On the maximum size of proteins to stay and fold in the cavity of GroEL underneath GroES.

GroEL encapsulates non-native protein in a folding cage underneath GroES (cis-cavity). Here we report the maximum size of the non-native protein to stay and fold in the cis-cavity. Using total soluble proteins of Escherichia coli in denatured state as binding substrates and protease resistance as the measure of polypeptide held in the cis-cavity, it was estimated that the cis-cavity can accommodate up to approximately 57-kDa non-native proteins. To know if a protein with nearly the maximum size can complete folding in the cis-cavity, we made a 54-kDa protein in which green fluorescent protein (GFP) and its blue fluorescent variant were fused tandem. This fusion protein was captured in the cis-cavity, and folding occurred there. Fluorescence resonance energy transfer proved that both GFP and blue fluorescent protein moieties of the same fused protein were able to fold into native structures in the cis-cavity. Consistently, simulated packing of crystal structures shows that two native GFPs just fit in the cis-cavity. A fusion protein of three GFPs (82 kDa) was also attempted, but, as expected, it was not captured in the cis-cavity.