TY - JOUR A1 - Wolff, Martin A1 - Gast, Klaus A1 - Evers, Andreas A1 - Kurz, Michael A1 - Pfeiffer-Marek, Stefania A1 - Schüler, Anja A1 - Seckler, Robert A1 - Thalhammer, Anja T1 - A Conserved Hydrophobic Moiety and Helix-Helix Interactions Drive the Self-Assembly of the Incretin Analog Exendin-4 JF - Biomolecules N2 - Exendin-4 is a pharmaceutical peptide used in the control of insulin secretion. Structural information on exendin-4 and related peptides especially on the level of quaternary structure is scarce. We present the first published association equilibria of exendin-4 directly measured by static and dynamic light scattering. We show that exendin-4 oligomerization is pH dependent and that these oligomers are of low compactness. We relate our experimental results to a structural hypothesis to describe molecular details of exendin-4 oligomers. Discussion of the validity of this hypothesis is based on NMR, circular dichroism and fluorescence spectroscopy, and light scattering data on exendin-4 and a set of exendin-4 derived peptides. The essential forces driving oligomerization of exendin-4 are helix–helix interactions and interactions of a conserved hydrophobic moiety. Our structural hypothesis suggests that key interactions of exendin-4 monomers in the experimentally supported trimer take place between a defined helical segment and a hydrophobic triangle constituted by the Phe22 residues of the three monomeric subunits. Our data rationalize that Val19 might function as an anchor in the N-terminus of the interacting helix-region and that Trp25 is partially shielded in the oligomer by C-terminal amino acids of the same monomer. Our structural hypothesis suggests that the Trp25 residues do not interact with each other, but with C-terminal Pro residues of their own monomers. KW - biophysics KW - diabetes KW - peptides KW - oligomerization KW - conformational change KW - molecular modeling KW - static and dynamic light scattering KW - spectroscopy Y1 - 2021 U6 - https://doi.org/10.3390/biom11091305 SN - 2218-273X VL - 11 IS - 9 PB - MDPI CY - Basel ER - TY - GEN A1 - Wolff, Martin A1 - Gast, Klaus A1 - Evers, Andreas A1 - Kurz, Michael A1 - Pfeiffer-Marek, Stefania A1 - Schüler, Anja A1 - Seckler, Robert A1 - Thalhammer, Anja T1 - A Conserved Hydrophobic Moiety and Helix-Helix Interactions Drive the Self-Assembly of the Incretin Analog Exendin-4 T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Exendin-4 is a pharmaceutical peptide used in the control of insulin secretion. Structural information on exendin-4 and related peptides especially on the level of quaternary structure is scarce. We present the first published association equilibria of exendin-4 directly measured by static and dynamic light scattering. We show that exendin-4 oligomerization is pH dependent and that these oligomers are of low compactness. We relate our experimental results to a structural hypothesis to describe molecular details of exendin-4 oligomers. Discussion of the validity of this hypothesis is based on NMR, circular dichroism and fluorescence spectroscopy, and light scattering data on exendin-4 and a set of exendin-4 derived peptides. The essential forces driving oligomerization of exendin-4 are helix–helix interactions and interactions of a conserved hydrophobic moiety. Our structural hypothesis suggests that key interactions of exendin-4 monomers in the experimentally supported trimer take place between a defined helical segment and a hydrophobic triangle constituted by the Phe22 residues of the three monomeric subunits. Our data rationalize that Val19 might function as an anchor in the N-terminus of the interacting helix-region and that Trp25 is partially shielded in the oligomer by C-terminal amino acids of the same monomer. Our structural hypothesis suggests that the Trp25 residues do not interact with each other, but with C-terminal Pro residues of their own monomers. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1161 KW - biophysics KW - diabetes KW - peptides KW - oligomerization KW - conformational change KW - molecular modeling KW - static and dynamic light scattering KW - spectroscopy Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-522081 SN - 1866-8372 IS - 9 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 - Damaschun, Gregor A1 - Damaschun, Hilde A1 - Gast, Klaus A1 - Misselwitz, Rolf A1 - Müller, Jürgen J. A1 - Pfeil, Wolfgang A1 - Zirwer, Dietrich T1 - Cold denaturation-induced conformational changes in phosphoglycerate kinase from yeast Y1 - 1993 ER - 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 - Borgia, Alessandro A1 - Zheng, Wenwei A1 - Buholzer, Karin A1 - Borgia, Madeleine B. A1 - Schüler, Anja A1 - Hofmann, Hagen A1 - Soranno, Andrea A1 - Nettels, Daniel A1 - Gast, Klaus A1 - Grishaev, Alexander A1 - Best, Robert B. A1 - Schuler, Benjamin T1 - Consistent View of Polypeptide Chain Expansion in Chemical Denaturants from Multiple Experimental Methods JF - Journal of the American Chemical Society N2 - 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. Y1 - 2016 U6 - https://doi.org/10.1021/jacs.6b05917 SN - 0002-7863 VL - 138 SP - 11714 EP - 11726 PB - American Chemical Society CY - Washington 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 - Modler, Andreas Johannes T1 - Dynamic and static light scattering of proteins Y1 - 2007 SN - 978-1-600-21704-3 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 - Herbst, R. A1 - Gast, Klaus A1 - Seckler, Robert T1 - Folding of firefly (Photinus pyralis) luciferase : aggregation and reactivation of unfolding intermediates Y1 - 1998 ER -