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Comparative Study
. 2015 Nov;1848(11 Pt A):2980-4.
doi: 10.1016/j.bbamem.2015.09.004. Epub 2015 Sep 2.

Protein transport across membranes: Comparison between lysine and guanidinium-rich carriers

Max Lein  1 , Brittany M deRonde  2 , Federica Sgolastra  2 , Gregory N Tew  3 , Matthew A Holden  4
Affiliations
Comparative Study

Protein transport across membranes: Comparison between lysine and guanidinium-rich carriers

Max Lein et al. Biochim Biophys Acta. 2015 Nov.

Abstract

The mechanism(s) by which certain small peptides and peptide mimics carry large cargoes across membranes through exclusively non-covalent interactions has been difficult to resolve. Here, we use the droplet-interface bilayer as a platform to characterize distinct mechanistic differences between two such carriers: Pep-1 and a guanidinium-rich peptide mimic we call D9. While both Pep-1 and D9 can carry an enzyme, horseradish peroxidase (HRP) across a lipid bilayer, we found that they do so by different mechanisms. Specifically, Pep-1 requires voltage or membrane asymmetry while D9 does not. In addition, D9 can facilitate HRP transport without pre-forming a complex with HRP. By contrast, complex formation is required by Pep-1. Both carriers are capable of forming pores in membranes but our data hints that these pores are not responsible for cargo transport. Overall, D9 appears to be a more potent and versatile transporter when compared with Pep-1 because D9 does not require an applied voltage or other forces to drive transport. Thus, D9 might be used to deliver cargo across membranes under conditions where Pep-1 would be ineffective.

Keywords: Arginine-rich peptide; Droplet-interface bilayer (DIB); Guanidine; Membrane translocation; Pep-1; Protein transduction domain mimic (PTDM).

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Figures

Figure 1
Figure 1
Carrier-mediated transport across a droplet-interface bilayer (DIB). (a) Carriers transport an enzyme (HRP) from the source to the capture droplet during a fixed interval. Electrodes within each droplet are used to record the ionic current and membrane capacitance. Following translocation the capture droplet is analyzed for enzyme activity via a fluorescence assay. (b) An NBD-labeled diguanidine polymer (9-mer) or Pep-1 was used to facilitate transport.
Figure 2
Figure 2
Concentration dependence of D9-NBD on HRP translocation across PC DIBs. The left data set (i) demonstrates D9-NBD’s activity as a protein carrier. Controls show reduced transport at (ii) lower concentration (iii) absence of carrier. Finally, droplets were contacted, then detached before DIB formation (iv). This demonstrated that merely contacting droplets does not transfer HRP. The third data set from the left was previously published30 and is included here for comparison.
Figure 3
Figure 3
D9-NBD facilitated transport as a function of voltage. Regardless of the presence or polarity of applied potential, little variation in the quantity of translocated HRP was observed.
Figure 4
Figure 4
D9-NBD facilitated transport as a function of membrane charge symmetry. 10 mol% of a PG lipid was added to either or both sides of DIBs to create negatively charged leaflet; the remaining lipids were neutral PC. Similar amounts of HRP were transported regardless of charge asymmetry.
Figure 5
Figure 5
Transport as a function of complex formation. Either Pep-1 or D9-NBD was used to transport HRP either from the same or the opposite side of the DIB. All trials were conducted at 0 mV. Remarkably, D9-NBD facilitates significantly more translocation when arranged opposite to the source droplet. No transport is observed with Pep-1. The second data set was published previously30 and the third data set is copied from Fig. 1 for comparison. Inset: 50 mM carboxyfluorescein was extracted from lipid vesicles as a function of D9-NBD concentration.
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