Probing the Internal pH and Permeability of a Carboxysome Shell

Abstract

The carboxysome is a protein-based nanoscale organelle in cyanobacteria and many proteobacteria, which encapsulates the key CO 2 -fixing enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase (CA) within a polyhedral protein shell. The intrinsic self-assembly and architectural features of carboxysomes and the semipermeability of the protein shell provide the foundation for the accumulation of CO 2 within carboxysomes and enhanced carboxylation. Here, we develop an approach to determine the interior pH conditions and inorganic carbon accumulation within an α-carboxysome shell derived from a chemoautotrophic proteobacterium Halothiobacillus neapolitanus and evaluate the shell permeability. By incorporating a pH reporter, pHluorin2, within empty α-carboxysome shells produced in Escherichia coli, we probe the interior pH of the protein shells with and without CA. Our in vivo and in vitro results demonstrate a lower interior pH of α-carboxysome shells than the cytoplasmic pH and buffer pH, as well as the modulation of the interior pH in response to changes in external environments, indicating the shell permeability to bicarbonate ions and protons. We further determine the saturated HCO 3 − concentration of 15 mM within α-carboxysome shells and show the CA-mediated increase in the interior CO 2 level. Uncovering the interior physiochemical microenvironment of carboxysomes is crucial for understanding the mechanisms underlying carboxysomal shell permeability and enhancement of Rubisco carboxylation within carboxysomes. Such fundamental knowledge may inform reprogramming carboxysomes to improve metabolism and recruit foreign enzymes for enhanced catalytical performance.

Synthetic engineering of a new biocatalyst encapsulating [NiFe]-hydrogenases for enhanced hydrogen production

“…1C ), confirming the EP-mediated encapsulation of HyaAB into the shells. Along with previous evidence revealing that CsoS2 C-terminus could drive the encapsulation of [FeFe]-hydrogenases with cofactors and fluorescence proteins, 19,29 we provide an efficient encapsulation approach using the CsoS2 C-terminus as an EP to encase foreign cargos within the α-carboxysome shells in synthetic biology.…”

“…1A) resulting in the formation of empty a-carboxysome shells. 19,29 Expression of the hyaAB-EP plasmid was induced by adding IPTG for 4 hours before arabinoseinduced shell expression, to ensure heterologous production of functional [NiFe]-hydrogenases prior to shell encapsulation (Fig. 1B).…”

“…In our previous studies, we demonstrated that the C-terminus of CsoS2 could serve as an encapsulation peptide (EP) to bind with shell proteins and efficiently recruit non-native cargos, for example fluorescent proteins, algal [FeFe]-hydrogenases, and ferredoxin-NADP + reductase, into the a-carboxysome shell; without the fusion with the CsoS2 C-terminus, foreign cargo proteins could not be incorporated within the shells. 19,29 The hyaAB-EP vector was then transformed into the E. coli BL21(DE3) cells comprising an a-carboxysome shell-expressing vector, which can express a-carboxysome shell proteins (CsoS4A, CsoS4B, CsoS1A, CsoS1B, CsoS1C, CsoS1D, Fig. 1A) resulting in the formation of empty a-carboxysome shells.…”

“…[12][13][14][15][16][17][18] Carboxysomes encapsulate the key CO 2 -fixing enzyme, ribulose-1,5bisphosphate carboxylase oxygenase (Rubisco), using a polyhedral protein shell; unlike virus capsids that do not possess permeability, the carboxysome shell is selectively permeable to HCO 3 À and protons while diminishing O 2 entry and CO 2 leakage. [19][20][21][22][23] This elaborate bacterial organelle provides elevated levels of CO 2 around Rubisco to enhance carbon fixation on the global scale and represents an attractive engineering objective in synthetic biology. [24][25][26][27][28] Recent studies have shown that direct encapsulation of O 2 -sensitive [FeFe]-hydrogenases into the cavity of a synthetic a-carboxysome shell resulted in improved hydrogen production and O 2 tolerance of the hybrid biocatalyst, taking advantage of the unique a-carboxysome shell permeability and confinement of cargo enzymes.…”

Electrostatic Theory of the Acidity of the Solution in the Lumina of Viruses and Virus-Like Particles

“…According to the theory, the dimensionless Donnan potential ϕ in the capsid lumen, defined as the Donnan potential energy scaled to the thermal energy, obeys the simple relation , ϕ = nobreak0em0.25em⁢ sinh − 1 nobreak0em.25em⁡ γ where the quantity γ ≡ Δρ/2ρ s is defined in terms of the difference Δρ = ρ + i – ρ – i of the number densities of positive and negative immobile charges ρ ± i , averaged over the volume of the lumen, and 2ρ s the overall density of mobile ionic species in the bulk solution (tacitly presumed to be monovalent). This quantity is dominated by the concentrations of added salt and buffer of the assembly mixture, and explains why in practice the Donnan potential and also the pH differential across the protein shell is virtually independent of the acidity of the solution, , unless some form of charge regulation takes place involving weakly ionic moieties on the cargo or the coat proteins themselves. , …”