Multistep protein unfolding during nanopore translocation

Abstract

Cells are divided into compartments and separated from the environment by lipid bilayer membranes. Essential molecules are transported back and forth across the membranes. We have investigated how folded proteins use narrow transmembrane pores to move between compartments. During this process, the proteins must unfold. To examine co-translocational unfolding of individual molecules, we tagged protein substrates with oligonucleotides to enable potential-driven unidirectional movement through a model protein nanopore, a process that differs fundamentally from extension during force spectroscopy measurements. Our findings support a four-step translocation mechanism for model thioredoxin substrates. First, the DNA tag is captured by the pore. Second, the oligonucleotide is pulled through the pore, causing local unfolding of the C terminus of the thioredoxin adjacent to the pore entrance. Third, the remainder of the protein unfolds spontaneously. Finally, the unfolded polypeptide diffuses through the pore into the recipient compartment. The unfolding pathway elucidated here differs from those revealed by denaturation experiments in solution, for which two-state mechanisms have been proposed.

Figure 1. Interaction of V5-C109-oligo(dC)₃₀ with the αHL pore

a) The pore is inserted in a lipid bilayer from the cis compartment and a potential is applied causing an ionic current to flow through the pore. b) Current trace at +140 mV in 2 M KCl in the absence of V5-C109-oligo(dC)₃₀. c) Current trace at +140 mV in 2 M KCl with V5-C109-oligo(dC)₃₀ (0.4 µM, cis). d) and e) Level 1: V5-C109-oligo(dC)₃₀ is in solution and the pore is unoccupied. Level 2: the oligonucleotide threads into the pore and pulls on the protein. Level 3: the pulling force causes partial unfolding allowing the oligonucleotide to traverse the pore and the unfolded segment of the polypeptide to enter. Level 4: the remainder of the polypeptide unfolds spontaneously, diffuses through the pore and leaves through the trans entrance.

Figure 2. Voltage-dependences of the rate constants for transitions between current levels

a) (), Voltage-dependence of the frequency of occurrence of interactions between the V5-C109-oligo(dC)₃₀ and the αHL pore (step 1→2, k₁₂). (), Voltage-dependence of the frequency of occurrence of interactions between a 96-mer oligonucleotide and the same wild-type αHL pore used here. b) Voltage-dependence of the rate constant for step 2→3 in the translocation of V5-C109-oligo(dC)₃₀ (k₂₃). c) Voltage-dependence of the rate constant for step 3→4 (k₃₄). d) Voltage-dependence of the rate constant for step 4→1 (k₄₁). Error bars represent the standard deviations between independent experiments (n = 6).

Figure 3. Oligonucleotide insertion in step 1→2

a) Histogram of the current blockade levels observed with V5-C109-oligo(dC)₃₀. b) Histogram of the current blockade levels in the presence of equal concentrations of V5-C109-oligo(dC)₃₀ and V5-C109-oligo(dA)₃₀. I_RES% = (I_RES/I_O) × 100, where I_RES is the current flowing during a blockade and I_O is the current through the unblocked pore.

Figure 4. Voltage-dependences of the rate constants for transitions between current levels in the presence of urea

a) The effect of urea on step 2→3 (k₂₃). b) The effect of urea on step 3→4 (k₃₄). c) The effect of urea on step 4→1 (k₄₁). Error bars represent the standard deviations between independent experiments (n = 3).

Figure 5. The effect of mutations on the rate constants for transitions between current levels

a) The thioredoxin structure with arrows to highlight the network of ionic interactions (green residues) that link the C-terminal α-helix (center) with the rest of the structure. Pro-22 and Val-23 (red) make Van der Waal’s interactions both with the C-terminal α-helix and the core of the protein. Stars, residues mutated in this work. b) Secondary structure of thioredoxin mapped onto the primary sequence. Arrow, direction of translocation (C to N). c), d), e) Voltage dependences of k₂₃, k₃₄ and k₄₁ for K96A-oligo(dC)₃₀ () and V5-C109-oligo(dC)₃₀ (). f), g), h) Voltage dependences of k₂₃, k₃₄ and k₄₁ for K96D/K90D-oligo(dC)₃₀ (Δ) and V5-C109-oligo(dC)₃₀ (). The y-axis in 'f' is on a logarithmic scale. i), j), k) Voltage dependences of k₂₃, k₃₄ and k₄₁ for P22A/V23I-oligo(dC)₃₀ (◊) and V5-C109-oligo(dC)₃₀ (). Error bars represent the standard deviations between independent experiments (n = 4).

Figure 6. Detection of events from the unfolded population in urea solution

Left. The two types of events that are detected by electrical recording (events with level 4 only, and events with levels 2, 3 and 4) and the variation of the percentage of each type with the urea concentration. The events that lack distinct current steps (level 4 only) represent the translocation of unfolded thioredoxin. Right. The data are fitted to a two-state reversible process (black) assuming that the protein is completely unfolded in 12 M urea. Data from CD measurements in bulk solution are also shown (red).