Winter, D. L. | bioRxiv Science

bioRxiv2023

Tuning the stator subunit of the flagellar motor with coiled-coil engineering.

Many bacteria swim driven by an extracellular filament rotated by the bacterial flagellar motor. This motor is powered by the stator complex, MotA5MotB2, a heterodimeric complex which forms an ion channel which couples energy from the ion motive force to torque generation. Recent structural work revealed that stator complex consists of a ring of five MotA subunits which rotate around a central dimer of MotB subunits. Transmembrane (TM) domains TM3 and TM4 from MotA combine with the single TM domain from MotB to form two separate ion channels within this complex. Much is known about the ion binding site and ion specificity; however, to date, no modelling has been undertaken to explore the MotB-MotB dimer stability and the role of MotB conformational dynamics during rotation. Here, we modelled the central MotB dimer using coiled-coil engineering and modelling principles and calculated free energies to identify stable states in the operating cycle of the stator. We found 3 stable coiled-coil states with dimer interface angles of 28{degrees}, 56{degrees} and 64{degrees}. We tested the effect of strategic mutagenesis on the comparative energy of the states and correlated motility with a specific hierarchy of stability between the three states. In general, our results indicate agreement with existing models describing a 36{degrees} rotation step of the MotA pentameric ring during the power stroke and provide an energetic basis for the coordinated rotation of the central MotB dimer based on coiled-coil modelling.

microbiology↗

bioRxiv2022

Single-step purification of functionalized protein nanostructures using multimodal chromatography

Protein nanostructures produced through the self-assembly of individual subunits are attractive scaffolds to attach and position functional molecules for applications in biomaterials, metabolic engineering, tissue engineering, and a plethora of nanomaterials. However, the assembly of multicomponent protein nanomaterials is generally a laborious process that requires each protein component to be separately expressed and purified prior to assembly. Moreover, excess components not incorporated into the final assembly must be removed from the solution and thereby necessitate additional processing steps. Here, we developed an efficient approach to purify functionalized protein filament assemblies directly from bacterial lysates in a single step through a type of multimodal chromatography that combines size-exclusion, hydrophilic interaction, and ion exchange to separate recombinant protein assemblies from excess free subunits and bacterial proteins. In this approach, the ultrastable filamentous protein gamma-prefoldin was employed as a material scaffold that can be functionalized with a variety of protein domains through SpyTag/SpyCatcher conjugation chemistry. The purification of recombinant gamma-prefoldin filaments from bacterial lysates using multimodal chromatography was optimized across a wide range of salt concentrations and pH. Subsequently, functionalized protein assemblies were purified from bacterial lysates using multimodal chromatography in a single step and shown to befree of unincorporated subunits. The assembly and purification of protein nanostructures with varying amounts of functionalization was confirmed using polyacrylamide gel electrophoresis, Forster resonance energy transfer, and transmission electron microscopy. We envision that the use of multimodal chromatography will increase the throughput of protein nanostructure prototyping as well as enable the upscaling of the bioproduction of protein nanodevices.

bioengineering↗