1Department of Chemistry, Wellesley College,
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Spinella, S. A., Nelson, R. B., Elmore, D. E. Measuring Peptide Translocation into Large Unilamellar Vesicles. J. Vis. Exp. (59), e3571, doi:10.3791/3571 (2012).
There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1. Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors1-3. Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms4,5, particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes6,7. In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides1,8. A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)9-13. LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies14,15. Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II16,17. In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties18, to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.
1. Preparation of Large Unilamellar Lipid Vesicles (LUVs)
2. Quantifying LUV Concentration
3. Preparing Peptide Solutions
4. Translocation Quantification
5. Generating a Quantitative Translocation Ratio
6. Representative Results
Figure 2 shows the results of this assay for a representative peptide that showed robust translocation. The signal in this experiment (black trace) shows a marked drop in FRET signal over time. However, it is important to control for the potential loss of FRET signal due to incomplete trypsin inhibition or other factors unrelated to translocation ability. To this end, we always also measure the FRET signal between peptide and LUVs containing both trypsin and Bowman-Birk trypsin inhibitor (gray trace, Figure 2). In our hands, it has been important to perform this control for every peptide every time an experiment is run. This allows us to clearly correct for any changes in signal due to any differences between lipid vesicle preparations or instrument noise, which can be significant for the relatively weak fluorescent signals typically observed at these concentrations.
The importance of the control experiment using LUVs containing trypsin inhibitor is highlighted by the data shown in Figure 3. In this case, the peptide signal decreased a similar amount to that in Figure 2 in the experimental sample (black trace). However, the control sample shows a more rapid decrease for this peptide, so its net translocation is lower.
Section 5 of our protocol describes a straightforward method for quantifying translocation from this experiment. Higher translocation ratios are indicative of peptides that translocate efficiently; the translocation ratio for the cell penetrating peptide shown in Figure 2 is 1.16, while weakly translocating peptides have translocation ratios closer to 1. In our experience, the standard error for three independent experiments performed with different vesicle preparations is in the range of 0.01 to 0.06.
Figure 1. Schematic of translocation assay. Experimental LUV samples (A) are doped with fluorescent dansyl POPE (black bars) and contain encapsulated trypsin (scissors). Trypsin inhibitor (red circle) is used to inhibit trypsin outside the LUVs. The LUVs are exposed to peptide (purple). Peptide association with LUV membranes yields a FRET signal (green) that decreases as translocting peptides encounter uninhibited internal trypsin. Both trypsin and trypsin inhibitor are encapsulated in control LUV samples (B) to measure decreases in FRET signal unrelated to translocation.
Figure 2. Representative data for a cell-penetrating peptide. A representative translocating antimicrobial pepide shows a significant drop in FRET signal compared to its control.
Figure 3. Representative data highlighting the importance of the control. Incomplete inhibition of tryptic digestion of a non-translocating peptide leads to a drop in FRET signal over time in both experimental and control LUV solutions. Because the difference between the control and experimental traces is relatively small, this mutant is characterized as a weakly translocating peptide.
The protocol presented here can be used to assess the relative change in the concentration of peptides inside and outside of lipid vesicles. These changes are related to translocation ability. This protocol can be used to identify cell-penetrating peptides with potential as drug delivery vectors. As interest in cell-penetrating peptides grows, it will be interesting to see how methods that directly measure the translocation event are developed and used in a quantitative manner.
In our lab, we have found that the consistency of the assay can be improved by carefully monitoring a few particular aspects of the experiment. First, quantification of both experimental and control vesicles improves the consistency of the results obtained using this protocol. This assay also depends on the ability to detect an accurate initial fluorescence prior to the beginning of protein translocation and digestion. Therefore, it is essential for the fluorescence signal collection to begin as soon as the protein or peptide is exposed to the LUV solution. This assay is also sensitive to the particular trypsin inhibitor used; to date, we have obtained the most consistent results with Bowman-Birk trypsin inhibitor (Sigma T-9777). Importantly, the degree of degradation observed in control scenarios seems to vary significantly between peptides (as highlighted in Figures 2 and 3) and in some cases between different vesicle preparations for the same peptide. This further emphasizes the need to run a control with each experimental replication. As an additional control, one could also measure the FRET response for peptide exposed to a vesicle sample containing neither trypsin nor trypsin inhibitor. However, this control does not provide any additional information that is necessary in order to evaluate data for peptide translocation.
One concern is that membrane-lytic peptides could permeabilize the membrane enough to allow trypsin or trypsin inhibitor to travel across the membrane. The peptides used for examples in this paper have been shown to cause little membrane permeabilization. Nonetheless, many membrane-lytic peptides also are amenable to this and similar translocation assays9,16,21,22. This is likely because the volume is much greater outside than inside the vesicles. Thus, any trypsin that leaks out of the vesicle can be inhibited by excess trypsin inhibitor, preventing any cleavage of peptide outside the vesicle. Thus, a positive result from this assay likely denotes a peptide that translocates while causing relatively minimal membrane disruption, preventing inhibitor from leaking into the vesicles. Regardless, a thorough characterization of peptide properties should include an assessment of membrane permeabilization.
This assay is amenable to adaptation for a wider variety of experimental circumstances. Given that translocation is measured as a function of FRET signal between protein and membrane, as described this assay is best-suited to proteins and peptides that spontaneously associate with anionic LUVs, contain one tryptophan residue and have trypsin cut sites near the tryptophan residue. The proximity of the tryptophan to a cut site ensures that the fragment containing tryptophan has a negligible membrane affinity, and thus a negligible FRET signal. However, one could also use peptides containing tyrosine or other chemically conjugated fluorescent moities along with vesicles containing an appropriate FRET acceptor. Similarly, trypsin could be replaced with another protease that targets alternative cut sites in a peptide of interest. However, altering the enzyme and inhibitor may require additional optimization since some commercially available trypsins and trypsin inhibitors led to aggregation or instability of vesicle samples. By altering the lipid composition of the LUVs, this assay may also be used to determine the role that lipid charge or structure plays in determining peptide translocation. Additionally, this assay could also be readily adapted to provide high-throughput measurements of translocation in an approach similar to that of Wimley and co-workers9.
No conflicts of interest declared.
The authors would like to thank Eleanor Fleming and Jessica Chen for helpful discussions. Funding was provided by National Institute of Allergy and Infectious Diseases (NIH-NIAID) Award R15AI079685 and a Research Corporation Cottrell College Science Award. Additional student support was provided by the Howard Hughes Medical Institute and the Staley fund.
|16:0-18:1 PG||Avanti Polar Lipid, Inc||840457C|
|1:18 Dansyl PE||Avanti Polar Lipid, Inc||810330C|
|16:0-18:1 PC||Avanti Polar Lipid, Inc||850457C|
|Bowman-Birk trypsin/chymotrypsin inhibitor||Sigma-Aldrich||T-9777|
|Mini-extruder||Avanti Polar Lipid, Inc||610000|
|Ammonium molybdate (para)||Alfa Aesar||10811|
|Hydrogen Peroxide, 30% solution||Mallinckrodt Baker Inc.||5240-05|