Utilizing ESEEM spectroscopy to locate the position of specific regions of membrane-active peptides within model membranes

Abstract

Membrane-active peptides participate in many cellular processes, and therefore knowledge of their mode of interaction with phospholipids is essential for understanding their biological function. Here we present a new methodology based on electron spin-echo envelope modulation to probe, at a relatively high resolution, the location of membrane-bound lytic peptides and to study their effect on the water concentration profile of the membrane. As a first example, we determined the location of the N-terminus of two membrane-active amphipathic peptides, the 26-mer bee venom melittin and a de novo designed 15-mer D,L-amino acid amphipathic peptide (5D-L9K6C), both of which are antimicrobial and bind and act similarly on negatively charged membranes. A nitroxide spin label was introduced to the N-terminus of the peptides and measurements were performed either in H2O solutions with deuterated model membranes or in D2O solutions with nondeuterated model membranes. The lipids used were dipalmitoyl phosphatidylcholine (DPPC) and phosphatidylglycerol (PG), (DPPC/PG (7:3 w/w)), egg phosphatidylcholine (PC) and PG (PC/PG (7:3 w/w)), and phosphatidylethanolamine (PE) and PG (PE/PG, 7:3w/w). The modulation induced by the 2H nuclei was determined and compared with a series of controls that produced a reference "ruler". Actual estimated distances were obtained from a quantitative analysis of the modulation depth based on a simple model of an electron spin situated at a certain distance from the bottom of a layer with homogeneously distributed deuterium nuclei. The N-terminus of both peptides was found to be in the solvent layer in both the DPPC/PG and PC/PG membranes. For PE/PG, a further displacement into the solvent was observed. The addition of the peptides was found to change the water distribution in the membrane, making it "flatter" and increasing the penetration depth into the hydrophobic region.

FIGURE 1

(a) The three-pulse ESEEM sequence and the ESEEM waveform with the definition of the modulation depth, K(²H). (b) A schematic representation of the various regions of the model membrane and the expected modulation depth for a spin label located within the different regions. The gradient in the gray color distinguishes between the hydrophilic and hydrophobic regions.

FIGURE 2

Schematic illustration of the phospholipids and the nitroxide spin probes used in this study. (a) PG, (b) DPPC, (c) 5DSA, (d) MTSSL, (e) proxy, and (f) CAT1.

FIGURE 3

Room temperature X-band EPR spectra of some of the samples studied as noted on the figure. The asterisks (*) mark the signal of free nitroxide impurity.

FIGURE 4

Room temperature X-band EPR spectra of 5DSA, 7DSA, and 16DSA in DPPC-d₁₃/PG LUVs without peptides (solid line), with 5D-L₉K₆C (dashed line) and with melittin (dotted line).

FIGURE 5

FT-ESEEM spectra of reference spin probes in (a) DPPC/PG/D₂O and (b) DPPC-d₁₃ /PG. 16DSA (solid line), 7DSA (dashed line), 5DSA (dotted-dashed line). The arrow marks the broad signal due to water H-bonded to the NO group.

FIGURE 6

The K(²H) (solid symbols, arrow points to left axis) and I(²H) (shaded symbols, right axis) values of all reference spin probes in (a) DPPC/PG/D₂O (▪) and PC/PG/D₂O (•) and (b) DPPC-d₁₃/PG.

FIGURE 7

The dependence of the frequency of the W-band Mims ENDOR ³¹P signal of 5DSA in PC/PG on the magnetic field. The solid line was calculated based on the gyromagnetic ratio of ³¹P. The inset on the left shows the echo detected EPR spectrum and the fields at which the ENDOR was measured. The inset on the right displays the ENDOR spectra of 5DSA and 7DSA in PC/PG recorded at a magnetic field corresponding maximum echo intensity.

FIGURE 8

Calculated K(²H) values as a function of z₀ (the distance of the electron spin from the bottom of the layer) for several layer thicknesses, a, and densities, d, (a) for a homogeneous distribution, d = 0.055 n/A³, a = 5 Å (▪), d = 0.045 n/A³, a = 6 Å (•), d = 0.033 n/A³, a = 8 Å (▴), d = 0.025 n/A³, a = 8 Å (▾), d = 0.0125 n/A³, a = 8 Å (♦). (b) For a bimodal distribution with a = 8 Å and d = 0.0125 n/A³ (•), d = 0.025 n/A³ (▴), d = 0.033 n/A³ (▾). Here one of the traces for a homogeneous distribution (d = 0.0125 n/A³, (▪)) is shown for comparison. The vertical lines define the range of the layer for a = 5, 6, and 8 Å and the horizontal lines the experimental K(²H) values of 5DSA (upper) and 7DSA (lower).

FIGURE 9

Comparison of the I(²H) values for 5DSA, 7DSA, and 16DSA in PG/PC/D₂O LUVs without peptides (▪), with melittin (▴), and with 5D-L₉K₆C (•).

FIGURE 10

(a) Comparison of the I(²H) (solid symbols, right axis) and K(²H) (shaded symbols, left axis) values for 5D-L₉K₆C-NO (LKC-NO) and melittin-NO (Mel-NO) in DPPC-d₁₃/PG with those of 5DSA, 7DSA, and 16DSA in DPPC-d₁₃/PG LUVs with 5D-L₉K₆C (left) and melittin (right). (b) Comparison of the I(²H) values for LKC-NO and Mel-NO in DPPC/PG/D₂O (squares) with those of 5DSA, 7DSA, and 16DSA in DPPC/PG/D₂O with 5D-L₉K₆C (left) and melittin (right). The circles (connected by a line) correspond to the same in a PC/PG/D2O system, including also melittin-NO and 5D-L₉K₆C-NO in PE/PG/D₂O.

FIGURE 11

A schematic representation of the relative location (•) of the nitroxide spin label in the reference probes on the labeled peptides with respect to the surface of the model membrane's layer.

FIGURE A1

The model for calculating K(²H) from a layer with a homogeneous distribution of deuterons.