TY - JOUR A1 - Seckler, Robert A1 - Jaenicke, R. T1 - Spontaneous versus assisted protein folding Y1 - 1999 SN - 90-5702-370-9 ER - TY - JOUR A1 - Schuler, Benjamin A1 - Rachel, Reinhard A1 - Seckler, Robert T1 - Formation of fibrous aggregates from a non-native intermediate : the isolated P22 tailspike -helix domain Y1 - 1999 ER - TY - JOUR A1 - Baxa, Ulrich A1 - Steinbacher, Stefan A1 - Weintraub, Andrej A1 - Huber, Robert A1 - Seckler, Robert T1 - Mutations improving the folding of phage P22 tailspike protein affect its receptor binfing activity Y1 - 1999 ER - TY - JOUR A1 - Schuler, Benjamin A1 - Fürst, Frank A1 - Osterroth, Frank A1 - Steinbacher, Stefan A1 - Huber, Robert A1 - Seckler, Robert T1 - Plasticity and steric strain in a parallel beta-helix: Rational mutations in P22 tailspike protein N2 - By means of genetic screens, a great number of mutations that affect the folding and stability of the tailspike protein from Salmonella phage P22 have been identified. Temperature-sensitive folding (tsf) mutations decrease folding yields at high temperature, but hardly affect thermal stability of the native trimeric structure when assembled at low temperature. Global suppressor (su) mutations mitigate this phenotype. Virtually all of these mutations are located in the central domain of tailspike, a large parallel beta-helix. We modified tailspike by rational single amino acid replacements at three sites in order to investigate the influence of mutations of two types: (1) mutations expected to cause a tsf phenotype by increasing the side-chain volume of a core residue, and (2) mutations in a similar structural context as two of the four known su mutations, which have been suggested to stabilize folding intermediates and the native structure by the release of backbone strain, an effect well known for residues that are primarily evolved for function and not for stability or folding of the protein. Analysis of folding yields, refolding kinetics and thermal denaturation kinetics in vitro show that the tsf phenotype can indeed be produced rationally by increasing the volume of side chains in the beta-helix core. The high-resolution crystal structure of mutant T326F proves that structural rearrangements only take place in the remarkably plastic lumen of the beta-helix, leaving the arrangement of the hydrogen-bonded backbone and thus the surface of the protein unaffected. This supports the notion that changes in the stability of an intermediate, in which the beta-helix domain is largely formed, are the essential mechanism by which tsf mutations affect tailspike folding. A rational design of su mutants, on the other hand, appears to be more difficult. The exchange of two residues in the active site expected to lead to a drastic release of steric strain neither enhanced the folding properties nor the stability of tailspike. Apparently, side-chain interactions in these cases overcompensate for backbone strain, illustrating the extreme optimization of the tailspike protein for conformational stability. The result exemplifies the view arising from the statistical analysis of the distribution of backbone dihedral angles in known three-dimensional protein structures that the adoption of straight phi/psi angles other than the most favorable ones is often caused by side-chain interactions. Y1 - 2000 UR - http://www3.interscience.wiley.com/cgi-bin/abstract/71001984/START ER - TY - JOUR A1 - Seckler, Robert T1 - Assembly of multi-subunit structures Y1 - 2000 ER - TY - JOUR A1 - Baxa, Ulrich A1 - Cooper, Alan A1 - Weintraub, N. A1 - Pfeil, Wolfgang A1 - Seckler, Robert T1 - Enthalpic barriers to the hydrophobic binding of oligosaccharides to phage P22 tailspike protein Y1 - 2001 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 - 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 - 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 - Kaufmann, B. A1 - Baxa, Ulrich A1 - Chipman, P. R. A1 - Rossmann, M. G. A1 - Modrow, Susanne A1 - Seckler, Robert T1 - Parvovirus B19 does not bind to membrane-associated globoside in vitro N2 - The glycosphingolipid globoside (globotetraosylceramide, Gb4Cer) has been proposed to be the cellular receptor of human parvovirus B19. Quantitative measurements of the binding of parvovirus B19 to Gb4Cer were performed to explore the molecular basis of the virus tropism. Solid-phase assays with fluorescence-labeled liposomes or (125)iodine-labeled empty capsids were used to characterize the specificity of binding. In addition, surface plasmon resonance on lipid layers, as well as isothermal titration microcalorimetry, was utilized for real-time analysis of the virus-receptor interaction. These studies did not confirm binding of Gb4Cer to recombinant B19 VP2 capsids, suggesting that Gb4Cer does not function on its own as the cellular receptor of human parvovirus B19, but might be involved in a more complex recognition event. The biochemical results were further confirmed by cryo-electron microscopy image reconstructions at 10 A resolution, in which the structures of empty capsids were compared with empty capsids incubated with Gb4Cer. (C) 2004 Elsevier Inc. All rights reserved Y1 - 2005 SN - 0042-6822 ER -