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Interactions of peptides with a protein pore.

ABSTRACT: The partitioning of polypeptides into nanoscale transmembrane pores is of fundamental importance in biology. Examples include protein translocation in the endoplasmic reticulum and the passage of proteins through the nuclear pore complex. Here we examine the exchange of cationic alpha-helical peptides between the bulk aqueous phase and the transmembrane beta-barrel of the alpha-hemolysin (alphaHL) protein pore at the single-molecule level. The experimental kinetic data suggest a two-barrier, single-well free energy profile for peptide transit through the alphaHL pore. This free energy profile is strongly voltage- and peptide-length-dependent. We used the Woodhull-Eyring formalism to rationalize the values measured for the association and dissociation rate constants k(on) and k(off), and to separate k(off) into individual rate constants for exit through each of the openings of the protein pore. The rate constants k(on), k(off)(cis), and k(off)(trans) decreased with the length of the peptide. At high transmembrane potentials, the alanine-based peptides, which include bulky lysine side chains, bind more strongly (formation constants K(f) approximately tens of M(-1)) than highly flexible polyethylene glycols (K(f) approximately M(-1)) to the lumen of the alphaHL protein pore. In contrast, at zero transmembrane potential, the peptides bind weakly to the lumen of the pore, and the affinity decreases with the peptide length, similar to the case of the polyethylene glycols. The binding is enhanced at increased transmembrane potentials, because the free energy contribution DeltaG = -zetadeltaFV/RT predominates with the peptides. We suggest that the alphaHL protein may serve as a robust and versatile model for examining the interactions between positively charged signal peptides and a beta-barrel pore.

SUBMITTER: Movileanu L

PROVIDER: S-EPMC1366589 | biostudies-literature | 2005 Aug

REPOSITORIES: biostudies-literature

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Interactions of peptides with a protein pore.

Movileanu Liviu L Schmittschmitt Jason P JP Scholtz J Martin JM Bayley Hagan H

Biophysical journal 20050527 2

The partitioning of polypeptides into nanoscale transmembrane pores is of fundamental importance in biology. Examples include protein translocation in the endoplasmic reticulum and the passage of proteins through the nuclear pore complex. Here we examine the exchange of cationic alpha-helical peptides between the bulk aqueous phase and the transmembrane beta-barrel of the alpha-hemolysin (alphaHL) protein pore at the single-molecule level. The experimental kinetic data suggest a two-barrier, sin ...[more]

PMID: 15923222

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Project description:Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

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Interactions of peptides with a protein pore.

Publications

Interactions of peptides with a protein pore.

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