Insights into SusCD-mediated glycan import by a prominent gut symbiont

In Bacteroidetes, one of the dominant phyla of the mammalian gut, active uptake of large nutrients across the outer membrane is mediated by SusCD protein complexes via a “pedal bin” transport mechanism. However, many features of SusCD function in glycan uptake remain unclear, including ligand binding, the role of the SusD lid and the size limit for substrate transport. Here we characterise the β2,6 fructo-oligosaccharide (FOS) importing SusCD from Bacteroides thetaiotaomicron (Bt1762-Bt1763) to shed light on SusCD function. Co-crystal structures reveal residues involved in glycan recognition and suggest that the large binding cavity can accommodate several substrate molecules, each up to ~2.5 kDa in size, a finding supported by native mass spectrometry and isothermal titration calorimetry. Mutational studies in vivo provide functional insights into the key structural features of the SusCD apparatus and cryo-EM of the intact dimeric SusCD complex reveals several distinct states of the transporter, directly visualising the dynamics of the pedal bin transport mechanism

Location Health Department Paediatric Unit, Colombo South Teaching Hospital.

Aim of study To study the epidemiological pattern and the effects of envenomation by the hump nosed viper () in children. All previous publications on this subject are based on the adult experience

Single-molecule analysis of protein-free U2-U6 snRNAs

Spliceosomes catalyze the maturation of precursor mRNAs in organisms ranging from yeast to humans. Their catalytic core comprises three small nuclear RNAs (U2, U5 and U6) involved in substrate positioning and catalysis. It has been postulated, but never shown experimentally, that the U2–U6 complex adopts at least two conformations that reflect different activation states. We have used single-molecule fluorescence to probe the structural dynamics of a protein-free RNA complex modeling U2–U6 from yeast and mutants of highly conserved regions of U2–U6. Our data show the presence of at least three distinct conformations in equilibrium. The minimal folding pathway consists of a two-step process with an obligatory intermediate. The first step is strongly magnesium dependent, and we provide evidence suggesting that the second step corresponds to the formation of the genetically conserved helix IB. Site-specific mutations in the highly conserved AGC triad and the U80 base in U6 suggest that the observed conformational dynamics correlate with residues that have an important role in splicing

Cryo-electron Microscopy Structures of Expanded Poliovirus with VHHs Sample the Conformational Repertoire of the Expanded State

By using cryo-electron microscopy, expanded 80S-like poliovirus virions (poliovirions) were visualized in complexes with four 80S-specific camelid VHHs (Nanobodies). In all four complexes, the VHHs bind to a site on the top surface of the capsid protein VP3, which is hidden in the native virus. Interestingly, although the four VHHs bind to the same site, the structures of the expanded virus differ in detail in each complex, suggesting that each of the Nanobodies has sampled a range of low-energy structures available to the expanded virion. By stabilizing unique structures of expanded virions, VHH binding permitted a more detailed view of the virus structure than was previously possible, leading to a better understanding of the expansion process that is a critical step in infection. It is now clear which polypeptide chains become disordered and which become rearranged. The higher resolution of these structures also revealed well-ordered conformations for the EF loop of VP2, the GH loop of VP3, and the N-terminal extensions of VP1 and VP2, which, in retrospect, were present in lower-resolution structures but not recognized. These structural observations help to explain preexisting mutational data and provide insights into several other stages of the poliovirus life cycle, including the mechanism of receptor-triggered virus expansion. IMPORTANCE When poliovirus infects a cell, it undergoes a change in its structure in order to pass RNA through its protein coat, but this altered state is short-lived and thus poorly understood. The structures of poliovirus bound to single-domain antibodies presented here capture the altered virus in what appear to be intermediate states. A careful analysis of these structures lets us better understand the molecular mechanism of infection and how these changes in the virus lead to productive-infection events

Single molecule FRET characterization of lage ribozyme folding

A procedure to investigate the folding of group II intron by single molecule Fluorescence Resonance Energy Transfer (smFRET) using total internal reflection fluorescence microscopy (TIRFM) is described in this chapter. Using our previous studies on the folding and dynamics of a large ribozyme in the presence of metal ions (i.e., Mg2+ and Ca2+) and/or the DEAD-box protein Mss116 as an example, we here describe step-by-step procedures to perform experiments. smFRET allows the investigation of individual molecules, thus, providing kinetic and mechanistic information hidden in ensemble averaged experiments