Excursion of a single polypeptide into a protein pore: simple physics, but complicated biology

Abstract

Despite its fundamental and critical importance in molecular biology and practical medical biotechnology, how a polypeptide interacts with a transmembrane protein pore is not yet comprehensively understood. Here, we employed single-channel electrical recordings to reveal the interactions of short polypeptides and small folded proteins with a robust beta-barrel protein pore. The short polypeptides were approximately 25 residues in length, resembling positively charged targeting presequences involved in protein import. The proteins were consisted of positively charged pre-cytochrome b2 fragments (pb2) fused to the small ribonuclease barnase (approximately 110 residues, Ba). Single-molecule experiments exploring the interaction of a folded pb2-Ba protein with a single beta-barrel pore, which contained negatively charged electrostatic traps, revealed the complexity of a network of intermolecular forces, including driving and electrostatic ones. In addition, the interaction was dependent on other factors, such as the hydrophobic content of the interacting polypeptide, the location of the electrostatic trap, the length of the pb2 presequence and temperature. This single-molecule approach together with protein design of either the interacting polypeptide or the pore lumen opens new opportunities for the exploration of the polypeptide-pore interaction at high temporal resolution. Such future studies are also expected to unravel the advantages and limitations of the nanopore technique for the detection and exploration of individual polypeptides.

Membrane Protein Structure, Function, and Dynamics: a Perspective from Experiments and Theory

“…Upon passage through membrane pores, peptides undergo conformational transitions and sample intermediates that block the transmembrane current (Dekker, 2013; Mohammad & Movileanu, 2008; Movileanu et al, 2000; Oukhaled et al, 2007; Pastoriza-Gallego et al, 2011; Payet et al, 2012; Stefureac et al, 2008) that would otherwise flow in an open pore under a potential drop. These intermediate states can be considered jammed states.…”

Enhanced translocation of single DNA molecules through α-hemolysin nanopores by manipulation of internal charge

“…Movileanu and colleagues (13,15) have explored the passage of positively charged peptides through ␣HL pores with additional negative charge in the lumen. Both the rate of capture and the translocation time were increased, which they explained in terms of reduced barriers to transit.…”

“…Polymers can also be analyzed from the characteristics of transit events through the ␣HL pore (11)(12)(13)(14)(15). Studies of nucleic acids have been especially intensive, following the demonstration of the transit of single strands in 1996 (16).…”

Dynamics and Energy Contributions for Transport of Unfolded Pertactin through a Protein Nanopore

“…Using this approach, it has been possible to describe the transport and structure of peptides, 7-11 protein transport dynamics through toxins 7,12,13 and mitochondrial channels, 4,14 and studying protein-pore 15,16 and protein-protein interactions, 17 Protein nanopores have also emerged as powerful tools to study protein unfolding transitions using chemical denaturing agents, 18,19 thermal denaturation 20 or an electric field 21,22 at the single molecule level. Recently, Akeson's group used the energy of ATP hydrolysis provided by the AAA+ unfoldase ClpX to transport folded proteins through α-hemolysin.…”