Spectroscopic Insights into Protein Folding at Birth: FRET Measurements on ApoHmpH

Co-translational protein folding is the physical process by which a polypeptide folds into a characteristic and functional three dimensional structure, upon emergence from the ribosomal exit tunnel. Currently, this process is poorly understood.  Deciphering how proteins fold will ultimately impact our ability to understand and control cellular function. My research goal is to apply Förster Resonance Energy Transfer (FRET) via lifetime measurements to determine the degree of compaction of a ribosome-bound nascent protein. I am exploring whether ApoHmpH, a prokaryotic globin, folds as a ribosome-bound species or after its release from the ribosome. I hypothesize that when only a portion of the nascent protein (i.e. from the N terminus to EF region) emerges out of the ribosomal tunnel, it has an unfolded or partially folded conformation, and little FRET is observed. Once the protein is released from the ribosome, it is expected to acquire its native conformation, bringing the N terminus in proximity to the EF helix, thereby increasing FRET efficiency.

I will employ E.coli cell-free systems to generate equilibrium populations of ribosome stalled ApoHmpH and measure the interaction between the A and EF helices by incorporating a donor fluorophore at the nascent chain N terminus and an acceptor fluorophore at position 67. Tris-MES gel electrophoresis shows co-translational incorporation of the donor and acceptor fluorophores. Preliminary results show that FRET efficiency increases upon nascent protein release from the ribosome. Future studies include investigations on the co-translational folding process and the formation of non-native interactions in the AB helical region.