Figure 4. The folding and aggregation states of full-length NosA, NosA_1-111 and its variants.

(A–D) The aggregation states detected by size-exclusion chromatography assay; In (D), gel filtration protein standard markers were highlighted with arabic numerals 1, 2, 3 and 4, representing thyroglobulin with a molecular weight (MW) of 670KDa, γ-globulin with a MW of 158 KDa, Ovalbumin with a MW of 44 KDa, myoglobin with MW of 17 KDa and vitamin B12 with a MW of 1.35 KDa, respectively. (E) The folding of NosA and its variants detected by circular dichroism (CD) spectroscopies respectively; (F) The folding of NosA K49A variant further confirmed by two-dimensional NMR ¹H-¹⁵N HSQC spectrum of the full-length NosA K49A variant (green), overlapped with that of wild-type NosA (red). In these two NMR experiments, the concentration of the WT NosA protein and its K49A variant is about 0.2 mM in NMR buffer.