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. 2021 Jan 22;10:e63356. doi: 10.7554/eLife.63356

Figure 4. FP oligomerization interfaces can open up to enable exchange monomer.

Figure 4.

(A) SEC analysis of FPhtΔ3 in 20 mM HEPES, 150 mM NaCl, pH 7.5 (black), or 100 mM glycine pH 2.3 (red), demonstrating that the two chains of FPhtΔ3 dissociate at low pH (top). Fractions from the SEC experiments were analyzed by SDS–PAGE and silver staining showing that at neutral pH the two fragments of FPhtΔ3 co-elute (bottom, left), whereas at low pH, FPtHis (25 kDa) elute first and FPhΔ3 (18 kDa) elute last (bottom, right). (B) Overview of FP constructs used in (C) and (D). (C) A silver-stained SDS–PAGE gel following the exchange of chains between FPc and FPhtΔ3. The exchange was monitored by the appearance of the FPh chain (25 kDa) from FPc and the disappearance of the FPhΔ3 chain, after pull-down on Ni-NTA beads using the His-tag on the C-terminus of FPtHis. (D) A silver-stained gel from SDS–PAGE demonstrating the exchange of FP chains between FP2 and FPhtΔ3. The exchange was monitored by the appearance of the full-length FP (50 kDa) after pull-down on Ni-NTA beads using the C-terminal His-tag of FPtHis from FPhtΔ3.