Table 2.

Characterization of rGFP3 circular permutants

Construct	Sequence Order	Fluorescent (+/−)	% Solubility
GFP-OPT	1-2-3-A-4-5-6-L-7-8-9-10-11	+	80
rGFP3	8-9-1-2-3-A-4-5-6-L-7-10-11	+	80
CP 9–8	9-1-2-3-A-4-5-6-L-7-10-11-8	+	75
CP 1–9	1-2-3-A-4-5-6-L-7-10-11-8-9	+	67
CP 2–1	2-3-A-4-5-6-L-7-10-11-8-9-1	+	20
CP 3–2	3-A-4-5-6-L-7-10-11-8-9-1-2	+	0
CP A-3	A-4-5-6-L-7-10-11-8-9-1-2-3	+	67
CP 4-A	4-5-6-L-7-10-11-8-9-1-2-3-A	+	50
CP 5–4	5-6-L-7-10-11-8-9-1-2-3-A-4	−	0
CP 6–5	6-L-7-10-11-8-9-1-2-3-A-4-5	+	67
CP 6-L	L-7-10-11-8-9-1-2-3-A-4-5-6	+	100
CP L-7	7-10-11-8-9-1-2-3-A-4-5-6-L	+	40
CP 11–10	11-8-9-1-2-3-A-4-5-6-L-7-10	+	20
CP 10–7	10-11-8-9-1-2-3-A-4-5-6-L-7	−	80

Summary of circular permutant expression data based on fluorescence data of purified constructs and visual inspection of SDS-PAGE of lysates (soluble) and pellets (insoluble) following French press (% soluble is defined by relative amount soluble to insoluble). In two cases, CP3-2 and CP5-4, the insoluble pellet protein was denatured and then allowed to refold on Ni-agarose column media (4 hrs at room temperature while his-tag immobilized on the column) before reassessing fluorescence (+/−). Sequence order refers to the secondary structure naming, based on the sequence of GFP, where numbers indicate β-strands going from N-to-C terminus, A is the α-helix, and L is the long unstructured loop connecting strands 6 and 7.