Mapping the energy landscape for second-stage folding of a single membrane protein

Abstract

Membrane proteins are designed to fold and function in a lipid membrane, yet folding experiments within a native membrane environment are challenging to design. Here we show that single-molecule forced unfolding experiments can be adapted to study helical membrane protein folding under native-like bicelle conditions. Applying force using magnetic tweezers, we find that a transmembrane helix protein, Escherichia coli rhomboid protease GlpG, unfolds in a highly cooperative manner, largely unraveling as one physical unit in response to mechanical tension above 25 pN. Considerable hysteresis is observed, with refolding occurring only at forces below 5 pN. Characterizing the energy landscape reveals only modest thermodynamic stability (ΔG = 6.5 kBT) but a large unfolding barrier (21.3 kBT) that can maintain the protein in a folded state for long periods of time (t1/2 ∼3.5 h). The observed energy landscape may have evolved to limit the existence of troublesome partially unfolded states and impart rigidity to the structure.

Figure 1. Cooperative unfolding and refolding of GlpG in bicelles

(a) Schematic of the single-molecule magnetic tweezers experiment for studying unfolding and refolding of a single GlpG protein. (b) Representative force-extension curves in each buffer condition. After several cycles of unfolding and refolding in bicelles (left), the bicelles were removed and the unfolding and refolding cycles were repeated (middle). In the buffer condition without bicelles, a very small amount of CHAPSO (0.0038%) was added to prevent nonspecific binding. After up to tens of pulling cycles, the bicelle condition could be restored by another round of microfluidic buffer exchange (right), and the unfolding behavior seen previously in bicelles was fully restored. (c) Representative force-extension curves showing multiple-step unfolding of single GlpG proteins. In b and c, scale bar represents 50 nm.

Figure 2. C- to N-terminus unfolding of single GlpG with two intermediates

(a) Representative extension traces at 21 pN for unfolding events (n = 295) with no intermediates (59.0%), one intermediate (33.2%) and two intermediates (7.8%). Statistics of unfolding step sizes are in Supplementary Figure 6. Scale bars, 1 s. (b) Dwell time analysis (n = 295). τ_U is the waiting time until complete unfolding (blue), and τ₁ and τ₂ are the dwell times in the intermediate states I₁ (green) and I₂ (yellow). (c) Dwell times in the intermediates normalized by τ_U. (d) Comparison of the normalized proportion of unfolding patterns with one intermediate between the WT and the L155A and A206G mutants. The normalized proportion is defined by P(X)/P(WT), where P(X) means the proportion of each unfolding pattern in the total number of traces for X = WT (n = 295), L155A (n = 81) or A206G (n = 97). The histograms for one intermediate with I₁ or I₂ are shown in red and blue, respectively. (e) GlpG structure showing the intermediate positions I₁ and I₂ (black). The mutation sites Leu155 and Ala206 are shown in ball-and-stick representation. Left, cytoplasmic view; right, side view showing a lipid bilayer. (f) Schematic diagram showing the mapping of Gaussian peak values to the intermediate residue positions. Arrow indicates unfolding direction. (g) Conceptual folding energy landscape at 21 pN. The arrows denote the structural transitions among the native state (N), the intermediate states (I₁ and I₂) and the unfolded state (U).

Figure 3. Folding energy landscape of GlpG

(a) Representative gradual pulling experiments measuring the unfolding force of single GlpG proteins. Scale bar, 50 nm. (b) Unfolded fraction versus force (n = 233) from which the zero-force unfolding rate (k_u0) and the distance from the native state to the transition state (Δx_f^†) were obtained. (c) Representative extension traces in force-cycle experiment for obtaining the folding kinetics. After unfolding at 21 pN, the force was lowered back to 0.9–7.3 pN and maintained for 3 min. The extent of refolding was then determined by restoring the 21-pN force and comparing the observed extension with the extensions observed for the native and unfolded states (N_{21 pN} and U_{21 pN} are shown as blue and red dashed lines, respectively). (d) Folded fraction versus force (n = 125), which was used to obtain the folding kinetic rate at zero force (k_f0) and the distance from the unfolded state to the transition state (Δx_u^†). (e) Putative folding energy landscape of GlpG. The energy difference between the native state and the unfolded state (ΔG) and the energy barriers (ΔG_u^†, ΔG_f^†) are denoted with red arrows. The error of the ΔG represents s.e.m., and the error of the energy barriers represent the error of the frequency factor k_w (Online Methods).

Figure 4. Comparison of kinetic and thermodynamic properties between WT and mutant GlpG

(a,b) Unfolded fraction (a) and folded fraction (b) as a function of force for the WT and mutant GlpG proteins. The total number of unfolding and refolding events are n = 233 and n = 125 for WT; n = 77 and n = 58 for the L155A mutant; and n = 95 and n = 87 for the A206G mutant. Fitting the data (Online Methods) yields kinetic rates for unfolding and folding at zero tension (k_u0 and k_f0) and distances from the folded (and unfolded) state to the transition state (Δx_f^† and Δx_u^†). (c) Comparison of the distance values (left, Δx_f^†; middle, Δx_u^†) and the transition state positions (right, β_f) of the WT and the mutants. (d,e) Comparison of the unfolding rate (d) and refolding rate (e) for the WT and mutant proteins, normalized to the WT rate. (f) The change in unfolding free energy of the mutants relative to the WT observed in forced unfolding experiments (ΔΔG = ΔG_WT − ΔG_mutant). All error bars represent s.e.m.

Figure 5. How the folding energy landscape of GlpG may prevent dangerous misfolded states

Cooperativity can limit the formation of stable off-pathway structures before completion of translation. The high kinetic barrier near the folded state prevents folded GlpG from returning to the unfolded state on a biologically relevant time scale, imparts rigidity and limits the existence of partially unfolded states that might be prone to inappropriate interactions.