The shape of the bacterial ribosome exit tunnel affects cotranslational protein folding

Abstract

The E. coli ribosome exit tunnel can accommodate small folded proteins, while larger ones fold outside. It remains unclear, however, to what extent the geometry of the tunnel influences protein folding. Here, using E. coli ribosomes with deletions in loops in proteins uL23 and uL24 that protrude into the tunnel, we investigate how tunnel geometry determines where proteins of different sizes fold. We find that a 29-residue zinc-finger domain normally folding close to the uL23 loop folds deeper in the tunnel in uL23 Δloop ribosomes, while two ~ 100 residue proteins normally folding close to the uL24 loop near the tunnel exit port fold at deeper locations in uL24 Δloop ribosomes, in good agreement with results obtained by coarse-grained molecular dynamics simulations. This supports the idea that cotranslational folding commences once a protein domain reaches a location in the exit tunnel where there is sufficient space to house the folded structure.

Cotranslational folding cooperativity of contiguous domains of α-spectrin

“…To obtain good estimates of the Bell parameters $ , D ‡ we started from the values obtained in 6 and performed a local optimization by requiring that k0 (the release rate at zero force) is close to kR measured for the "zero-force" construct R16 nL27, Fig. 4a, and that the titin I27, spectrin R16, and ADR1a force profiles in 12 are well reproduced by the molecular dynamics simulation also described in 12 . These conditions are both fulfilled by setting k0 = 3 x 10 -4 s -1 (i.e., the same value as in 6 and equal to kR for R16 nL27, Fig.…”

“…It is not exactly the same thing as the ensemble average force calculated from MD simulations, 〈 LM 〉 = P P + ) ) , where P()) and P()) are, respectively, the population of, and force exerted by, the unfolded (folded) state in the simulation. In previous work 12 , we calculated both fFL as well as 〈 LM 〉 directly from MD simulations. In Supplementary Fig.…”

“…The coarse-grained molecular dynamics (MD) simulations of cotranslational folding of R16 and ADR1a constructs in WT, DuL23, and DuL24 ribosomes used here were originally reported in 12 . For each construct and each linker length, these simulations were used to -16 -calculate the ensemble average force 〈 LM 〉 = P P + ) ) , where P()) and P()) are, respectively, the population of, and force exerted by, the unfolded (folded) state in the simulation.…”

“…For each ∆ ‡ value, we calculated an average kR over unfolded and folded states as " = P $ exp[ P ∆ ‡ /`] + ) $ exp[ ) ∆ ‡ / ] and then obtained fFL (setting Dt = 550 s) from equation [2] in the main text. The simulated fFL values (i.e, the simulated force profiles) obtained in this way were then compared with the experimental force profiles reported in 12 for the I27 and R16 domains expressed with WT, DuL23, and DuL24 ribosomes, and for the ADR1a domain expressed with WT and DuL24 ribosomes. The parameter values k0 = 3 x 10 -4 s -1 , Dx ‡ = 0.65 nm were found to give the best fit between the simulated and experimental force profiles (see Supplementary Fig.…”

“…The discovery 2,3 and engineering 4,5 of translational arrest peptides (APs) has provided us with a new tool to examine how proteins fold cotranslationally. To date, the folding of several small proteins and protein domains has been studied using the force-sensitive AP from the E. coli SecM protein with a method we have called Force-Profile Analysis (FPA) [6][7][8][9][10][11][12][13][14][15] . Recently, we demonstrated that FPA faithfully picks up cotranslational protein folding events observed by direct biophysical measurements 15 .…”