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. 2016 May 20;5:e15312. doi: 10.7554/eLife.15312

Figure 2. dGBP1 destabilizing mutations can be transferred to Nbs derived from different species to create antigen-dependent stability.

(A) Protein alignment of Nbs against GFP (GBP1), HIV-1 CA (αCA) and E.coli DHFR (αDHFR). Amino acid positions numbered according to the ImMunoGeneTics information system (IMGT). FR, framework; CDR, complementarity determining region. Purple- and green-highlighted residues indicate dGBP1 mutation position. Green-highlighted residues (3maj) are most destabilizing when mutated. The underlined serine was a cysteine in the original GBP1. (B–E). Transfer of dGBP1 mutations to other Nbs. Destabilized, but not unmodified αCA and αDHFR showed antigen-dependent fluorescence (B,D) as well as protein level (C,E). DsRed (red) indicates transfected cells in (B,D) and is only shown when TagBFP (gray) results are negative. Scale bar, 50 μm. (F) A dNb generated by mutation transfer was degraded by the UPS. αCA-dNb6mut-TagBFP showed an increase in protein level when transfected 293T cells were treated with MG132 for 6 hr. Results representative of 3 independent experiments. (G) Heat map showing median TagBFP fluorescence intensity of Nb-TagBFP fusions. All Nbs shown recognize epitopes of intracellular origin. Fluorescent readings were normalized to that of unmodified Nb (no antigen) condition, which was set to 100. n = 3 biological replicates pooled from 3 independent experiments per Nb. Each biological replicate result is shown as a horizontal bar in the heat map. Bar graphs indicate median and maximum-to-minimum range. Additional data related to mutation transfer of destabilizing mutations are shown in Figure 2—figure supplements 1–3. Source data for % Nb-TagBFP fluorescence values are shown in Figure 2—source data 1.

DOI: http://dx.doi.org/10.7554/eLife.15312.006

Figure 2—source data 1. Source data for fluorescence quantifications of Nb-TagBFP tests.
This excel file contains numerical values for the % Nb-TagBFP fluorescence parameter shown as a colored heat map in Figure 2G.
DOI: http://dx.doi.org/10.7554/eLife.15312.007
elife-15312-fig2-data1.xlsx (38.5KB, xlsx)
DOI: 10.7554/eLife.15312.007

Figure 2.

Figure 2—figure supplement 1. Mapping of mutations necessary for dGBP1 destabilization.

Figure 2—figure supplement 1.

(A) Representative images showing expression of GBP1 variants tagged with mCherry in 293T cells. Images taken 17 hr post-transfection. Scale bar, 50 μm. (B) Semi-quantitative summary of mCherry fluorescence intensity as well as cellular solubility phenotype. Sol, soluble. Agg, aggregate. Plot is mean ± standard deviation. Asterisk indicates mutations that showed clear increase in fluorescence compared to dGBP1-mCherry. n = 4–5 per condition. Consistent results were obtained in at least 3 biological replicates (transfected wells) in at least 3 independent experiments.
DOI: http://dx.doi.org/10.7554/eLife.15312.008
Figure 2—figure supplement 2. Mapping mutations sufficient for dGBP1 destabilization.

Figure 2—figure supplement 2.

(A) Representative images showing expression of GBP1 variants tagged with mCherry in 293T cells. Images taken 17 hr post-transfection. Scale bar, 50 μm. (B) Semi-quantitative summary of mCherry fluorescence intensity as well as cellular solubility phenotype. Sol, soluble. Agg, aggregate. Plot is mean ± standard deviation. Asterisk indicates clearly destabilized mutations compared to GBP1-mCherry. n = 6 per condition except GBP1 control (n = 3). Consistent results were obtained in at least 3 biological replicates (transfected wells) in at least 3 independent experiments.
DOI: http://dx.doi.org/10.7554/eLife.15312.009
Figure 2—figure supplement 3. Generation of dNbs by mutation transfer.

Figure 2—figure supplement 3.

(A) Conservation of dGBP1 mutations across 76 Nbs derived from Camelus dromedarius, Llama glama and Vicugna pacos. (B) Mapping of Nb destabilizing positions in relation to binding interfaces across Nb-Antigen complexes. (C) dGBP1-TagBFP expression does not have a clear effect on YFP protein level. Transfected 293T cells were harvested 1 day post-transfection. Whole cell lysates were blotted for anti-YFP as well as anti-βgal. Densitometry measurements of western blot bands were plotted as the relative density of anti-TagBFP bands compared to that of anti-βgal bands in the same lane. a.u. is arbitrary unit. n = 3. Plot shows median and range. Results are representative of at least 3 independent experiments.
DOI: http://dx.doi.org/10.7554/eLife.15312.010