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Multistep protein unfolding during nanopore translocation

Nature Nanotechnology volume 8, pages 288–295 (2013)Cite this article

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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.

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Figure 1: Interaction of V5-C109–oligo(dC)30 with the αHL pore.
Figure 2: Voltage dependences of the rate constants for transitions between current levels.
Figure 3: Oligonucleotide insertion in step 1 → 2.
Figure 4: Voltage dependences of the rate constants for transitions between current levels in the presence of urea.
Figure 5: Effect of mutations on the rate constants for transitions between current levels.
Figure 6: Detection of events from the unfolded population in urea solution.

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Acknowledgements

The authors thank E. Mikhailova for the αHL protein prepared by in vitro transcription and translation. D.R-L. is a recipient of an EMBO Long-Term Fellowship. This work was also supported by a grant from Oxford Nanopore Technologies.

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  1. Department of Chemistry, University of Oxford, Oxford, OX1 3TA, UK

    David Rodriguez-Larrea & Hagan Bayley

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D.R-L. and H.B. planned the research. D.R-L. perfomed the experiments and data analysis. D.R-L. and H.B. wrote the paper.

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Correspondence to Hagan Bayley.

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Rodriguez-Larrea, D., Bayley, H. Multistep protein unfolding during nanopore translocation. Nature Nanotech 8, 288–295 (2013). https://doi.org/10.1038/nnano.2013.22

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