Influence of Electrostatics on Small Molecule Flux through a Protein Nanoreactor

Abstract

Nature uses protein compartmentalization to great effect for control over enzymatic pathways, and the strategy has great promise for synthetic biology. In particular, encapsulation in nanometer-sized containers to create nanoreactors has the potential to elicit interesting, unexplored effects resulting from deviations from well-understood bulk processes. Self-assembled protein shells for encapsulation are especially desirable for their uniform structures and ease of perturbation through genetic mutation. Here, we use the MS2 capsid, a well-defined porous 27 nm protein shell, as an enzymatic nanoreactor to explore pore-structure effects on substrate and product flux during the catalyzed reaction. Our results suggest that the shell can influence the enzymatic reaction based on charge repulsion between small molecules and point mutations around the pore structure. These findings also lend support to the hypothesis that protein compartments modulate the transport of small molecules and thus influence metabolic reactions and catalysis in vitro.

A systems-level model reveals that 1,2-Propanediol utilization microcompartments enhance pathway flux through intermediate sequestration

“…Similarly, if the permeability of the MCP to propionaldehyde is sufficiently low, the permeability to 1,2-PD can be increased to saturate PduP/Q. This putative trapping mechanism is in congruence with the in vitro observation that small molecule efflux from a protein nanoreactor can be affected by the chemical character of the reactor pores [35]. Studies of substrate channeling in enzyme scaffolds also emphasize the importance of creating a high substrate concentration in the vicinity of downstream enzymes [28, 36].…”

“…The permeability we predict is much lower than would be estimated based on free diffusion through an area equivalent to the cumulative area of the pores in the MCP shell (on the order of 1 − 10 cm/s). The phenomenon of surprisingly low apparent permeability of a protein shell has been predicted previously [35], and is likely rooted in the fact that the Debye length, governing the distance at which electronic interactions occur between the amino acid residues lining the pore and the client small molecules, extends across the entire pore area. Excluded-volume models of apparent diffusivity in the pore, therefore, do not apply.…”

pH determines the energetic efficiency of the cyanobacterial CO 2 concentrating mechanism

“…However, recent structures of carboxysome shell proteins offer a potential mechanism for differential permeability of CO 2 and HCO 3 − : The pores of shell proteins typically carry positive charge, which might increase the rate of HCO 3 − transit relative to CO 2 (12,49). Indeed, recent experimental evidence suggests that protein compartments can be selectively permeable (50,51). Intuitively, it seems that a very high HCO 3 − permeability and a very low CO 2 permeability would be best for the efficient operation of the CCM.…”

Engineering of Metabolic Pathways Using Synthetic Enzyme Complexes

“…Tagging the enzymes with SpyCatcher and bacteriophage MS2 capsid protein with SpyTag resulted in assembly of particles in E. coli that increased indigo production by 60% compared to controls (Giessen and Silver, 2016). Pores of BMCs and capsids can be engineered to control substrate uptake specificity (Glasgow et al, 2015). Isolated capsids showed that the enzymes were markedly more stable in vitro because of the covalent linkages.…”