In vivo solubility assay. Insoluble (I) and soluble (S) fractions for each left out segment, labeled along the top. Triplicate experiments were done.

(a) TOPS13 secondary structure element diagram for GFP. Triangles are beta strands; up or down indicates strand direction. Gray fill shading reflects solubility in single expression experiment when the secondary structure element is left out. Green border shading reflects RF in dual expression experiments. (b) A working hypothesis to explain variable solubility of LOO constructs. U is the unfolded state. F is folded. Arrow thickness represents the expected decreasing rate of aggregation (down) and increasing peptide (s) binding affinity (up) as folding proceeds, based on the dynamic competition model for folding/aggregation13 (c) Sequence of circularized GFP, with left out segments in yellow highlighting, numbered. For each construct, the N-terminal tag sequence MTHHHHHHSSG replaces the yellow segment, and the C-terminus is the last position before the left out segment.

FPLC results using three purified LOO constructs. The LOO11m peak with highest retention time is monomeric. Aggregates in this image refers to soluble, native aggregates. Soluble LOO-GFPs exist in a variety of oligomeric states, all having some fluorescence. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]