Genetic Code Expansion in Rhodobacter sphaeroides to Incorporate Non-canonical Amino Acids into Photosynthetic Reaction Centers

Abstract

was evaluated by measuring the absorbance of hemoglobin released from erythrocytes after incubation with 0.01 g/L of nanoparticles at 37 C. Nanoparticle interactions with V exo and V cyto vesicles were drastically different. V exo vesicles showed significant leakage after exposure to hydroxyl-and amine-modified nanoparticles, but were not disrupted by PEGylated nanoparticles. Conversely, none of the particles caused significant leakage in V cyto vesicles. GUV images indicated visual disruption of V exo vesicles by nanoparticles. Interestingly, hemolysis of erythrocytes by nanoparticles was consistent with leakage assays using V exo vesicles. Hemolysis was observed after erythrocyte incubation with hydroxyl-and amine-modified particles, but not after incubation with PEGylated particles. In conclusion, these results suggest that artificial vesicles mimicking the lipid composition of the exofacial leaflet of the cell plasma membrane might be a useful tool in predicting nanoparticle-induced membrane damage in cells.

Photosynthetic Reaction Center Variants Made via Genetic Code Expansion Show Tyr at M210 Tunes the Initial Electron Transfer Mechanism

“…The mass shifts of the M-subunit peak correspond to the respective mass of the incorporated ncAAs (within a mass accuracy of ±1 Da for a 30 kDa subunit) (Fig. S2), while the H-and Lsubunits remained identical to their WT mass as shown in previous work (36). This shows the general adaptability of our previous methods for genetic code expansion in R. sphaeroides.…”

“…4) based on DADS/SADS and an ET competition model proposed previously (19). With our introduction of amber suppression into R. sphaeroides, we hope to site-specifically introduce ncAAs containing nitrile vibrational probes (36,(69)(70)(71) which can act as true ET spectators whose response will not be obfuscated by interchromophoric electronic coupling or electronic absorption shifts from the formation and decay of charged intermediates.…”

“…In previous work, we took an amber suppression system developed for halotyrosine (3-chlorotyrosine, 3-bromotyrosine, 3iodotyrosine) incorporation in E. coli (35) and transferred this system from E. coli to R. sphaeroides gene control, optimizing it for RC expression (36). Here, incorporation of 3-methyltyrosine (37) is also performed using this previously created system (SI Secs.…”

“…S1.1 and S1.3-S1.4). We apply the same strategy used earlier (36) to a recently created amber suppression system (38) and genetically encode 3nitrotyrosine incorporation for the first time in R. sphaeroides (SI Secs. S1.1-S1.4).…”