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. 2024 Dec 10;13:RP99854. doi: 10.7554/eLife.99854

Figure 2. Expression, purification, and analysis of tetrameric SthKCterm.

Figure 2.

(A) In-gel protein fluorescence showing selective Acd incorporation into SthKCterm in the absence and presence of S361Acd TAG site, Acd aminoacyl tRNA synthetase/tRNA (RS/tRNA), and unnatural amino acid Acd. (B) Structures of cysteine modified by acceptor compounds [Cu(TETAC)]2+ (cyan), [Fe(phenM)3]2+ (magenta), and [Ru(bpy)2phenM]2+ (orange) along with their corresponding Förster curves of FRET efficiency as a function of distance from Acd, and their R0 values specified and marked with dashed lines. (C) SthKCterm cartoon as tetramer and monomer showing WT subunits in gray and cysteine-containing Acd-labeled subunits in tan. (D) SEC traces (absorbance at 280 nm in black and 425 nm fluorescence emission for Acd in blue) of isolated WT-Acd-heterotetrameric protein (closed triangle) vs monomeric WT protein (open triangle). (E) Mass photometry histogram data showing primarily tetramers (*), with single Gaussian fits (blue, 29±7.3 kDa and 104.5±16.8 kDa , means ± SD).

Figure 2—source data 1. Original uncropped protein gel image (Figure 2A).
Figure 2—source data 2. Labeled cropped protein gel image (Figure 2A).
Figure 2—source data 3. Excel data for size exclusion chromatography (Figure 2D) and mass photometry data (Figure 2E).
elife-99854-fig2-data3.xlsx (392.2KB, xlsx)