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 - 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 - 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 T2 - Postprints der Universität Potsdam Mathematisch-Naturwissenschaftliche Reihe N2 - Purpose: Comparison of the dissociation kinetics of rapid-acting insulins lispro, aspart, glulisine and human insulin under physiologically relevant conditions. Methods: 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. Results: 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. Conclusion: Knowledge of the diverging dissociation mechanisms of lispro and aspart compared to glulisine will be helpful for optimizing formulation conditions of rapid-acting insulins. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 795 KW - circular dichroism KW - dissociation kinetics KW - insulin analog KW - light scattering KW - rapid-acting Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-431572 SN - 1866-8372 IS - 795 SP - 2270 EP - 2286 ER - TY - JOUR A1 - Mishra, Rajesh A1 - Seckler, Robert A1 - Bhat, Rajiv T1 - Efficient refolding of aggregation-prone citrate synthase by polyol osmolytes : how well are protein folding and stability aspects coupled? N2 - Efficient refolding of proteins and prevention of their aggregation during folding are of vital importance in recombinant protein production and in finding cures for several diseases. We have used citrate synthase ( CS) as a model to understand the mechanism of aggregation during refolding and its prevention using several known structure-stabilizing cosolvent additives of the polyol series. Interestingly, no parallel correlation between the folding effect and the general stabilizing effect exerted by polyols was observed. Although increasing concentrations of polyols increased protein stability in general, the refolding yields for CS decreased at higher polyol concentrations, with erythritol reducing the folding yields at all concentrations tested. Among the various polyols used, glycerol was the most effective in enhancing the CS refolding yield, and a complete recovery of enzymatic activity was obtained at 7 M glycerol and 10 mu g/ml protein, a result superior to the action of the molecular chaperones GroEL and GroES in vitro. A good correlation between the refolding yields and the suppression of protein aggregation by glycerol was observed, with no aggregation detected at 7 M. The polyols prevented the aggregation of CS depending on the number of hydroxyl groups in them. Stopped-flow fluorescence kinetics experiments suggested that polyols, including glycerol, act very early in the refolding process, as no fast and slow phases were detectable. The results conclusively demonstrate that both the thermodynamic and kinetic aspects are critical in the folding process and that all structure-stabilizing molecules need not always help in productive folding to the native state. These findings are important for the rational design of small molecules for efficient refolding of various aggregation-prone proteins of commercial and medical relevance Y1 - 2005 SN - 0021-9258 ER - TY - JOUR A1 - Freiberg, Alexander A1 - Machner, M. P. A1 - Pfeil, Wolfgang A1 - Schubert, W. D. A1 - Heinz, Dirk W. A1 - Seckler, Robert T1 - Folding and stability of the leucine-rich repeat domain of internalin B from Listeria monocytogenes N2 - Internalin B (InlB), a surface protein of the human pathogen Listeria monocytogenes, promotes invasion into various host cell types by inducing phagocytosis of the entire bacterium. The N-terminal half of InlB (residues 36-321, InlB(321)), which is sufficient for this process, contains a central leucine-rich repeat (LRR) domain that is flanked by a small a-helical cap 2 and an immunoglobulin (Ig)-like domain. Here we investigated the variant lacking the Ig-like domain (lnlB(248)). The circular dichroism spectra of both protein variants in the far ultraviolet region are very similar, with a characteristic minimum found at similar to200 nm, possibly resulting from the high 3(10)-helical content in the LRR domain. Upon addition of chemical denaturants, both variants unfold in single transitions with unusually high cooperativity that are fully reversible and best described by two-state equilibria. The free energies of GdmCl-induced unfolding determined from transitions at 20degreesC are 9.9(+/- 0.8)kcal/mol for InlB(321) and 5.4(+/- 0.4) kcal/mol for InlB(248). InlB(321) is also more stable against thermal denaturation, as observed by scanning calorimetry. This suggests, that the Ig-like domain, which presumably does not directly interact with the host cell receptor during bacterial invasion, plays a critical role for the in vivo stability of InlB. (C) 2004 Elsevier Ltd. All rights reserved Y1 - 2004 SN - 0022-2836 ER - TY - JOUR A1 - Scheich, Christoph A1 - Niesen, F. H. A1 - Seckler, Robert A1 - Bussow, K. T1 - An automated in vitro protein folding screen applied to a human dynactin subunit N2 - The preparation of proteins for structural and functional analysis using the Escherichia coli expression system is often hampered by the formation of insoluble intracellular protein aggregates (inclusion bodies). Transferring those proteins into their native states by in vitro protein folding requires screening for the best buffer conditions and suitable additives. However, it is difficult to assess the success of such a screen if no biological assay is available. We established a fully automated folding screen and a system to detect folded protein that is based on analytical hydrophobic interaction chromatography and tryptophan fluorescence spectroscopy. The system was evaluated with two model enzymes (carbonic anhydrase II and malate dehydrogenase), and was successfully applied to the folding of the p22 subunit of human dynactin, which is expressed in inclusion bodies in E. coli. The described screen allows for high-throughput folding analysis of inclusion body proteins for structural and functional analyses Y1 - 2004 SN - 0961-8368 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 - Thalhammer, Anja A1 - Hundertmark, Michaela A1 - Popova, Antoaneta V. A1 - Seckler, Robert A1 - Hincha, Dirk K. T1 - Interaction of two intrinsically disordered plant stress proteins (COR15A and COR15B) with lipid membranes in the dry state N2 - COR15A and COR15B form a tandem repeat of highly homologous genes in Arabidopsis thaliana. Both genes are highly cold induced and the encoded proteins belong to the Pfam LEA_4 group (group 3) of the late embryogenesis abundant (LEA) proteins. Both proteins were predicted to be intrinsically disordered in solution. Only COR15A has previously been characterized and it was shown to be localized in the soluble stroma fraction of chloroplasts. Ectopic expression of COR15A in Arabidopsis resulted in increased freezing tolerance of both chloroplasts after freezing and thawing of intact leaves and of isolated protoplasts frozen and thawed in vitro. In the present study we have generated recombinant mature COR15A and COR15B for a comparative study of their structure and possible function as membrane protectants. CD spectroscopy showed that both proteins are predominantly unstructured in solution and mainly a-helical after drying. Both proteins showed similar effects on the thermotropic phase behavior of dry liposomes. A decrease in the gel to liquid-crystalline phase transition temperature depended on both the unsaturation of the fatty acyl chains and lipid headgroup structure. FTIR spectroscopy indicated no strong interactions between the proteins and the lipid phosphate and carbonyl groups, but significant interactions with the galactose headgroup of the chloroplast lipid monogalactosyldiacylglycerol. These findings were rationalized by modeling the secondary structure of COR15A and COR15B. Helical wheel projection indicated the presence of amphipathic a-helices in both proteins. The helices lacked a clear separation of positive and negative charges on the hydrophilic face, but contained several hydroxylated amino acids. Y1 - 2010 UR - http://www.sciencedirect.com/science/journal/00052736 U6 - https://doi.org/10.1016/j.bbamem.2010.05.015 SN - 0005-2736 ER - TY - JOUR A1 - Hoffmann, Armin S. A1 - Kane, Avinash S. A1 - Nettels, Daniel A1 - Hertzog, David E. A1 - Baumgärtel, Peter A1 - Lengefeld, Jan A1 - Reichardt, Gerd A1 - Horsley, David A. A1 - Seckler, Robert A1 - Bakajin, Olgica A1 - Schuler, Benjamin T1 - Mapping protein collapse with single molecule fluorescence and kinetic synchrotron radiation circular dichroism spectroscopy Y1 - 2007 UR - http://www.mendeley.com/research/mapping-protein-collapse-with-singlemolecule-fluorescence-and-kinetic- synchrotron-radiation-circular-dichroism-spectroscopy/# SN - 0027-8424 ER - TY - JOUR A1 - Küster, Frank A1 - Seckler, Robert T1 - Pea seed lectin folds and oligomerizes via an intermediate not represented in the structural hierarchy N2 - Large oligomeric proteins are usually thought to fold and assemble hierarchically: Domains fold and coalesce to form the subunits, and folded subunits can then associate to form the multimeric structure. We have investigated the refolding pathway of the ;-sheet protein pea seed lectin using spectroscopic and hydrodynamic techniques. In vivo, it is proteolytically processed post-translationally, so that the single-domain subunits of the initial homodimer themselves become heterodimers of intertwined fragment polypeptide chains. Despite this complex topology, mature pea seed lectin reassembles with considerable efficiency at low total protein concentration (10 ;g/mL) and low temperature (10 °C), albeit very slowly (t1/2 ; 2 days). Contrary to expectations, the primary assembly product is not the intact ;-sheet domain, but the larger fragment chains first dimerize to form the native-like subunit interface. The smaller fragment chains then associate with this preformed dimer. Y1 - 2008 UR - http://pubs.acs.org/doi/abs/10.1021/bi7019047 U6 - https://doi.org/10.1021/Bi7019047 SN - 0006-2960 ER -