TY - JOUR A1 - Barbirz, Stefanie A1 - Müller, Jürgen J. A1 - Uetrecht, Charlotte A1 - Clark, Alvin J. A1 - Heinemann, Udo A1 - Seckler, Robert T1 - Crystal structure of Escherichia coli phage HK620 tailspike : podoviral tailspike endoglycosidase modules are evolutionarily related N2 - Bacteriophage HK620 infects Escherichia coli H and is closely related to Shigella phage Sf6 and Salmonella phage P22. All three Podoviridae recognize and cleave their respective host cell receptor polysaccharide by homotrimeric tailspike proteins. The three proteins exhibit high sequence identity in the 110 residues of their N-terminal particle- binding domains, but no apparent sequence similarity in their major, receptor-binding parts. We have biochemically characterized the receptor-binding part of HK620 tailspike and determined its crystal structure to 1.38 Å resolution. Its major domain is a right-handed parallel ;-helix, as in Sf6 and P22 tailspikes. HK620 tailspike has endo-N- acetylglucosaminidase activity and produces hexasaccharides of an O18A1-type O-antigen. As indicated by the structure of a hexasaccharide complex determined at 1.6 Å resolution, the endoglycosidase-active sites are located intramolecularly, as in P22, and not between subunits, as in Sf6 tailspike. In contrast, the extreme C-terminal domain of HK620 tailspike forms a ;-sandwich, as in Sf6 and unlike P22 tailspike. Despite the different folds, structure-based sequence alignments of the C-termini reveal motifs conserved between the three proteins. We propose that the tailspike genes of P22, Sf6 and HK620 have a common precursor and are not mosaics of unrelated gene fragments. Y1 - 2008 UR - http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2008.06311.x/pdf SN - 0950-382X ER - TY - JOUR A1 - Kane, Avinash S. A1 - Hoffmann, Armin S. A1 - Baumgärtel, Peter A1 - Seckler, Robert A1 - Reichardt, Gerd A1 - Horsley, David A. A1 - Schuler, Benjamin A1 - Bakajin, Olgica T1 - Microfluidic mixers for the investigation of rapid protein folding kinetics using synchrotron radiation circular dichroism spectroscopy N2 - We have developed a microfluidic mixer optimized for rapid measurements of protein folding kinetics using synchrotron radiation circular dichroism (SRCD) spectroscopy. The combination of fabrication in fused silica and synchrotron radiation allows measurements at wavelengths below 220 nm, the typical limit of commercial instrumentation. At these wavelengths, the discrimination between the different types of protein secondary structure increases sharply. The device was optimized for rapid mixing at moderate sample consumption by employing a serpentine channel design, resulting in a dead time of less than 200 ;s. Here, we discuss the design and fabrication of the mixer and quantify the mixing efficiency using wide-field and confocal epi-fluorescence microscopy. We demonstrate the performance of the device in SRCD measurements of the folding kinetics of cytochrome c, a small, fast-folding protein. Our results show that the combination of SRCD with microfluidic mixing opens new possibilities for investigating rapid conformational changes in biological macromolecules that have previously been inaccessible. Y1 - 2008 UR - http://pubs.acs.org/doi/abs/10.1021/ac801764r SN - 0003-2700 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 - TY - JOUR A1 - Landström, Jens A1 - Nordmark, Eva-Lisa A1 - Eklund, Robert A1 - Weintraub, Andrej A1 - Seckler, Robert A1 - Widmalm, Göran T1 - Interaction of a Salmonella enteritidis O-antigen octasaccharide with the phage P22 tailspike protein by NMR spectroscopy and docking studies N2 - The tailspike protein P22 recognizes an octasaccharide derived from the O-antigen polysaccharide of Salmonella enteritidis in a shallow groove and molecular docking successfully identifies this binding region on the protein surface. Analysis by 2D 1H,1H-T-ROESY and transferred NOESY NMR experiments indicate that the bound octasaccharide ligand has a conformation similar to that observed in solution. The results from a saturation transfer difference NMR experiment show that a large number of protons in the octasaccharide are in close contact with the protein as a result of binding. A comparison of the crystal structure of the complex and a molecular dynamics simulation of the octasaccharide with explicit water molecules suggest that only minor conformational changes are needed upon binding to the tailspike protein. Y1 - 2008 UR - http://www.springerlink.com/content/w3146138p25r2456/ U6 - https://doi.org/10.1007/s10719-007-9065-9 SN - 0282-0080 ER - TY - JOUR A1 - Müller, Jürgen J. A1 - Barbirz, Stefanie A1 - Heinle, Karolin A1 - Freiberg, Alexander A1 - Seckler, Robert A1 - Heinemann, Udo T1 - An intersubunit active site between supercoiled parallel beta helices in the trimeric tailspike endorhamnosidase of Shigella flexneri phage Sf6 N2 - Sf6 belongs to the Podoviridae family of temperate bacteriophages that infect gram-negative bacteria by insertion of their double-stranded DNA. They attach to their hosts specifically via their tailspike proteins. The 1.25 Å crystal structure of Shigella phage Sf6 tailspike protein (Sf6 TSP) reveals a conserved architecture with a central, right-handed ; helix. In the trimer of Sf6 TSP, the parallel ; helices form a left-handed, coiled;; coil with a pitch of 340 Å. The C-terminal domain consists of a ; sandwich reminiscent of viral capsid proteins. Further crystallographic and biochemical analyses show a Shigella cell wall O-antigen fragment to bind to an endorhamnosidase active site located between two ;-helix subunits each anchoring one catalytic carboxylate. The functionally and structurally related bacteriophage, P22 TSP, lacks sequence identity with Sf6 TSP and has its active sites on single subunits. Sf6 TSP may serve as an example for the evolution of different host specificities on a similar general architecture. Y1 - 2008 UR - http://www.cell.com/structure/abstract/S0969-2126%2808%2900106-8 U6 - https://doi.org/10.1016/j.str.2008.01.019 ER - TY - JOUR A1 - Walter, Monica A1 - Fiedler, Christian A1 - Grassl, Renate A1 - Biebl, Manfred A1 - Rachel, Reinhard A1 - Hermo-Parrado, Lois X. A1 - Llamas-Saiz, Aantonio L. A1 - Seckler, Robert A1 - Miller, Stefan A1 - Raaij van, Mark J. T1 - Structure of the receptor-binding protein of bacteriophage Det7 : a podoviral tailspike in a myovirus Y1 - 2008 UR - http://jvi.asm.org/cgi/content/abstract/82/5/2265 SN - 0022-538X ER -