Fig. 3.

FRET properties of Alexa Fluor 488 C5-maleimide dye labeled wild type and portal A316C proheads. (A) Typhoon image and Coomassie stained image of dye labeled wild type and A316C proheads, with gp20-portal and gp23* major capsid protein (mcp) positions indicated. (B) Typhoon image of specific tetra-cysteine peptide binding ReAsH dye labeled CT-terminase, without (left lane) and with (right lane) added dye. (C) Nuclease protection assay gel showing packaged DNA in wild type and A316C proheads. (D) FRET-FCS analysis as described in methods of the Alexa 488 maleimide labeled A316C proheads and CT-gp17 labeled with ReAsH dyes revealing a C-terminal terminase to portal clip distance of 7.5 nm. (E) Diffusion coefficient of the A316C proheads (~2 µm²/s) acceptor channel from the correlated emissions. (F) FRET-FCS analysis of the Alexa 488 maleimide labeled Wt proheads and CT-gp17 labeled with ReAsH dyes showing a diffusion coefficient of ~280 µm²/s in the acceptor channel from less correlated emissions; (G) acceptor channel counts for the A316C proheads with CT-ReAsH; (H) acceptor channel counts for the wild type proheads with CT-ReAsH.