Translocation of amino acyl residues from the membrane interface to the hydrophobic core: thermodynamic model and experimental analysis using ATR-FTIR spectroscopy

Figures & data

Figure 1.  Sequence and helical wheel diagram of LAH₄X₄ peptides. Whereas the hydrophobic side B consists of leucines and alanines, face A is assembled from the amino acids X and flanked by two histidines on each side.

Figure 2.  Model of the topological states of LAH₄-like polypeptides (cf. text for details).

Figure 3.  The fraction of transmembrane aligned peptide, p_TM, as a function of pH at different transfer energies ΔG/RT. The titration curves were simulated with pK_a values 6.0 for the histidine side chains. The simulations shown represent the following values of ΔG/RT: -8 (solid line), -2 (hatched line), 0 (dotted line), and 2 (hatched-dotted line).

Figure 4.  The calculated shifts for the in-plane to transmembrane transition pH as compared to the pK_a value common to all histidines is shown as a function of changes in hydrophobicity of the X-amino acids, ΔG/RT. Whereas the solid line corresponds to the pH where half the maximum of p_TM is reached, the dotted line indicates the pH value where half the peptides are in a transmembrane state, p_TM=50%. The dashed line represents the linear approximation obtained for very hydrophobic peptides (large negative ΔG). See text for details.

Figure 5.  The amide region of ATR-FTIR spectra of LAH₄F₄ reconstituted into oriented POPC membranes is shown at pH 8 (A) and at pH 3 (B). The incident light was polarized in a parallel (dotted lines) or in a perpendicular orientation (hatched lines). The difference spectra (solid lines) are obtained by subtracting the perpendicular polarized spectrum from the parallel polarized spectrum. For this Figure the latter was multiplied by a factor 1.35 before subtraction, which corrects for the difference of strength of the evanescent power for the parallel and perpendicular polarisations as discussed in detail in reference [17].

Figure 6.  CD spectra of 10 µM LAH₄L₄ in the presence of 1000 µM small unilamellar POPC vesicles at pH 4.0 (dotted line) pH 5.0 (dash-dot line), pH 6.0 (dashed line) and pH 7 (solid line)

Figure 7.  The dichroic ratio R^ATR of LAH₄X₄ peptides reconstituted into oriented POPC phospholipids bilayers are shown as a function of pH. A. LAH₄F₄, B. LAH₄L₄, C. LAH₄V₄, D. LAH₄W₄, E. LAH₄I₄., F. LAH₄A₃L. The experiments were performed in duplicate.

Table I.  Analysis of the pH-dependent dichroic ratio of LAH₄X₄ peptides as observed in oriented ATR-FTIR experiments. A fit of the experimental data was obtained using Equations 11 and 12.

	Rmin	Rmax	ΔG/RT complete peptide	ΔG (kJ/mol) per amino acid relative to leucin
LAH4L4	1.36±0.06	2.25±0.05	−6.78±0.73	0.00±0.44
LAH4F4	1.43±0.07	2.73±0.08	−4.99±0.40	1.09±0.24
LAH4V4	1.41±0.06	3.37±0.04	−1.17±0.51	3.41±0.31
LAH4W4	1.26±0.03	2.17±0.14	−1.27±0.46	3.35±0.28
LAH4I4			−5.73±1.11	0.64±0.67
LAH4A3L	1.52±0.05	2.43±0.04	−6.00±0.48	0.63±0.39
LAH4G3L	1.52±0.04	2.29±0.07	−4.33±0.54	1.98±0.44
LAH4S3L	1.38±0.09	2.51±0.08	−5.31±0.63	1.19±0.51
LAH4T3L	1.43±0.04	2.08±0.07	−4.22±0.66	2.07±0.53
LAH4Y2L2	1.51±0.05	3.06±0.25	−1.42±0.46	6.51±0.56

Figure 8.  Summary of the relative contributions of the amino acid X to ΔG of transfer from in-plane to transmembrane alignments (cf. text for details). The values are scaled to take into account the influence of one X amino acid side chain within the peptide. The LAH₄L₄ peptide is taken as a reference. The boxes indicate the experimental errors obtained during the line fit analyis of ΔG/RT (Table I). The top scale indicates values obtained when calculating the difference ΔG_{(aqueous→octanol)} - ΔG_{(aqueous→membraneinterface)} as published in [6].

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