Biology subjects

Lamb, D. C.

Publications and source records attributed to Lamb, D. C..

3 recordsLinked to original sources

bioRxiv2018

Dynamic interactions of type I cohesin modules fine-tune the structure of the cellulosome of Clostridium thermocellum

Efficient degradation of plant cell walls by selected anaerobic bacteria is performed by large extracellular multienzyme complexes termed cellulosomes. The spatial arrangement within the cellulosome is organized by a protein called scaffoldin, which recruits the cellulolytic subunits through interactions between cohesin modules on the scaffoldin and dockerin modules on the enzymes. Although many structural studies of the individual components of cellulosomal scaffoldins have been performed, the role of interactions between individual cohesin modules and the flexible linker regions between them are still not entirely understood. Here, we report single-molecule measurements using Forster resonance energy transfer to study the conformational dynamics of a bimodular tandem cohesin segment of the scaffoldin protein CipA of Clostridium thermocellum. Our data reveal the existence of compacted structures in solution that persist on the timescale of milliseconds. The compacted conformation is found to be in dynamic equilibrium with an extended state that shows distance fluctuations on the microsecond timescale. Shortening of the inter-cohesin linker does not significantly alter the structural dynamics. Upon addition of dockerin-containing enzymes, an extension of the flexible state is observed but the cohesin-cohesin interactions persist. This suggests that the dockerin-binding interfaces are not involved in cohesin-cohesin interactions. The formation of cohesin-cohesin interactions is also observed in all-atom molecular dynamics simulations of the system. From the simulations, we identify possible inter-cohesin binding modes, none of which show obstruction of the cohesin-dockerin binding interfaces. Our results go beyond the view of scaffoldin as \"beads on a string\". We propose that both the flexibility and cohesin-cohesin interactions are important factors for the precise spatial arrangement of the enzymatic subunits in the cellulosome that leads to the high catalytic synergy in these assemblies. Hence, the flexibility of the linker region and cohesin-cohesin interactions should be considered when designing cellulosomes for industrial applications.

biophysics↗

bioRxiv2018

Stability of local secondary structure determines selectivity of viral RNA chaperones

To maintain genome integrity, segmented double-stranded RNA viruses of the Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA-RNA interactions between single-stranded RNA segment precursors. These are mediated by virus-encoded non-structural proteins with RNA chaperone-like activities, such as rotavirus NSP2 and avian reovirus {sigma}NS. Here, we compared the abilities of NSP2 and {sigma}NS to mediate sequence-specific interactions between rotavirus genomic segment precursors. Despite their similar activities, NSP2 successfully promotes inter-segment association, while {sigma}NS fails to do so. To understand the mechanisms underlying such selectivity in promoting inter-molecular duplex formation, we compared RNA-binding and helix-unwinding activities of both proteins. We demonstrate that octameric NSP2 binds structured RNAs with high affinity, resulting in efficient intramolecular RNA helix disruption. Hexameric {sigma}NS oligomerises into an octamer that binds two RNAs, yet it exhibits only limited RNA-unwinding activity compared to NSP2. Thus, the formation of intersegment RNA-RNA interactions is governed by both helix-unwinding capacity of the chaperones and stability of RNA structure. We propose that this protein-mediated RNA selection mechanism may underpin the high fidelity assembly of multi-segmented RNA genomes in Reoviridae.

biophysics↗

bioRxiv2018

Dimer arrangement and monomer flattening determine actin filament formation

Actin filament dynamics underlie key cellular processes, such as cell motility. Although actin filament elongation has been extensively studied under the past decades, the mechanism of filament nucleation remains unclear. Here, we immobilized gelsolin, a pointed-end nucleator, at the bottom of zero-mode waveguides to directly monitor the early steps of filament assembly. Our data revealed extensive dynamics and that only one, of two populations, elongates. Annalysis of the kinetics revealed a more stable trimer but a less stable tetramer in the elongating population compared to the non-elongating one. Furthermore, blocking flattening, the conformational change associated with filament formation, prevented the formation of both types of assemblies. Thus, flattening and the initial monomer arrangement determine gelsolin-mediated filament initiation.

biophysics↗