Interactions between Charged Polypeptides and Nonionic Surfactants Helen Sjögren, Caroline A. Ericsson, Johan Evenäs, Stefan Ulvenlund  Biophysical Journal  Volume 89, Issue 6, Pages 4219-4233 (December 2005) DOI: 10.1529/biophysj.105.065342 Copyright © 2005 The Biophysical Society Terms and Conditions

Figure 1 Chemical structure of three different alkylglycosides: n-octyl-β-D-glucoside (β-C8G1), n-dodecyl-α-D-maltoside (α-C12G2), and n-dodecyl-β-D-maltotrioside (β-C12G3). Note the difference between alkylglycosides (surfactants with an undefined number of sugar units in the headgroup) and alkylglucosides (surfactants with one sugar unit in the headgroup). Biophysical Journal 2005 89, 4219-4233DOI: (10.1529/biophysj.105.065342) Copyright © 2005 The Biophysical Society Terms and Conditions

Figure 2 CD spectra for (KF)n with (dotted trace) and without (dashed trace) addition of 0.2M β-C8G1. The reference spectra for 100% α-helix and 100% random coil (solid traces) are given for comparison. A (KF)n concentration of 25μg/ml (which equals 0.14mM of amino acid residues) was used in the CD measurements. All measurements were performed in a 20-mM acetate buffer at pH 4.9. Biophysical Journal 2005 89, 4219-4233DOI: (10.1529/biophysj.105.065342) Copyright © 2005 The Biophysical Society Terms and Conditions

Figure 3 Surface tension (▴) and the fraction of (KF)n in α-helix conformation (Xα, ♦) as a function of β-C12G2 concentration. A (KF)n concentration of 25μg/ml (which equals 0.14mM of amino acid residues) was used in the CD measurements. All measurements were performed in a 20-mM acetate buffer at pH 4.9. The kink in the tensiometric data indicates the CMC of β-C12G2. The solid traces are added as guides for the eye. Biophysical Journal 2005 89, 4219-4233DOI: (10.1529/biophysj.105.065342) Copyright © 2005 The Biophysical Society Terms and Conditions

Figure 4 The fraction of (KF)n in α-helix conformation, Xα, as a function of surfactant concentration for α-C12G2 (●), β-C12G2 (■), β-C9G1 (▴), and C12E5 (♦). All curves are normalized to the CMC of the specific surfactant. A (KF)n concentration of 25μg/ml (which equals 0.14mM of amino acid residues) was used, and the measurements were performed in a 20-mM acetate buffer at pH 4.9. The solid traces are added as guides for the eye. Biophysical Journal 2005 89, 4219-4233DOI: (10.1529/biophysj.105.065342) Copyright © 2005 The Biophysical Society Terms and Conditions

Figure 5 Increase in peptide α-helix conformation, ΔXα, (determined with CD spectroscopy) as a function of surfactant headgroup size, for β-C12GX surfactants (▴) and C12EX surfactants (♦). A (KF)n concentration of 25μg/ml (which equals 0.14mM of amino acid residues) was used, and the measurements were performed in a 20-mM acetate buffer at pH 4.9. Since the Krafft point of β-C12G1 is above room temperature, it cannot be used for comparison. The glucosides are therefore represented by β-C9G1. The data points represent the mean value from at least three independent measurements, and the error bars correspond to 1 SD. Biophysical Journal 2005 89, 4219-4233DOI: (10.1529/biophysj.105.065342) Copyright © 2005 The Biophysical Society Terms and Conditions

Figure 6 Increase in peptide α-helix conformation, ΔXα, as determined with CD spectroscopy, as a function of surfactant hydrocarbon chain length, for β-CXG1 surfactants (▴) and β-CXG2 surfactants (♦). Data represent systems with a surfactant concentration of ≥10×CMC and a (KF)n concentration of 25μg/ml (which equals 0.14mM of amino acid residues) in a 20-mM acetate buffer at pH 4.9. The solid traces are added as guides for the eye. The data points represent the mean value from at least three independent measurements, and the error bars correspond to 1 SD. Biophysical Journal 2005 89, 4219-4233DOI: (10.1529/biophysj.105.065342) Copyright © 2005 The Biophysical Society Terms and Conditions

Figure 7 Increase in peptide α-helix conformation, ΔXα, (♦) (measured with CD spectroscopy) and the effective hydrodynamic diameter, dH, (▴) (measured with DLS) as a function of (A) temperature for C12E6 and (B) hydrocarbon chain length for β-CXG1. A surfactant concentration of ≥10×CMC and a (KF)n concentration of 25μg/ml (which equals 0.14mM of amino acid residues) were used. The measurements were performed in a 20-mM acetate buffer at pH 4.9. The DLS measurements were performed on solutions of pure surfactant (no peptide added). The solid traces are added as guides for the eye. Biophysical Journal 2005 89, 4219-4233DOI: (10.1529/biophysj.105.065342) Copyright © 2005 The Biophysical Society Terms and Conditions

Figure 8 Number of bound β-C8G1 molecules per amino acid residue in (KF)n (β) as a function of free β-C8G1 concentration [Sfree]. [Sfree] as determined by equilibrium dialysis and subsequent NMR assay of surfactant concentration. The data points represent the average of values calculated from signals in three different regions in the NMR spectra, and the error bars correspond to 1 SD. The solid trace is a fit to the Hill equation (Eq. 5), whereas the dashed trace is a fit to the Scatchard equation (noncooperative binding, Eq. 5 with n=1) and the dotted line to the Satake-Yang equation (Eq. 4). Biophysical Journal 2005 89, 4219-4233DOI: (10.1529/biophysj.105.065342) Copyright © 2005 The Biophysical Society Terms and Conditions

Figure 9 Expansion of the aromatic region of 1D 1H NMR spectra acquired at 500MHz for (A) 80mM (KF)n, (B) 80mM (KF)n+40mM β-C12G2, (C) 80mM (K4Y)n, and (D) 80mM (K4Y)n+40mM β-C12G2. All concentrations are given as amino acid residue concentrations. All solutions were prepared in 20mM deuterated acetate buffer, pD 5. Biophysical Journal 2005 89, 4219-4233DOI: (10.1529/biophysj.105.065342) Copyright © 2005 The Biophysical Society Terms and Conditions

Figure 10 Expansion of 1D 1H NMR spectra recorded at 500MHz showing the sugar proton resonances for (A) 40mM β-C12G2, (B) 40mM β-C12G2+80mM (KF)n, and (C) 40mM β-C12G2+80mM (K4Y)n. Hα peaks of lysine and phenylalanine are indicated by * and **, respectively, in B. Similarly, in C, * and ** indicate the Hα peaks of lysine and tyrosine. For the peptides, all concentrations are given as the amino acid residue concentrations. All solutions were prepared in 20mM deuterated acetate buffer, pD 5. Biophysical Journal 2005 89, 4219-4233DOI: (10.1529/biophysj.105.065342) Copyright © 2005 The Biophysical Society Terms and Conditions

Figure 11 Expansions of the 2D 1H-13C HSQC spectrum (top) and the 2D 1H NOESY spectrum, using a mixing time of 15ms (bottom), of a mixture of 80mM (KF)n and 40mM β-C12G2 solution in 20mM deuterated acetate buffer, pD 5. For peptides, all concentrations are given as the amino acid residue concentrations. The atom-specific assignment is indicated with the numbering presented in Fig. 12. The NOESY spectrum shows NOE crosspeaks between the aromatic ring protons of the phenylalanine residues and the protons in the aliphatic hydrocarbon chain of β-C12G2. Atom-specific assignments of the 1H peaks are presented in the corresponding regions of the 1D 1H spectrum displayed along the edges of the 2D spectrum. Biophysical Journal 2005 89, 4219-4233DOI: (10.1529/biophysj.105.065342) Copyright © 2005 The Biophysical Society Terms and Conditions

Figure 12 1H chemical shift difference for 40mM β-C12G2 in acetate buffer observed upon addition of 80mM (KF)n. (solid bars) and 9.3mM naphthylacetate (open bars). For peptides, all concentrations are given as the amino acid residue concentrations. Small chemical shift differences (≤∼0.04ppm) could simply be explained by subtle sample variations (e.g., pH and ionic strength) rather than significant peptide-surfactant interactions. Biophysical Journal 2005 89, 4219-4233DOI: (10.1529/biophysj.105.065342) Copyright © 2005 The Biophysical Society Terms and Conditions

Figure 13 (A) Number of bound β-C8G1 molecules per (KF)n monomers (β, ×) as determined by equilibrium dialysis and the fraction of (KF)n in α-helix conformation (Xα, ♦; determined by CD spectroscopy) as a function of free β-C8G1 concentration [Sfree]. (B) The number of bound β-C8G1 molecules per (KF)n amino acid monomers (β) as a function of the fraction of (KF)n in α-helix conformation (Xα) plotted for the same free β-C8G1 concentration. Biophysical Journal 2005 89, 4219-4233DOI: (10.1529/biophysj.105.065342) Copyright © 2005 The Biophysical Society Terms and Conditions

Figure 14 Schematic view of the surfactant-peptide binding. The upper left panel illustrates the surfactant micelle-peptide binding above CMC, yielding an increased fraction of peptide in α-helix conformation. The enlargements of the β-C12G2 micelle and the (KF)n-β-C12G2 complex (lower left and right) are drawn to scale. Previously reported (72,73) dimensions of the oblate ellipsoid β-C12G2 micelle have been used. Thus, the radii of the hydrocarbon region (rsmall=14.1Å and rlarge=28.2Å) and the thickness of the headgroup region (6.2Å) as well as the micelle aggregation number (132, assuming an unchanged aggregation number upon peptide binding) are taken from literature data on the “free” micelle. In the lower left panel, the peptide-micelle complex is sketched based on the NMR results and by using the number of amino acid (3.6) and the pitch (5.4Å) per α-helix turn (74), and an approximate amino acid side-chain length (for a fully stretched chain) of 6Å, assuming that the side-chain length of phenylalanine is the same as for lysine side chain as presented in the literature (75). For clarity, only part of the micelle and the peptide chain is drawn for the peptide-micelle complex in the lower left panel. Biophysical Journal 2005 89, 4219-4233DOI: (10.1529/biophysj.105.065342) Copyright © 2005 The Biophysical Society Terms and Conditions