Watching helical membrane proteins fold reveals a common N-to-C-terminal folding pathway

Abstract

To understand membrane protein biogenesis, we need to explore folding within a bilayer context. Here, we describe a single-molecule force microscopy technique that monitors the folding of helical membrane proteins in vesicle and bicelle environments. After completely unfolding the protein at high force, we lower the force to initiate folding while transmembrane helices are aligned in a zigzag manner within the bilayer, thereby imposing minimal constraints on folding. We used the approach to characterize the folding pathways of the Escherichia coli rhomboid protease GlpG and the human β₂-adrenergic receptor. Despite their evolutionary distance, both proteins fold in a strict N-to-C-terminal fashion, accruing structures in units of helical hairpins. These common features suggest that integral helical membrane proteins have evolved to maximize their fitness with cotranslational folding.

Fig. 1.. Physicochemical search for refolding conditions of polytopic helical membrane proteins.

(A) Schematic of single-molecule MT folding experiment for a single GlpG protein reconstituted in a bicelle. (B) FEC of single GlpG proteins averaged over 28 cycles of mechanical stretching and relaxation (black heat map). To show individual unfolding events, representative raw traces are overlaid above 20 pN tension (blue traces). Yellow trace shows the mean extension value in the relaxation phase. F_h,start and F_h,end indicates the force levels at which the coil-to-helix transition starts and ends. Theoretical FECs for the N, U_c and U_h states are shown as red, light blue and pink dashed lines, respectively. Upper inset: a close-up view of the unfolding events. Lower inset: a magnified view of the FEC between 8 and 4 pN force. (C) Average F_h,start and F_h,end values determined under different refolding conditions. n = 34, 42, 58 for the 0, 10 and 30 mol% DMPG cases. n = 16 for the no bicelle case. n = 6, 10 for the A206G and L155A single-point GlpG mutant cases. All error bars represent mean ± s.d. (D) Refolding probability determined using a simple force jump experiment (see fig. S3) at different applied force levels (n = 125, 147, 111 for the 0, 10 and 30 mol% PG cases, respectively). Inset shows the refolding probability normalized to the 0 mol% PG case. (E) Allan deviation of the magnetic bead fluctuation at different force levels. Inset: Representative trace showing the Brownian fluctuation of a magnetic bead at different force levels (black trace: raw data at 1.2 kHz sampling, yellow trace: a median-filtered data with a 5 Hz window).

Fig. 2.. Direct observation of single GlpG folding.

(A) Designed mechanical cycle for inducing refolding of single GlpG proteins. The gray and black traces are 1.2-kHz raw data and 5 Hz median-filtered data, respectively. (B) Representative time-resolved traces comparing the extensions following slow force relaxation and force jump. Lower inset shows the extension difference (ΔzUz) at indicated force levels. Right inset shows a closed-up trace of the force jump that takes ~300 ms. (C) Representative folding traces for WT, A206G and L155A GlpG under 6 pN tension. Right insets show close-up traces exhibiting reversible transitions among the U_z, I₁, and I₂ states. (D) BIC values for indicated number of states. n = 20, 21 and 11 for the WT, A206G and L155A cases, respectively. (E) Positions of the intermediate states in the normalized extension space (at 6 pN, n is same as (D); at 5 pN, n = 21, 24 and 14 for WT, A206G and L155A, respectively). Error bars represent s.e.m. (F) Transition kinetics between the neighboring states at indicated force levels. In N-to-I₂ transition, both slow (inset, black) and fast (red) rates are displayed. Error bars represent s.e.m.

Fig. 3.. Characterization of folding properties for vesicle- and bicelle-reconstituted single GlpGs.

(A) Schematic of single-molecule MT folding experiments for GlpGs reconstituted in vesicle membranes. (B) Representative FECs showing successive stretching cycles applied to a single GlpG protein in a vesicle membrane. (C) FEC of vesicle-reconstituted single GlpG proteins averaged over 5 cycles of mechanical stretching and relaxation (black heat map). To show individual unfolding events, representative raw traces during stretching are overlaid (blue traces). Other definitions are the same as in Fig. 1B. Upper inset shows the average F_h,start and F_h,end values. Lower inset: a magnified view of the FEC between 5 and 2 pN force. (D) Refolding probability determined under different membrane conditions. n is the number of trials. (E) Representative traces of the folding protocol that directly induces the U_z state. Inset shows the extension changes (Δz_Unfolding) during unfolding at 8 pN under indicated membrane conditions. Pink dotted line is the expected extension change when reaching the U_h state. (F) Normalized transition rates determined for the A206G and L155A single-point mutants relative to the WT case (dashed line). Right illustrations show an anticipated conformational status of GlpG in each indicated state. Error bars represent s.e.m (G) Representative force-jump experiments applied for the intermediate states. Each inset shows the distribution of extension values recorded during high-force unfolding. Estimated extensions for individual states are shown as the dashed lines. (H) Folding energy landscapes of a single GlpG protein along the molecular extension reconstructed based on the Bell-Zhrukov (black trace) and the Dudko-Hummer-Szabo models (green trace). Inset: detailed structural segments in the folding pathways of GlpG. Each structurally segment is denoted in distinct colors. Black-colored amino acids correspond to the boundaries of the intermediate states. Faint colors around the boundaries represent the measurement errors (s.d.).

Fig. 4.. Direct observation of the complete folding pathway of human β₂AR.

(A) Schematic diagram of single-molecule MT folding experiment for β₂AR. (B) FEC of single β₂AR proteins averaged over 6 cycles of mechanical stretching and relaxation (black heat map). To show individual unfolding events, representative raw traces during stretching are overlaid (blue traces). Other definitions are the same as in Fig. 1B. Inset: an enlarged view of the FEC between 6 and 4 pN force. (C) Designed mechanical cycle for inducing refolding of human β₂AR at low force levels. (D) Representative time-resolved traces for β₂AR folding under 5 pN tension (with 2 mM TCEP). Right insets show close-up trajectories. Red traces show the transitions between four intermediates identified the HMM. (E) BIC values for indicated numbers of states (n = 18). (F) Transition kinetics between the neighboring states at 5 pN. In N-to-I_f4 transition, both slow (inset, black) and fast (red) rates are displayed. Error bars represent s.e.m. (G, H) Representative traces for the force jump experiments applied to individual folding intermediates (G) and the native state (H). Each inset shows an extension distribution during high-force unfolding. (I, J) Extension distribution during high-force unfolding initiated from the native N state. (n = 29, 13 for the cases with 2 mM TCEP and without TCEP, respectively). The peaks indicate the fit centers of multiple Gaussian functions (colored for each function). Upper insets show structural diagrams of β₂AR to guide mapping onto the structure. (K) Representative β₂AR folding trace at 5 pN with no TCEP (n = 10). HMM analysis finds three intermediate states (I_f1^´, I_f2^´, I_f3^´). (L) Normalized extensions for β₂AR folding intermediates under no TCEP condition. Dashed lines are anticipated extensions for the intermediates under 2 mM TCEP condition. Error bars are s.d. (M) Representative folding traces for β₂AR at 5 pN in the presence of 2.5 μM carazolol (with 2 mM TCEP). Right inset shows the structure of carazolol-bound human β₂AR. Yellow circle indicates interaction regions between carazolol and β₂AR. (N) Normalized rates determined for carazolol-bound β₂AR relative to the apo β₂AR case (dashed line). (O) Representative FECs of β₂AR showing high-force cooperative unfolding in the presence (orange) and absence (black) of 2.5 μM carazolol (with 2 mM TCEP). N and U_c are defined the same as in (B). Inset: distributions of unfolding forces with (orange, n = 26) and without (black, n = 35) carazolol. Error bars are s.d. (P) Detailed structural segments in the folding pathways of β₂AR. Each structurally segment is denoted in distinct colors. Other notations are same as inset in Fig. 3H.