syrian hamster prion protein (SHaPrP(90- 232)) were investigated by static and dynamic light scattering, size exclusion chromatography and electron microscopy. Changes in secondary structure were monitored by Fourier transform infrared spectroscopy and by circular dichroism. Protofibril formation of the two proteins is found to be a two-stage process. At the beginning, an ensemble of critical oligomers is built lip. These critical oligomeric states possess a predominant beta-sheet structure and do not interact considerably with monomers. Initial oligomerization and transition to beta-sheet structure are coupled events differing in their details for both proteins. Intermediate oligomeric states (dimers, trimers, etc.) are populated in case of PGK, whereas SHaPrP(90-232) behaves according to oil apparent two-state reaction between monomers and octamers rich in beta- structure with a reaction order varying between 2 and 4. All oligomers coalesce to PGK protofibrils in the second stage, while SHaPrP(90-232) protofibrils are only formed by a subpopulation. The rates of both growth stages can be tuned in case of PGK by different salts preserving the underlying generalized diffusion-collision mechanism. The different kinetics of the early misfolding and oligomerization events of the two proteins argue against a common mechanism of protofibril formation. A classification scheme for misassembly, mechanisms of proteins based on energy landscapes is presented. It includes scenarios of downhill polymerization to which protofibril formation of PGK and SHaPrP(90-232) belong Y1 - 2004 ER - TY - JOUR A1 - Hanisch, Uwe-Karsten A1 - Van Rossum, Denise A1 - Xie, Yiheng A1 - Misselwitz, Rolf A1 - Auriola, Seppo A1 - Goldstein, Gundars A1 - Koistinaho, Jari A1 - Kettemann, Helmut A1 - Möller, Thomas A1 - Gast, Klaus T1 - The microglia-activating potential of thrombin : the protease is not involved in the induction of proinflammatory cytokines and chemokines N2 - The serine protease thrombin is known as a blood coagulation factor. Through limited cleavage of proteinase- activated receptors it can also control growth and functions in various cell types, including neurons, astrocytes, and microglia ( brain macrophages). A number of previous studies indicated that thrombin induces the release of proinflammatory cytokines and chemokines from microglial cells, suggesting another important role for the protease beyond hemostasis. In the present report, we provide evidence that this effect is not mediated by any proteolytic or non- proteolytic mechanism involving thrombin proper. Inhibition of the enzymatic thrombin activity did not affect the microglial release response. Instead the cyto-/chemokine-inducing activity solely resided in a high molecular weight protein fraction that could be isolated in trace amounts even from apparently homogenous alpha- and gamma-thrombin preparations. High molecular weight material contained thrombin-derived peptides as revealed by mass spectrometry but was devoid of thrombin-like enzymatic activity. Separated from the high molecular weight fraction by fast protein liquid chromatography, enzymatically intact alpha- and gamma-thrombin failed to trigger any release. Our findings may force a revision of the notion that thrombin itself is a direct proinflammatory release signal for microglia. In addition, they could be relevant for the study of other cellular activities and their assignment to this protease Y1 - 2004 ER - TY - JOUR A1 - Nettels, Daniel A1 - Müller-Späth, Sonja A1 - Küster, Frank A1 - Hofmann, Hagen A1 - Haenni, Domminik A1 - Rüegger, Stefan A1 - Reymond, Luc A1 - Hoffmann, Armin S. A1 - Kubelka, Jan A1 - Heinz, Benjamin A1 - Gast, Klaus A1 - Best, Robert B. A1 - Schuler, Benjamin T1 - Single-molecule spectroscopy of the temperature-induced collapse of unfolded proteins N2 - We used single-molecule FRET in combination with other biophysical methods and molecular simulations to investigate the effect of temperature on the dimensions of unfolded proteins. With singlemolecule FRET, this question can be addressed even under nearnative conditions, where most molecules are folded, allowing us to probe a wide range of denaturant concentrations and temperatures. We find a compaction of the unfolded state of a small cold shock protein with increasing temperature in both the presence and the absence of denaturant, with good agreement between the results from single-molecule FRET and dynamic light scattering. Although dissociation of denaturant from the polypeptide chain with increasing temperature accounts for part of the compaction, the results indicate an important role for additional temperaturedependent interactions within the unfolded chain. The observation of a collapse of a similar extent in the extremely hydrophilic, intrinsically disordered protein prothymosin suggests that the hydrophobic effect is not the sole source of the underlying interactions. Circular dichroism spectroscopy and replica exchange molecular dynamics simulations in explicit water show changes in secondary structure content with increasing temperature and suggest a contribution of intramolecular hydrogen bonding to unfolded state collapse. Y1 - 2009 UR - http://www.pnas.org/content/106/49/20740.full.pdf+html SN - 0027-8424 ER - TY - JOUR A1 - Walter, Juliane K. A1 - Castro, Victor Manuel A1 - Voss, M. A1 - Gast, Klaus A1 - Rueckert, C. A1 - Piontek, J. A1 - Blasig, Ingolf E. T1 - Redox sensitivity of the dimerization of occludin N2 - Occludin is a self-associating transmembrane tight junction protein affected in oxidative stress. However, its function is unknown. The cytosolic C-terminal tail contains a coiled coil-domain forming dimers contributing to the self- association. Studying the hypothesis that the self-association is redox-sensitive, we found that the dimerization of the domain depended on the sulfhydryl concentration of the environment in low-millimolar range. Under physiological conditions, monomers and dimers were detected. Masking the sulfhydryl residues in the domain prevented the dimerization but affected neither its helical structure nor cylindric shape. Incubation of cell extracts containing full-length occludin with sulfhydryl reagents prevented the dimerization; a cysteine/alanine exchange mutant also did not show dimer formation. This demonstrates, for the first time, that disulfide bridge formation of the domain is involved in the occludin dimerization. It is concluded that the redox-dependent dimerization of occludin may play a regulatory role in the tight junction assembly under physiological and pathological conditions. Y1 - 2009 UR - http://www.springerlink.com/content/a0w10t7jgn01lk6h/ SN - 1420-682X ER - TY - JOUR A1 - Fabian, Heinz A1 - Gast, Klaus A1 - Laue, Michael A1 - Misselwitz, Rolf A1 - Uchanska-Ziegler, Barbara A1 - Ziegler, Andreas A1 - Naumann, Dieter T1 - Early stages of misfolding and association of beta2-microglobulin : insights from infrared spectroscopy and dynamic light scattering N2 - Conformational changes associated with the assembly of recombinant ;2-microglobulin in vitro under acidic conditions were investigated using infrared spectroscopy and static and dynamic light scattering. In parallel, the morphology of the different aggregated species obtained under defined conditions was characterized by electron microscopy. The initial salt-induced aggregate form of ;2-microglobulin, composed of small oligomers (dimers to tetramers), revealed the presence of ;-strands organized in an intramolecular-like fashion. Further particle growth was accompanied by the formation of intermolecular ;-sheet structure and led to short curved forms. An increase in temperature by only 25 °C was able to disaggregate these assemblies, followed by the formation of longer filamentous structures. In contrast, a rise in temperature up to 100 °C was associated with a reorganization of the short curved forms at the level of secondary structure and the state of assembly, leading to a species with a characteristic infrared spectrum different from those of all the other aggregates observed before, suggesting a unique overall structure. The infrared spectral features of this species were nearly identical to those of ;2-microglobulin assemblies formed at low ionic strength with agitation, indicating the presence of fibrils, which was confirmed by electron microscopy. The observed spectroscopic changes suggest that the heat-triggered conversion of the short curved assemblies into fibrils involves a reorganization of the ;-strands from an antiparallel arrangement to a parallel arrangement, with the latter being characteristic of amyloid fibrils of ;2-microglobulin. Y1 - 2008 UR - http://pubs.acs.org/doi/abs/10.1021/bi800279y ER - TY - JOUR A1 - Gast, Klaus A1 - Modler, Andreas Johannes T1 - Dynamic and static light scattering of proteins Y1 - 2007 SN - 978-1-600-21704-3 ER - TY - JOUR A1 - Gast, Klaus A1 - Modler, Andreas Johannes T1 - Studying protein folding and aggregation by LASER light scattering Y1 - 2005 SN - 3-527-30784-2 ER - TY - JOUR A1 - Lilie, Hauke A1 - Bär, Dorit A1 - Kettner, Karina A1 - Weininger, Ulrich A1 - Balbach, Jochen A1 - Naumann, Manfred A1 - Müller, Eva-Christina A1 - Otto, Albrecht A1 - Gast, Klaus A1 - Golbik, Ralph T1 - Yeast hexokinase isoenzyme ScHxk2 : stability of a two-domain protein with discontinuous domains N2 - The hexokinase isoenzyme 2 of Saccharomyces cerevisiae (ScHxk2) represents an archetype of a two-domain protein with the active site located in a cleft between the two domains. Binding of the substrate glucose results in a rigid body movement of the two domains leading to a cleft closure of the active site. Both domains of this enzyme are composed of discontinuous peptide sequences. This structural feature is reflected in the stability and folding of the ScHxk2 protein. Structural transitions induced by urea treatment resulted in the population of a thermodynamically stable folding intermediate, which, however, does not correspond to a molecule with one domain folded and the other unfolded. As demonstrated by different spectroscopic techniques, both domains are structurally affected by the partial denaturation. The intermediate possesses only 40% of the native secondary structural content and a substantial increase in the Stokes radius as judged by circular dichroism and dynamic light scattering analyses. One-dimensional 1H NMR data prove that all tryptophan residues are in a non-native environment in the intermediate, indicating substantial changes in the tertiary structure. Still, the intermediate possesses quite a high stability for a transition intermediate of about ;G = ;22 kJ mol;1. Y1 - 2011 UR - http://peds.oxfordjournals.org/content/24/1-2/79.long SN - 0269-2139 ER - TY - JOUR A1 - Gast, Klaus T1 - Dynamic and static light scattering Y1 - 2010 SN - 978-0-470-34341-8 ER - TY - JOUR A1 - Gast, Klaus A1 - Damaschun, Gregor A1 - Desmadril, Michel A1 - Minard, Philippe A1 - Müller-Frohne, Marlies A1 - Pfeil, Wolfgang A1 - Zirwer, Dietrich T1 - Cold denaturation of yeast phosphoglycerate kinase : which domain is more stable? Y1 - 1995 ER - TY - JOUR A1 - Walter, Juliane K. A1 - Castro, Victor Manuel A1 - Voss, Martin A1 - Gast, Klaus A1 - Rueckert, Christine A1 - Piontek, Jörg A1 - Blasig, Ingolf E. T1 - Redox-sensitivity of the dimerization of occludin N2 - Occludin is a self-associating transmembrane tight junction protein affected in oxidative stress. However, its function is unknown. The cytosolic C-terminal tail contains a coiled coil-domain forming dimers contributing to the self- association. Studying the hypothesis that the self-association is redox-sensitive, we found that the dimerization of the domain depended on the sulfhydryl concentration of the environment in low-millimolar range. Under physiological conditions, monomers and dimers were detected. Masking the sulfhydryl residues in the domain prevented the dimerization but affected neither its helical structure nor cylindric shape. Incubation of cell extracts containing full-length occludin with sulfhydryl reagents prevented the dimerization; a cysteine/alanine exchange mutant also did not show dimer formation. This demonstrates, for the first time, that disulfide bridge formation of the domain is involved in the occludin dimerization. It is concluded that the redox-dependent dimerization of occludin may play a regulatory role in the tight junction assembly under physiological and pathological conditions. Y1 - 2009 UR - http://www.springerlink.com/content/101193 U6 - https://doi.org/10.1007/s00018-009-0150-z SN - 1420-682X ER - TY - JOUR A1 - Walter, Juliane K. A1 - Rückert, Christine A1 - Voss, Martin A1 - Müller, Sebastian L. A1 - Piontek, Joerg A1 - Gast, Klaus A1 - Blasig, Ingolf E. T1 - The oligomerization of the coiled coil-domain of occluddin is redox sensitive N2 - The transmembrane tight junction protein occludin is sensitive to oxidative stress. Occludin oligomerizes; however, its function in the tight junction is unknown. The cytosolic C-terminal tail contains a coiled coil-domain and forms dimers contributing to the oligomerization. The regulation of the oligomerization remains unclear. As the domain area contains sulfhydryl residues, we tested the hypothesis that the dimerization of the coiled coil-domain depends on these residues. We showed that the dimerization is modulated by the thiol concentration in the low-millimolar range, which is relevant both for physiological and pathophysiological conditions. Masking the sulfhydryl residues in the fragment by covalent binding of 4-vinyl pyridine prevented the dimerization but did not affect its helical structure and cylindric shape. The data demonstrate, for the first time, that disulfide bridge formation of murine cystein 408 is involved in the dimerization. This process is redox-sensitive but the secondary structure of the domain is not. It is concluded that the dimerization of occludin may play a regulatory role in the tight junction assembly under physiological and pathological conditions. Y1 - 2009 UR - http://www3.interscience.wiley.com/cgi-bin/issn?DESCRIPTOR=PRINTISSN&VALUE=0077-8923 U6 - https://doi.org/10.1111/j.1749-6632.2009.04058.x SN - 0077-8923 ER - TY - JOUR A1 - Fabian, Heinz A1 - Gast, Klaus A1 - Laue, Michael A1 - Jetzschmann, Katharina J. A1 - Naumann, Dieter A1 - Ziegler, Andreas A1 - Uchanska-Ziegler, Barbara T1 - IR spectroscopic analyses of amyloid fibril formation of beta(2)-microglobulin using a simplified procedure for its in vitro generation at neutral pH JF - Biophysical chemistry : an international journal devoted to the physical chemistry of biological phenomena N2 - beta(2)-microglobulin (beta(2)m) is known to be the major component of fibrillar deposits in the joints of patients suffering from dialysis-related amyloidosis. We have developed a simplified procedure to convert monomeric recombinant beta(2)m into amyloid fibrils at physiological pH by a combination of stirring and heating, enabling us to follow conformational changes associated with the assembly by infrared spectroscopy and electron microscopy. Our studies reveal that fibrillogenesis begins with the formation of relatively large aggregates, with secondary structure not significantly altered by the stirring-induced association. In contrast, the conversion of the amorphous aggregates into amyloid fibrils is associated with a profound re-organization at the level of the secondary and tertiary structures, leading to non-native like parallel arrangements of the beta-strands in the fully formed amyloid structure of beta(2)m. This study highlights the power of an approach to investigate the formation of beta(2)m fibrils by a combination of biophysical techniques including IR spectroscopy. KW - Amyloid fibril KW - beta(2)-microglobulin KW - Amyloidogenesis KW - IR spectroscopy Y1 - 2013 U6 - https://doi.org/10.1016/j.bpc.2013.05.001 SN - 0301-4622 VL - 179 IS - 5 SP - 35 EP - 46 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Reschke, Stefan A1 - Sigfridsson, Kajsa G. V. A1 - Kaufmann, Paul A1 - Leidel, Nils A1 - Horn, Sebastian A1 - Gast, Klaus A1 - Schulzke, Carola A1 - Haumann, Michael A1 - Leimkühler, Silke T1 - Identification of a bis-molybdopterin intermediate in molybdenum cofactor biosynthesis in escherichia coli JF - The journal of biological chemistry N2 - The molybdenum cofactor is an important cofactor, and its biosynthesis is essential for many organisms, including humans. Its basic form comprises a single molybdopterin (MPT) unit, which binds a molybdenum ion bearing three oxygen ligands via a dithiolene function, thus forming Mo-MPT. In bacteria, this form is modified to form the bis-MPT guanine dinucleotide cofactor with two MPT units coordinated at one molybdenum atom, which additionally contains GMPs bound to the terminal phosphate group of the MPTs (bis-MGD). The MobA protein catalyzes the nucleotide addition to MPT, but the mechanism of the biosynthesis of the bis-MGD cofactor has remained enigmatic. We have established an in vitro system for studying bis-MGD assembly using purified compounds. Quantification of the MPT/molybdenum and molybdenum/phosphorus ratios, time-dependent assays for MPT and MGD detection, and determination of the numbers and lengths of Mo-S and Mo-O bonds by X-ray absorption spectroscopy enabled identification of a novel bis-Mo-MPT intermediate on MobA prior to nucleotide attachment. The addition of Mg-GTP to MobA loaded with bis-Mo-MPT resulted in formation and release of the final bis-MGD product. This cofactor was fully functional and reconstituted the catalytic activity of apo-TMAO reductase (TorA). We propose a reaction sequence for bis-MGD formation, which involves 1) the formation of bis-Mo-MPT, 2) the addition of two GMP units to form bis-MGD on MobA, and 3) the release and transfer of the mature cofactor to the target protein TorA, in a reaction that is supported by the specific chaperone TorD, resulting in an active molybdoenzyme. Y1 - 2013 U6 - https://doi.org/10.1074/jbc.M113.497453 SN - 0021-9258 SN - 1083-351X VL - 288 IS - 41 SP - 29736 EP - 29745 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda ER -