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Growing recognition of the importance of long-distance dispersal (LDD) of plant seeds for various ecological and evolutionary processes has led to an upsurge of research into the mechanisms underlying LDD. We summarize these findings by formulating six generalizations stating that LDD is generally more common in open terrestrial landscapes, and is typically driven by large and migratory animals, extreme meteorological phenomena, ocean currents and human transportation, each transporting a variety of seed morphologies. LDD is often associated with unusual behavior of the standard vector inferred from plant dispersal morphology, or mediated by nonstandard vectors. To advance our understanding of LDD, we advocate a vector-based research approach that identifies the significant LDD vectors and quantifies how environmental conditions modify their actions.
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.
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.
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.
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.
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.
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.