@article{BlasigWinklerLassowskietal.2006, author = {Blasig, Ingolf E. and Winkler, Lars and Lassowski, Birgit and M{\"u}ller, Sandra L. and Zuleger, Nikolaj and Krause, Eberhard and Krause, Gerd and Gast, Klaus and Kolbe, Michael and Piontek, J{\"o}rg}, title = {On the self-association potential of transmembrane tight junction proteins}, issn = {1420-682X}, doi = {10.1007/s00018-005-5472-x}, year = {2006}, abstract = {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}, language = {en} } @article{BorgiaZhengBuholzeretal.2016, author = {Borgia, Alessandro and Zheng, Wenwei and Buholzer, Karin and Borgia, Madeleine B. and Sch{\"u}ler, Anja and Hofmann, Hagen and Soranno, Andrea and Nettels, Daniel and Gast, Klaus and Grishaev, Alexander and Best, Robert B. and Schuler, Benjamin}, title = {Consistent View of Polypeptide Chain Expansion in Chemical Denaturants from Multiple Experimental Methods}, series = {Journal of the American Chemical Society}, volume = {138}, journal = {Journal of the American Chemical Society}, publisher = {American Chemical Society}, address = {Washington}, issn = {0002-7863}, doi = {10.1021/jacs.6b05917}, pages = {11714 -- 11726}, year = {2016}, abstract = {There has been a long-standing controversy regarding the effect of chemical denaturants on the dimensions of unfolded and intrinsically disordered proteins: A wide range of experimental techniques suggest that polypeptide chains expand with increasing denaturant concentration, but several studies using small-angle X-ray scattering (SAXS) have reported no: such increase of the radius of gyration (R-g). This inconsistency challenges our current understanding of the mechanism of chemical denaturants, which are widely employed to investigate protein folding and stability. Here, we use a combination Of single-molecule Forster resonance energy transfer (FRET), SAXS, dynamic light scattering (DLS), and two-focus fluorescence correlation spectroscopy (2f-FCS) to characterize the denaturant dependence of the unfolded state of the spectrin domain R17 and the intrinsically disordered protein ACTR in two different denaturants. Standard analysis of the primary data clearly indicates an expansion of the unfolded state with increasing denaturant concentration irrespective of the protein, denaturant, or experimental method used. This is the first case in which SAXS and FRET have yielded even qualitatively consistent results regarding expansion in denaturant when applied to the same proteins. To more directly illustrate this self-consistency, we used both SAXS and FRET data in a Bayesian procedure to refine structural ensembles representative of the observed unfolded state. This analysis demonstrates that both of these experimental probes are compatible with a common ensemble of protein configurations for each denaturant concentration. Furthermore, the resulting ensembles reproduce the trend of increasing hydrodynamic radius, with denaturant concentration obtained by 2f-FCS,and DLS. We were thus able to reconcile the results from all four experimental techniques quantitatively, to obtain a comprehensive structural picture of denaturant;induced unfolded state expansion, and to identify the Most likely sources of earlier discrepancies.}, language = {en} } @article{DamaschunDamaschunGastetal.1993, author = {Damaschun, Gregor and Damaschun, Hilde and Gast, Klaus and Misselwitz, Rolf and M{\"u}ller, J{\"u}rgen J. and Pfeil, Wolfgang and Zirwer, Dietrich}, title = {Cold denaturation-induced conformational changes in phosphoglycerate kinase from yeast}, year = {1993}, language = {en} } @article{FabianGastFilimonovetal.2005, author = {Fabian, H. and Gast, Klaus and Filimonov, Vladimir V. and Zamyatkin, D. F. and Rogov, V. V.}, title = {Thermal unfolding of two designed monomeric lambda Cro repressor variants}, issn = {0924-2031}, year = {2005}, abstract = {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}, language = {en} } @article{FabianGastLaueetal.2013, author = {Fabian, Heinz and Gast, Klaus and Laue, Michael and Jetzschmann, Katharina J. and Naumann, Dieter and Ziegler, Andreas and Uchanska-Ziegler, Barbara}, title = {IR spectroscopic analyses of amyloid fibril formation of beta(2)-microglobulin using a simplified procedure for its in vitro generation at neutral pH}, series = {Biophysical chemistry : an international journal devoted to the physical chemistry of biological phenomena}, volume = {179}, journal = {Biophysical chemistry : an international journal devoted to the physical chemistry of biological phenomena}, number = {5}, publisher = {Elsevier}, address = {Amsterdam}, issn = {0301-4622}, doi = {10.1016/j.bpc.2013.05.001}, pages = {35 -- 46}, year = {2013}, abstract = {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.}, language = {en} } @article{FabianGastLaueetal.2008, author = {Fabian, Heinz and Gast, Klaus and Laue, Michael and Misselwitz, Rolf and Uchanska-Ziegler, Barbara and Ziegler, Andreas and Naumann, Dieter}, title = {Early stages of misfolding and association of beta2-microglobulin : insights from infrared spectroscopy and dynamic light scattering}, year = {2008}, abstract = {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.}, language = {en} } @article{Gast2010, author = {Gast, Klaus}, title = {Dynamic and static light scattering}, isbn = {978-0-470-34341-8}, year = {2010}, language = {en} } @article{GastDamaschunDesmadriletal.1995, author = {Gast, Klaus and Damaschun, Gregor and Desmadril, Michel and Minard, Philippe and M{\"u}ller-Frohne, Marlies and Pfeil, Wolfgang and Zirwer, Dietrich}, title = {Cold denaturation of yeast phosphoglycerate kinase : which domain is more stable?}, year = {1995}, language = {en} } @article{GastModler2007, author = {Gast, Klaus and Modler, Andreas Johannes}, title = {Dynamic and static light scattering of proteins}, isbn = {978-1-600-21704-3}, year = {2007}, language = {en} } @article{GastModler2005, author = {Gast, Klaus and Modler, Andreas Johannes}, title = {Studying protein folding and aggregation by LASER light scattering}, isbn = {3-527-30784-2}, year = {2005}, language = {en} }