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 (c is-cavity). Here we report the maximum size of the non-native protein to st ay and fold in the cis-cavity, Using total soluble proteins of Escherichia coli in denatured state as binding substrates and protease resistance as th e measure of polypeptide held in the cis-cavity, it was estimated that the cis-cavity can accommodate up to similar to 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 (GF P) and its blue fluorescent variant were fused tandem. This fusion protein was captured in the cis-cavity, and folding occurred there. Fluorescence re sonance energy transfer proved that both GFP and blue fluorescent protein m oieties of the same fused protein were able to fold into native structures in the cis-cavity, Consistently, simulated packing of crystal structures sh ows that two native GFPs just fit in the cis-cavity, A fusion protein of th ree GFPs (82 kDa) was also attempted, but, as expected, it was not captured in the cis-cavity.