Free-energy landscapes of membrane co-translocational protein unfolding

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 αhemolysin 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 k B T, 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.

The emerging landscape of single-molecule protein sequencing technologies

“…Other proteins have the potential to control protein translocation through nanopores, beyond secretases and unfoldases, including chaperones (Hsp70), via processes resembling protein translocation into the mitochondrial matrix 71 . Recently, Rodriguez-Larrea's group has discussed how protein refolding at the entry and exit compartments can oppose and promote protein translocation, respectively 72,73 , and the use of deep learning networks to analyze raw ionic current signals for accurate classification of single point mutations in a translocating protein 74 . In addition, Cardozo et al built a library of approximately 20 proteins that are orthogonally barcoded with an intrinsic peptide sequence and successfully read them with nanopore sensors 75 .…”

Biological Nanopores: Engineering on Demand

“…Protein nanopores can be used to examine folding/unfolding pathways [165][166][167] and to probe for the presence of characteristic conformers [168]. The latter requires a pore that is sufficiently large to trap the enzyme in its lumen.…”

Herding cats: Label-based approaches in protein translocation through nanopore sensors for single-molecule protein sequence analysis

“…Hence, attaching an oligonucleotide strand to a protein is a straightforward way to facilitate electrophoresis-driven protein translocation ( Figure 4 A, Left). Using this approach, Bayley and colleagues have demonstrated controlled directional translocation of proteins through an α-hemolysin nanopore ( Rodriguez-Larrea and Bayley, 2013 , 2014 ; Rosen et al., 2020 ). The group used genetically engineered thioredoxin as a model protein and linked a C-terminal cysteine side chain to a 30-mer oligo(dC) nucleotide via a disulfide bond.…”