Abstract
Protein post-translational translocation is found at the plasma membrane of prokaryotes and protein import into organellae. Translocon structures are becoming available, however the dynamics of proteins during membrane translocation remain largely obscure. Here we study, at the single-molecule level, the folding landscape of a model protein while forced to translocate a transmembrane pore. We use a DNA tag to drive the protein into the α-hemolysin pore under a quantifiable force produced by an applied electric potential. Using a voltage-quench approach we find that the protein fluctuates between the native state and an intermediate in the translocation process at estimated forces as low as 1.9 pN. The fluctuation kinetics provide the free energy landscape as a function of force. We show that our stable, ≈15 kBT, substrate can be unfolded and translocated with physiological membrane potentials and that selective divalent cation binding may have a profound effect on the translocation kinetics.
Highlights
Protein post-translational translocation is found at the plasma membrane of prokaryotes and protein import into organellae
During the initial threading of the oligonucleotide into the pore at +120 mV, when the protein was in the native state, a characteristic low-conductance level was observed with Ires,% = 15.1 ± 0.5% [mean ± standard deviation (SD)], where is Ires,% is the Ires expressed as a percentage of the open pore current
In the α-HL pore, most of the membrane potential drops across the transmembrane barrel[46,47], and the ~10 negative charges on the phosphodiester bonds of the leader oligo, which are accommodated within the barrel[48], sense the field producing the pulling force that drives unfolding
Summary
Protein post-translational translocation is found at the plasma membrane of prokaryotes and protein import into organellae. We develop a voltage-quench approach that allows observation of the dynamics of a thioredoxin substrate as it is pulled through the α-hemolysin pore at voltages as low as +30 mV, exploring a force-regime comparable to that found in mitochondria and other important compartments such as the endoplasmic reticulum and the plasma membrane. By this means, we observe folding-unfolding transitions of single thioredoxin molecules, providing free energy measurements and the folding and unfolding kinetics. Our general model provides a framework with which to understand the energetics of additional protein posttranslational translocation systems
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