Artificial Forisomes Are Ideal Models of Forisome Assembly and Activity That Allow the Development of Technical Devices

Abstract

Forisomes are protein polymers found in leguminous plants that have the remarkable ability to undergo reversible "muscle-like" contractions in the presence of divalent cations and in extreme pH environments. To gain insight into the molecular basis of forisome structure and assembly, we used confocal laser scanning microscopy to monitor the assembly of fluorescence-labeled artificial forisomes in real time, revealing two distinct assembly processes involving either fiber elongation or fiber alignment. We also used scanning and transmission electron microscopy and X-ray diffraction to investigate the ultrastructure of forisomes, finding that individual fibers are arranged into compact fibril bundles that disentangle with minimal residual order in the presence of calcium ions. To demonstrate the potential applications of artificial forisomes, we created hybrid protein bodies from forisome subunits fused to the B-domain of staphylococcal protein A. This allowed the functionalization of the artificial forisomes with antibodies that were then used to target forisomes to specific regions on a substrate, providing a straightforward approach to develop forisome-based technical devices with precise configurations. The functional contractile properties of forisomes are also better preserved when they are immobilized via affinity reagents rather than by direct contact to the substrate. Artificial forisomes produced in plants and yeast therefore provide an ideal model for the investigation of forisome structure and assembly and for the design and testing of tailored artificial forisomes for technical applications.

Forizymes – functionalised artificial forisomes as a platform for the production and immobilisation of single enzymes and multi-enzyme complexes

“…MtSEO-F1 and MtSEO-F4 subunits can assemble into artificial forisomes in S. cerevisiae and can be functionalised by the translational fusion of small tags. This offers promising applications for the development of technical devices 20 24 27 . In order to functionalise artificial forisomes with larger enzyme tags, we first identified the most appropriate combination of MtSEO-F1 and MtSEO-F4 subunits as well as the best translational fusion site.…”

“…Translational fusions can influence protein folding, and functional differences between N-terminal and C-terminal fusions of the same protein components have been reported previously 29 . This suggests that C-terminal translational fusions inhibit interactions between or the assembly of MtSEO-F proteins and thus the overall forisome structure 27 . Because the N-terminal fusion strategy was generally more successful, all the C-terminal fusion constructs were excluded from further analysis.…”

“…The function of MtSEO-F3 remains unclear 26 . Artificial forisomes produced by expressing MtSEO-F1 or MtSEO-F4 in the yeast Saccharomyces cerevisiae also self-assemble into micro-scale forisome structures even when small additional tags are translationally fused to the forisome subunits 24 27 . However, the potential to display more complex tags such as enzymes on artificial forisomes has not been investigated.…”

Calcium powered phloem protein ofSEOgene family “Forisome” functions in wound sealing and act as biomimetic smart materials

“…Electropotential waves are being studied with calcium influx, because there is a strong link between calcium signaling and physiological processes. 50,51 In general 2 forms of EPWs have been known i.e., action potential and variation potential. EPWs comprise of action potential, which travels along phloem and variation potential travels from xylem toward phloem.…”

Similar Intracellular Location and Stimulus Reactivity, but Differential Mobility of Tailless (Vicia faba) and Tailed Forisomes (Phaseolus vulgaris) in Intact Sieve Tubes

“…Ca 2+ induced a 3 to 6-fold volume increase within 10 to 15 seconds [ 14 , 16 ] ( S4 Fig ). Isolation of forisomes further enabled in vitro testing of their biotechnological potential for microfluidics [ 16 ] and other applications [ 17 ]. In this way, the occlusion ability of dispersed forisomes has been demonstrated using silicon-based tubular systems with sieve-element diameters [ 16 ].…”