Co-translational folding properties of the beta-sheet protein green fluorescent protein (GFP)

Protein sequences have evolved to fold quickly and efficiently to their native, active conformation in the complex crowded environment of the cell. A role for co-translational folding in efficient protein folding has been proposed. Yet, the mechanisms by which co-translational folding might affect protein folding efficiency are not completely understood, primarily because only a small number of co-translational folding studies have been performed. The current study describes co-translational folding of green fluorescent protein (GFP), a Ìøå¢-sheet protein with complex native topology. The amino acid residues that make contacts in native Ìøå¢-sheet proteins are often far apart in the primary sequence. Formation of native-like contacts for N-terminal portions of such polypeptides will be delayed during vectorial synthesis on the ribosome, because the C-terminal contact residues have not yet been synthesized, or are sterically inaccessible in the ribosome exit tunnel. The degree of co-translational folding of these N-terminal regions, and the overall ability of the nascent chain to evade erroneous conformation after its complete synthesis, are still unknown. Studies with ribosome-bound GFP showed that GFP can fold completely to its native structure on the ribosomal surface. However, complete folding is only possible if the entire GFP sequence is exposed outside of the ribosome exit tunnel. Importantly, GFP nascent chains that have their C-terminus buried in the ribosome exit tunnel remain folding competent and fold to a native conformation with high efficiency after release from the ribosome; this efficiency is significantly higher than the folding efficiency of chemically denatured GFP.

Time resolved anisotropy measurements revealed low conformational flexibility for the GFP nascent chains with C-terminus in the ribosome exit tunnel. Similar low conformational flexibility was determined and for completely folded ribosome-bound GFP chains, but not for truncated ribosome-bound GFP chains. In addition, a protease resistance assay revealed the presence of a stable GFP nascent chain fragment with a size close to the size of full length GFP.

These results suggest that co-translational folding promotes the acquisition of on pathway, native-like conformations of the growing GFP nascent chains, resulting in high folding efficiency for an otherwise highly aggregation-prone Ì_å_-sheet protein.