TY - JOUR A1 - Baxa, Ulrich A1 - Weintraub, Andrej A1 - Seckler, Robert T1 - Self-competitive inhibition of the bacteriophage P22 Tailspike endorhamnosidase by O-antigen oligosaccharides JF - Biochemistry N2 - The P22 tailspike endorhamnosidase confers the high specificity of bacteriophage P22 for some serogroups of Salmonella differing only slightly in their O-antigen polysaccharide. We used several biophysical methods to study the binding and hydrolysis of O-antigen fragments of different lengths by P22 tailspike protein. O-Antigen saccharides of defined length labeled with fluorophors could be purified with higher resolution than previously possible. Small amounts of naturally occurring variations of 0antigen fragments missing the nonreducing terminal galactose could be used to determine the contribution of this part to the free energy of binding to be similar to 7 kJ/mol. We were able to show via several independent lines of evidence that an unproductive binding mode is highly favored in binding over all other possible binding modes leading to hydrolysis. This is true even under circumstances under which the O-antigen fragment is long enough to be cleaved efficiently by the enzyme. The high-affinity unproductive binding mode results in a strong self-competitive inhibition in addition to product inhibition observed for this system. Self-competitive inhibition is observed for all substrates that have a free reducing end rhamnose. Naturally occurring O-antigen, while still attached to the bacterial outer membrane, does not have a free reducing end and therefore does not perform self-competitive inhibition. Y1 - 2020 U6 - https://doi.org/10.1021/acs.biochem.0c00872 SN - 0006-2960 VL - 59 IS - 51 SP - 4845 EP - 4855 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Wolff, Martin A1 - Schüler, Anja A1 - Gast, Klaus A1 - Seckler, Robert A1 - Evers, Andreas A1 - Pfeiffer-Marek, Stefania A1 - Kurz, Michael A1 - Nagel, Norbert A1 - Haack, Torsten A1 - Wagner, Michael A1 - Thalhammer, Anja T1 - Self-Assembly of Exendin-4-Derived Dual Peptide Agonists is Mediated by Acylation and Correlated to the Length of Conjugated Fatty Acyl Chains JF - Molecular pharmaceutics N2 - Dual glucagon-like peptide-1/glucagon receptor agonists have emerged as promising candidates for the treatment of diabetes and obesity. Issues of degradation sensitivity and rapid renal clearance are addressed, for example, by the conjugation of peptides to fatty acid chains, promoting reversible albumin binding. We use combined dynamic and static light scattering to directly measure the self-assembly of a set of dual peptide agonists based on the exendin-4 structure with varying fatty acid chain lengths in terms of apparent molecular mass and hydrodynamic radius (R-S). We use NMR spectroscopy to gain an insight into the molecular architecture of the assembly. We investigate conformational changes of the monomeric subunits resulting from peptide self-assembly and assembly stability as a function of the fatty acid chain length using circular dichroism and fluorescence spectroscopy. Our results demonstrate that self-assembly of the exendin-4-derived dual agonist peptides is essentially driven by hydrophobic interactions involving the conjugated acyl chains. The fatty acid chain length affects assembly equilibria and the assembly stability, although the peptide subunits in the assembly retain a dynamic secondary structure. The assembly architecture is characterized by juxtaposition of the fatty acyl side chains and a hydrophobic cluster of the peptide moiety. This cluster experiences local conformational changes in the assembly compared to the monomeric unit leading to a reduction in solvent exposure. The N-terminal half of the peptide and a C-terminal loop are not in contact with neighboring peptide subunits in the assemblies. Altogether, our study contributes to a thorough understanding of the association characteristics and the tendency toward self-assembly in response to lipidation. This is important not only to achieve the desired bioavailability but also with respect to the physical stability of peptide solutions. KW - dual GLP-1/glucagon receptor agonist KW - self-assembly KW - light scattering KW - molecular architecture KW - lipidation KW - exendin-4 Y1 - 2020 U6 - https://doi.org/10.1021/acs.molpharmaceut.9b01195 SN - 1543-8384 SN - 1543-8392 VL - 17 IS - 3 SP - 965 EP - 978 PB - American Chemical Society CY - Washington ER - 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 - JOUR A1 - Wolff, Martin A1 - Schüler, Anja A1 - Gast, Klaus A1 - Seckler, Robert A1 - Evers, Andreas A1 - Pfeiffer-Marek, Stefania A1 - Kurz, Michael A1 - Nagel, Norbert A1 - Haack, Torsten A1 - Wagner, Michael A1 - Thalhammer, Anja T1 - Self-Assembly of Exendin-4-Derived Dual Peptide Agonists is Mediated by Acylation and Correlated to the Length of Conjugated Fatty Acyl Chains JF - Molecular pharmaceutics N2 - Dual glucagon-like peptide-1/glucagon receptor agonists have emerged as promising candidates for the treatment of diabetes and obesity. Issues of degradation sensitivity and rapid renal clearance are addressed, for example, by the conjugation of peptides to fatty acid chains, promoting reversible albumin binding. We use combined dynamic and static light scattering to directly measure the self-assembly of a set of dual peptide agonists based on the exendin-4 structure with varying fatty acid chain lengths in terms of apparent molecular mass and hydrodynamic radius (R-S). We use NMR spectroscopy to gain an insight into the molecular architecture of the assembly. We investigate conformational changes of the monomeric subunits resulting from peptide self-assembly and assembly stability as a function of the fatty acid chain length using circular dichroism and fluorescence spectroscopy. Our results demonstrate that self-assembly of the exendin-4-derived dual agonist peptides is essentially driven by hydrophobic interactions involving the conjugated acyl chains. The fatty acid chain length affects assembly equilibria and the assembly stability, although the peptide subunits in the assembly retain a dynamic secondary structure. The assembly architecture is characterized by juxtaposition of the fatty acyl side chains and a hydrophobic cluster of the peptide moiety. This cluster experiences local conformational changes in the assembly compared to the monomeric unit leading to a reduction in solvent exposure. The N-terminal half of the peptide and a C-terminal loop are not in contact with neighboring peptide subunits in the assemblies. Altogether, our study contributes to a thorough understanding of the association characteristics and the tendency toward self-assembly in response to lipidation. This is important not only to achieve the desired bioavailability but also with respect to the physical stability of peptide solutions. KW - dual GLP-1/glucagon receptor agonist KW - self-assembly KW - light scattering KW - molecular architecture KW - lipidation KW - exendin-4 Y1 - 2020 U6 - https://doi.org/10.1021/acs.molpharmaceut.9b01195 SN - 1543-8384 VL - 17 IS - 3 SP - 965 EP - 978 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Gast, Klaus A1 - Schüler, Anja A1 - Wolff, Martin A1 - Thalhammer, Anja A1 - Berchtold, Harald A1 - Nagel, Norbert A1 - Lenherr, Gudrun A1 - Hauck, Gerrit A1 - Seckler, Robert T1 - Rapid-acting and human insulins BT - Hexamer Dissociation Kinetics upon Dilution of the Pharmaceutical Formulation JF - Pharmaceutical research N2 - Comparison of the dissociation kinetics of rapid-acting insulins lispro, aspart, glulisine and human insulin under physiologically relevant conditions. Dissociation kinetics after dilution were monitored directly in terms of the average molecular mass using combined static and dynamic light scattering. Changes in tertiary structure were detected by near-UV circular dichroism. Glulisine forms compact hexamers in formulation even in the absence of Zn2+. Upon severe dilution, these rapidly dissociate into monomers in less than 10 s. In contrast, in formulations of lispro and aspart, the presence of Zn2+ and phenolic compounds is essential for formation of compact R6 hexamers. These slowly dissociate in times ranging from seconds to one hour depending on the concentration of phenolic additives. The disadvantage of the long dissociation times of lispro and aspart can be diminished by a rapid depletion of the concentration of phenolic additives independent of the insulin dilution. This is especially important in conditions similar to those after subcutaneous injection, where only minor dilution of the insulins occurs. Knowledge of the diverging dissociation mechanisms of lispro and aspart compared to glulisine will be helpful for optimizing formulation conditions of rapid-acting insulins. KW - circular dichroism KW - dissociation kinetics KW - insulin analog KW - light scattering KW - rapid-acting Y1 - 2017 U6 - https://doi.org/10.1007/s11095-017-2233-0 SN - 0724-8741 SN - 1573-904X VL - 34 IS - 795 SP - 2270 EP - 2286 PB - Springer CY - New York ER - TY - JOUR A1 - Seul, Anait A1 - Müller, Jürgen J. A1 - Andres, Dorothee A1 - Stettner, Eva A1 - Heinemann, Udo A1 - Seckler, Robert T1 - Bacteriophage P22 tailspike: structure of the complete protein and function of the interdomain linker JF - Acta crystallographica : Section D, Biological crystallography N2 - Attachment of phages to host cells, followed by phage DNA ejection, represents the first stage of viral infection of bacteria. Salmonella phage P22 has been extensively studied, serving as an experimental model for bacterial infection by phages. P22 engages bacteria by binding to the sugar moiety of lipopolysaccharides using the viral tailspike protein for attachment. While the structures of the N-terminal particle-binding domain and the major receptor-binding domain of the tailspike have been analyzed individually, the three-dimensional organization of the intact protein, including the highly conserved linker region between the two domains, remained unknown. A single amino-acid exchange in the linker sequence made it possible to crystallize the full-length protein. Two crystal structures of the linker region are presented: one attached to the N-terminal domain and the other present within the complete tailspike protein. Both retain their biological function, but the mutated full-length tailspike displays a retarded folding pathway. Fitting of the full-length tailspike into a published cryo-electron microscopy map of the P22 virion requires an elastic distortion of the crystal structure. The conservation of the linker suggests a role in signal transmission from the distal tip of the molecule to the phage head, eventually leading to DNA ejection. Y1 - 2014 U6 - https://doi.org/10.1107/S1399004714002685 SN - 1399-0047 VL - 70 SP - 1336 EP - 1345 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Hundertmark, Michaela A1 - Dimova, Rumiana A1 - Lengefeld, Jan A1 - Seckler, Robert A1 - Hincha, Dirk K. T1 - The intrinsically disordered late embryogenesis abundant protein LEA18 from Arabidopsis thaliana modulates membrane stability through binding and folding JF - Biochimica et biophysica acta : Biomembranes N2 - Intrinsically disordered proteins (IDPs) constitute a substantial part of cellular proteomes. late embryogenesis abundant (LEA) proteins are mostly predicted to be IDPs associated with dehydration tolerance in many plant, animal and bacterial species. Their functions, however, are largely unexplored and also their structure and interactions with potential target molecules have only recently been experimentally investigated in a small number of proteins. Here, we report on the structure and interactions with membranes of the Pfam LEA_1 protein LEA18 from the higher plant Arabidopsis thaliana. This functionally uncharacterized positively charged protein specifically aggregated and destabilized negatively charged liposomes. Isothermal titration calorimetry showed binding of the protein to both charged and uncharged membranes. LEA18 alone was largely unstructured in solution. While uncharged membranes had no influence on the secondary structure of LEA18, the protein partially folded into beta-sheet structure in the presence of negatively charged liposomes. These data suggest that LEA18 does not function as a membrane stabilizing protein, as suggested for other LEA proteins. Instead, a possible function of LEA18 could be the composition-dependent modulation of membrane stability, e.g., during signaling or vesicle-mediated transport. KW - Intrinsically disordered protein KW - Late embryogenesis abundant protein KW - Membrane stability KW - Protein-membrane interaction KW - Protein folding Y1 - 2011 U6 - https://doi.org/10.1016/j.bbamem.2010.09.010 SN - 0005-2736 VL - 1808 IS - 1 SP - 446 EP - 453 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Popova, Antoaneta V. A1 - Hundertmark, Michaela A1 - Seckler, Robert A1 - Hincha, Dirk K. T1 - Structural transitions in the intrinsically disordered plant dehydration stress protein LEA7 upon drying are modulated by the presence of membranes JF - Biochimica et biophysica acta : Biomembranes N2 - Dehydration stress-related late embryogenesis abundant (LEA) proteins have been found in plants, invertebrates and bacteria. Most LEA proteins are unstructured in solution, but some fold into amphipathic a-helices during drying. The Pfam LEA_4 (Group 3) protein LEA7 from the higher plant Arabidopsis thaliana was predicted to be 87% alpha-helical, while CD spectroscopy showed it to be largely unstructured in solution and only 35% alpha-helical in the dry state. However, the dry protein contained 15% beta-sheets. FTIR spectroscopy revealed the (beta-sheets to be largely due to aggregation. beta-Sheet content was reduced and alpha-helix content increased when LEA7 was dried in the presence of liposomes with secondary structure apparently influenced by lipid composition. Secondary structure was also affected by the presence of membranes in the fully hydrated state. A temperature-induced increase in the flexibility of the dry protein was also only observed in the presence of membranes. Functional interactions of LEA7 with membranes in the dry state were indicated by its influence on the thermotropic phase transitions of the lipids and interactions with the lipid headgroup phosphates. KW - Desiccation KW - CD spectroscopy KW - FTIR spectroscopy KW - LEA protein KW - Protein-membrane interactions KW - Protein secondary structure Y1 - 2011 U6 - https://doi.org/10.1016/j.bbamem.2011.03.009 SN - 0005-2736 VL - 1808 IS - 7 SP - 1879 EP - 1887 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Hundertmark, Michaela A1 - Popova, Antoaneta V. A1 - Rausch, Saskia A1 - Seckler, Robert A1 - Hincha, Dirk K. T1 - Influence of drying on the secondary structure of intrinsically disordered and globular proteins JF - Biochemical and biophysical research communications N2 - Circular dichroism (CD) spectroscopy of five Arabidopsis late embryogenesis abundant (LEA) proteins constituting the plant specific families LEA_5 and LEA_6 showed that they are intrinsically disordered in solution and partially fold during drying. Structural predictions were comparable to these results for hydrated LEA_6, but not for LEA_5 proteins. FTIR spectroscopy showed that verbascose, but not sucrose, strongly affected the structure of the dry proteins. The four investigated globular proteins were only mildly affected by drying in the absence, but strongly in the presence of sugars. These data highlight the larger structural flexibility of disordered compared to globular proteins and the impact of sugars on the structure of both disordered and globular proteins during drying. KW - Desiccation KW - CD spectroscopy KW - FTIR spectroscopy KW - Intrinsically disordered proteins KW - LEA proteins KW - Protein secondary structure Y1 - 2012 U6 - https://doi.org/10.1016/j.bbrc.2011.11.067 SN - 0006-291X VL - 417 IS - 1 SP - 122 EP - 128 PB - Elsevier CY - San Diego ER - TY - JOUR A1 - Andres, Dorothee A1 - Roske, Yvette A1 - Doering, Carolin A1 - Heinemann, Udo A1 - Seckler, Robert A1 - Barbirz, Stefanie T1 - Tail morphology controls DNA release in two Salmonella phages with one lipopolysaccharide receptor recognition system JF - Molecular microbiology N2 - Bacteriophages use specific tail proteins to recognize host cells. It is still not understood to molecular detail how the signal is transmitted over the tail to initiate infection. We have analysed in vitro DNA ejection in long-tailed siphovirus 9NA and short-tailed podovirus P22 upon incubation with Salmonella typhimurium lipopolysaccharide (LPS). We showed for the first time that LPS alone was sufficient to elicit DNA release from a siphovirus in vitro. Crystal structure analysis revealed that both phages use similar tailspike proteins for LPS recognition. Tailspike proteins hydrolyse LPS O antigen to position the phage on the cell surface. Thus we were able to compare in vitro DNA ejection processes from two phages with different morphologies with the same receptor under identical experimental conditions. Siphovirus 9NA ejected its DNA about 30 times faster than podovirus P22. DNA ejection is under control of the conformational opening of the particle and has a similar activation barrier in 9NA and P22. Our data suggest that tail morphology influences the efficiencies of particle opening given an identical initial receptor interaction event. Y1 - 2012 U6 - https://doi.org/10.1111/j.1365-2958.2012.08006.x SN - 0950-382X VL - 83 IS - 6 SP - 1244 EP - 1253 PB - Wiley-Blackwell CY - Hoboken ER -