Measurement of the Membrane Dipole Electric Field in DMPC Vesicles Using Vibrational Shifts of p-Cyanophenylalanine and Molecular Dynamics Simulations

Abstract

The magnitude of the membrane dipole field was measured using vibrational Stark effect (VSE) shifts of nitrile oscillators placed on the unnatural amino acid p-cyanophenylalanine (p-CN-Phe) added to a peptide sequence at four unique positions. These peptides, which were based on a repeating alanine-leucine motif, intercalated into small unilamellar DMPC vesicles which formed an α-helix as confirmed by circular dichroic (CD) spectroscopy. Molecular dynamics simulations of the membrane-intercalated helix containing two of the nitrile probes, one near the head-group region of the lipid (αLAX(25)) and one buried in the interior of the bilayer (αLAX(16)), were used to examine the structure of the nitrile with respect to the membrane normal, the assumed direction the dipole field, by quantifying both a small tilt of the helix in the bilayer and conformational rotation of the p-CN-Phe side chain at steady-state. Vibrational absorption energies of the nitrile oscillator at each position showed a systematic blue shift as the nitrile was stepped towards the membrane interior; for several different concentrations of peptide, the absorption energy of the nitrile located in the middle of the bilayer was ~3 cm−1 greater than that of the nitrile closest to the surface of the membrane. Taken together, the measured VSE shifts and nitrile orientations within the membrane resulted in a value of 8 – 11 MV/cm for the dipole field, at the high end of the range of possible values that have been accumulated from a variety of indirect measurements. Implications for this are discussed.

Site-selective characterization of Src homology 3 domain molecular recognition with cyanophenylalanine infrared probes

“…4). 8,12-18 Although the line shapes are best described as Voigt due to contributions from both homogeneous and inhomogeneous line broadening mechanisms, the bands are reasonably well fit with a single Gaussian function (Supplementary Information). All absorption bands also appear symmetrical about the center frequency.…”

Direct Measurement of the Effect of Cholesterol and 6-Ketocholestanol on the Membrane Dipole Electric Field Using Vibrational Stark Effect Spectroscopy Coupled with Molecular Dynamics Simulations

“…51 Upon incorporation of a small amount of sterol up to ~10 mol% into the bilayer, the membrane field increases for both sterols. At this low concentration, according to our simulations, the sterols are more concentrated near the helix while at higher concentrations their distribution is more uniform (Figure 8).…”

“…50,51 In our earlier work with 1,2-dimyristoyl- sn -glycero-3-phosphocholine (DMPC) vesicles, we placed a nitrile oscillator into the bilayer interior by intercalating an α -helical transmembrane peptide containing an unnatural amino acid, p -cyanophenylalanine ( p -CN-Phe). 51 The nitrile molecule served as an excellent VSE probe, as is described extensively in earlier publications. 48–52 We controlled the location of nitrile inside the bilayer by changing the position of p -CN-Phe along the amino acid sequence of the peptide.…”

“…The four polypeptide sequences that we used in this study are given in Table 1. 50,51 The peptide α LAX(25) places the nitrile near the lipid head group-water interface, where it was exposed to a very heterogeneous chemical environment containing charged groups and polar water molecules. At the other extreme, peptide α LAX(16) places the nitrile in a homogenous environment near the middle of the membrane that is mainly comprised of hydrocarbon chains.…”

“…We confirmed the chemical differences in the local vicinity of these probes both experimentally (through differences in vibrational absorption line widths), and computationally (through MD simulations) and determined the magnitude of the dipole electric field in a DMPC bilayer to be 8 – 11 MV/cm, depending on a variety of factors such as vesicle size, probe concentration, and nitrile orientation. 51 …”