Cotranslational folding of a periplasmic protein domain inEscherichia coli

Abstract

In Gram-negative bacteria, periplasmic domains in inner membrane proteins are cotranslationally translocated across the inner membrane through the SecYEG translocon. To what degree such domains also start to fold cotranslationally is generally difficult to determine using currently available methods. Here, we apply Force Profile Analysis (FPA - a method where a translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide - to follow the cotranslational translocation and folding of the large periplasmic domain of the E. coli inner membrane protease LepB in vivo. Membrane insertion of LepB′s two N-terminal transmembrane helices is initiated when their respective N-terminal ends reach 45-50 residues away from the peptidyl transferase center (PTC) in the ribosome. The main folding transition in the periplasmic domain involves all but the ~15 most C-terminal residues of the protein and happens when the C-terminal end of the folded part is ~70 residues away from the PTC; a smaller putative folding intermediate is also detected. This implies that wildtype LepB folds post-translationally in vivo, and shows that FPA can be used to study both co- and post-translational protein folding in the periplasm.

Cotranslational folding of human growth hormone in vitro and in Escherichia coli

“…The translational arrest can be overcome if a strong enough pulling force is exerted on the AP, essentially pulling it out of its binding site in the exit tunnel [8][9][10][11]. APs can be employed as sensitive "molecular force sensors" to report on various cotranslational events such as protein folding [12][13][14][15][16], protein translocation [17], and the membrane protein integration [9,18,19].…”