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Influence of Phosphocholine Alkyl Chain Length on Peptide-Micelle Interactions and Micellar Size and Shape Christoph Go¨bl,† Martin Dulle,‡ Walter Hohlweg,† Jo¨rg Grossauer,† S. Fabio Falsone,† Otto Glatter,‡ and Klaus Zangger*,† Department of Chemistry/Organic and Bioorganic Chemistry and Department of Chemistry/Physical Chemistry, UniVersity of Graz Heinrichstrasse 28, A-8010 Graz, Austria ReceiVed: December 1, 2009; ReVised Manuscript ReceiVed: February 9, 2010
The interaction with biological membranes is of functional importance for many peptides and proteins. Structural studies on such membrane-bound biomacromolecules are often carried out in solutions containing small membrane-mimetic assemblies of detergent molecules. To investigate the influence of the hydrophobic chain length on the structure, diffusional and dynamical behavior of a peptide bound to micelles, we studied the binding of three peptides to n-phosphocholines with n ranging from 8 to 16. The peptides studied are the 15 residue antimicrobial peptide CM15, the 25-residue transmembrane helix 7 of yeast V-ATPase (TM7), and the 35-residue bacterial toxin LdrD. To keep the dimension of the peptide-membrane-mimetic assembly small, micelles are typically used when studying membrane-bound peptides and proteins, for example, by solution NMR spectroscopy. Since they are readily available in deuterated form most often sodiumdodecylsulfate (SDS) and dodecylphosphocholine (DPC) are used as the micelle-forming detergent. Using NMR, CD, and SAXS, we found that all phosphocholines studied form spherical micelles in the presence and absence of small bound peptides and the diameters of the micelles are basically unchanged upon peptide binding. The size of the peptide relative to the micelle determines to what extent the secondary structure can form. For small peptides (up to ∼25 residues) the use of shorter chain phosphocholines is recommended for solution NMR studies due to the favorable spectral quality and since they are as well-structured as in DPC. In contrast, larger peptides are better structured in micelles formed by detergents with chain lengths longer than DPC. Introduction Membrane-bound peptides and proteins play a key role in most biological processes. The determination of high-resolution structures of membrane proteins is still quite difficult both for X-ray crystallography and NMR spectroscopy. Less than 2% of all structures in the Protein Data Bank (PDB) are membranebound proteins although they account for about 30% of all proteins. A series of detergents have been used to solubilize membrane-bound proteins and peptides in functional form.1-3 Structural studies of membrane-bound peptides have been carried out almost exclusively by NMR spectroscopy. For solution-state NMR spectroscopy the size of the molecular assembly to be studied has to be kept small enough to avoid excessive line broadening. Therefore, small micelles are typically employed for such studies. Most commonly SDS (sodium dodecyl-sulfate) or DPC (dodecyl-phosphocholine) has been used due to their commercial availability in deuterated form. SDS is a negatively charged detergent that has been used mainly in studies of antimicrobial peptides as a model for bacterial membranes.4-7 However, it was also shown that it has the potential to influence the secondary structure, especially when studying larger peptides or proteins3 and it can induce secondary structures that were not found in DPC.8 On the other hand the zwitterionic DPC9 structurally resembles the components of major eukaryotic biological membranes. It can well preserve * To whom correspondence should be addressed. E-mail: klaus.zangger@ uni-graz.at. Tel: ++43 316 380-8673. Fax: ++43 316 380-9840. † Department of Chemistry/Organic and Bioorganic Chemistry. ‡ Department of Chemistry/Physical Chemistry.
the 3D structure of bound peptides10-12 and proteins13 as well as the catalytic activity of membrane-bound enzymes.14,15 The effect of a series of different phospholipids on the structure and self-diffusion of a membrane-bound peptide has been studied by Keifer et al.16 They found that while there is little effect on the structure of a membrane-bound helical peptide the diffusion is in general faster when using shorter chain length lipids. In contrast the translational diffusion of the 39 kDa enzyme diacylglycerol kinase (DAGK) was almost invariant in different types of detergents.15 In this case the large size of the protein is obviously the dominant factor. To better understand the peptide-micelle interactions as a function of hydrophobic chain length we investigated structural and dynamic properties of three membrane-binding peptides in a series of phosphocholine micelles by varying the alkyl-chain length between 8 and 16 carbons. Therefore, the peptides CM15 (a 15 residue antimicrobial peptide), TM7 (the 25 residue peptide which constitutes the seventh transmembrane helix of subunit a of the yeast V0H+-ATPase), and LdrD (a 35 residue toxic component of a bacterial toxin-antitoxin system) were studied by NMR, circular dichroism (CD), and small-angle X-ray scattering (SAXS) to obtain information about their secondary structure, translational diffusion, and transverse relaxation as well as size and shape of the micelles in the presence and absence of the peptides. For all three peptides tested, the self-diffusion coefficient increases steadily by going to smaller chain length detergents and at the same time the transverse relaxation slows down. This higher mobility in solution is corroborated by the smaller micelle size found by SAXS. Since the secondary structure and NMR
10.1021/jp9114089 2010 American Chemical Society Published on Web 03/12/2010
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spectral dispersion is little or not affected, smaller chain length detergents are advantageous for NMR studies of micelle-bound peptides. Especially for peptides, which are usually prepared by solid-phase peptide synthesis where isotopic labeling is prohibitively expensive, smaller and thus more mobile micelles are essential for solution-state NMR studies. There are contradicting reports about detergent and lipid micelle shapes in the literature. While some molecular modeling studies indicated spherical shapes for phosphocholine micelles17 others postulated prolate ellipsoids.2 Our SAXS data show that phosphocholines with hydrophobic chain lengths between 10 and 16 carbon atoms can be described to a very good approximation by spherical micelles whose radii are almost unchanged upon binding of a peptide. The radii obtained from SAXS measurements correlate very well with the ones obtained by NMR self-diffusion measurements.
ln(I) ) a - bdG2
where I ) signal intensity, G ) the gradient strength, and D ) self-diffusion coefficient. All other parameters were combined to the instrument-dependent constants a and b, which are obtained through a calibration on a system where D is known. This calibration was carried out with a sample containing 90% H2O and 10% D2O and the temperature-corrected H2O diffusion coefficient of 2.49 × 10-9 m2/s.18,19 A solution of a micelleforming detergent contains free-detergent molecules at a concentration of the cmc according to the phase separation model. Therefore, the diffusion coefficient is a combination of the free detergent and the micelles. Since we are interested only in the latter, we need to subtract the diffusion of the free monomeric detergent from the one of micelles according to20
Experimental Section Materials. CM15 (KWKLFKKIGAVLKVL), TM7 (KKSHTASYLRLWALSLAHAQLSSKK), and LdrD (MTLAQFAMIFWHDLAAPILAGIITAAIVSWWRNRK) were made by FMOC-based solid-phase peptide synthesis and purchased from Peptide Specialty Laboratories GmbH (Heidelberg, Germany). Phosphocholines with chain lengths between 8 and 16 carbons, which will be called PC-8 to PC-16 from now on (and are also known as fos-choline-8 to fos-choline-16) were bought from Anatrace Inc. (Maumee, OH) at a purity level higher than 97%. All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) in the highest purity available. NMR Spectroscopy. All NMR spectra were acquired on a Bruker Avance DRX 500 MHz spectrometer using a TXI tripleresonance probe with z-axis gradients at 301.2 K. For all spectra, 8 k data points were acquired and the FID multiplied with a 60° phase-shifted squared sine-bell window function prior to Fourier transformation. The solvent signal was suppressed by excitation sculpting. A 2D NOESY spectrum consisting of 2048 × 512 (F2 × F1) data points was acquired for LdrD using again 60° phase-shifted squared sine-bell window function in both dimensions. The NOESY mixing time was 150 ms. Solutions of 0.6, 0.4, and 0.3 mM of CM15, TM7, and LdrD, respectively, were used in 50 mM KPi (pH 5.0) including 10% D2O and 0.02% sodium azide. The concentrations of the detergents were 200 mM of PC-8 and 100 mM of PC-10, PC-12, PC14, or PC16. A higher concentration had to be used for PC-8 due to its cmc of around 114 mM (Table 1). Proton T2 relaxation times were obtained from series of 1D CPMG spectra. Depending on the size of T2 different series of relaxation delays were used. For faster relaxing signals, we used nine relaxation delays of 4, 12, 20, 36, 52, 68, 84, 100, and 116 ms and for the more slowly relaxing ones delays of 4, 24, 48, 72, 100, 160, 200, 280, and 400 ms were used. The CPMG interpulse delay was 10 ms. Self-diffusion coefficients were obtained by measuring a series of nine PGSE spectra with variations of gradientstrengths. The signal intensity was least-squares fitted to the equation
(1)
(D )
[cmc] ( [total] )D ) [cmc] (1 - ( [total] ))
obs
Dmic
-
free
Dmic is the weighted diffusion coefficient for micelles only, Dobs corresponds to the measured diffusion coefficient, and Dfree is the self-diffusion coefficient of a detergent solution below the cmc. To use this equation, we assumed a two-site exchange model between free and micelle-bound detergent molecules. Taking into account the diffusion of free detergent has to be done only for PC-8 to PC-12 as due to the low cmc for higher chain length detergents the monomer contribution to the overall diffusion coefficient is negligible. The diffusion coefficient of the free detergent Dfree is obtained by measurements at various concentrations below the cmc and extrapolating to zero concentration.21 The hydrodynamic radii of the micelles were calculated from the selfdiffusion constants via the Einstein Stokes equation. CD Spectroscopy. Far-UV circular dichroism (CD) measurements were performed on a J-715 spectrapolarimeter with a PTC343 peltier unit (Jasco, Tokyo, Japan). Spectra were recorded at room temperature in 50 mM sodium phosphate buffer (pH 5.0), containing 0.02% sodium azide, using a 0.1 cm quartz cuvette, and a 0.12, 0.20, and 0.15 (CM15, TM7, and LdrD) mmolar peptide concentration, 200 mmolar PC-8 and 100 mmolar PC-12 and PC-16, respectively. Spectra were recorded with a response time of 1 s and a step resolution of 0.2 nm. Three scans were averaged to obtain smooth spectra. All spectra were background corrected. Density and Viscosity Measurements. The viscosity at 301.2 K was determined using a AMV 200 rolling ball microviscosimeter (Anton Paar, Graz, Austria) after calibration on an aqueous solution of known viscosity (0.848 ( 0.011 mPa · s). The dynamic viscosity was calculated via the equation
η ) ct∆F
(3)
TABLE 1: Sample Composition for NMR and SAXS Measurements alkyl chain length (number of carbons) 8 10 12 14 16
(2)
aggregation number32
CMC32 [mM]
MW (g/mol)
concentration [mM]
micelle/pept ratio CM15 | TM7 | LdrD
unknown ∼24 ∼54 ∼108 ∼178
114 11 1.5 0.12 0.013
295.4 323.4 351.4 379.5 407.5
200 100 100 100 100
>5.6 | > 9.8 | >14 6.8 | 9.8 | 13 3.2 | 4.2 | 6.9 1.6 | 2.5 | 3.2 0.9 | 1.3 | 2.0
Phosphocholine Alkyl Chain Length on Peptide-Micelle Interactions where η is the dynamic viscosity of the solution, c is a constant determined by calibration, t is the duration of the ball rolling, and ∆F is the density difference of the rolling ball and the solution. The density was measured using a DSA 48 vibrating tube densimeter (Anton Paar, Graz, Austria) after calibration with a water sample of known density (0.9964 g/mL). SAXS Measurements. The structural characterization of all micelles formed by phosphocholines (PC-8 to PC16) and in case of PC-8 and PC-12 with the addition of CM15 and TM7 was carried out at 298 K. The concentrations of the detergents and peptides were the same as in the NMR experiments. The SAXS equipment is composed of a SAXSess camera (Anton Paar, Austria), attached to a PW 1730/10 X-ray generator (Phillips) with a sealed-tube anode (Cu-KR wavelength of 0.154 nm). The generator was operated at 40 kV and 50 mA. The SAXSess camera is equipped with a focusing multilayer optics and a block collimator to obtain a monochromatic and intense primary beam with low background. A semitransparent beam stop is used to enable measurements of an attenuated primary beam for the exact definition of the zero-scattering vector and transmission correction. Samples were enclosed into vacuum-tight, reusable 1 mm quartz capillary to attain the same scattering volume and background contribution. The sample temperature was adjusted with a thermocontrolled sample holder unit (TCS 120, Anton Paar). The 2D scattering pattern was recorded with imaging plates or with a CCD detector and integrated into a onedimensional scattering function with SAXSQuant software from Anton Paar, Graz, Austria. The image plate reader was a Fuji BAS1800 (from Raytest, Straubenhardt, Germany). The CCD camera from Princeton Instruments (Trenton, NJ) is equipped with a PI-SCX fused fiber optic taper. The CCD camera features a 2048 × 2048 array with a pixel size of 24 × 24 µm (chip size: 50 × 50 mm) at a sample detector distance of 311 mm. All 2D scattering patterns were converted to one-dimensional scattering curves as a function of the magnitude of the scattering vector q ) (4π/λ)sin(θ/2), where θ is the total scattering angle. All scattering patterns were transmission corrected by putting the attenuated scattering intensity at q ) 0 to unity and corrected for the scattering of the sample cell and the solvent. To get the scattering patterns on absolute scale, water was used as a secondary standard.22 In case of monodisperse globular particle systems consisting of n particles in the unit volume, the q dependent total scattering intensity I(q) is in general given by
I(q) ) nP(q)S(q)
(4)
were P(q) is the averaged form factor of the globular particle and S(q) is the static structure factor describing the spatial distribution of the particles. The form factor P(q) is the Fourier transform of the PDDF, the pair distance distribution function p(r)
P(q) ) 4π
∫0∞ p(r) sinqrqr dr
(5)
The structure factor is the Fourier transform of the total correlation function h(r) ) g(r)-1 as
S(q) ) 1 + 4πn
∫0∞ [g(r) - 1]r2 sinqrqr dr
(6)
where g(r) is the pair correlation function. The SAXS data of the micelles as well as the micelle peptide mixtures were
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Figure 1. Structures of phosphocholines. The hydrophobic chain length varied between 8 and 16 saturated carbons.
evaluated using the generalized indirect Fourier transformation (GIFT) routine.23-25 The concept of this technique is the simultaneous determination of form and structure factor with very few assumptions about the system. In the present study, we were mostly interested in the form factors of our samples and therefore we used a very simple structure factor model of monodisperse hard spheres. This structure factor model yields not the correct values for volume fraction or interaction radii but still allows the calculation of accurate p(r) functions. Results and Discussion The peptides CM15 and TM7 are soluble and unstructured in aqueous solution with diffusion coefficients of 2.2 × 10-10 ( 8.7 × 10-12 m2/s and 2.3 × 10-10 ( 8.8 × 10-11 m2/s, respectively. Upon addition of DPC, CM15 and TM7 form helical structures and become immersed rather deeply into the micelles.26,27 LdrD is basically insoluble in water and its structure in DPC micelles could not be solved due to extensive linebroadening and signal overlap in the NMR spectra. However, the HR signals center around 4 ppm as typically found in R-helices. In addition some of the NOEs that could be assigned for LdrD (data not shown) are between protons that are 3-4 residues apart, which is also typical for R-helical segments.28 Because of extensive signal overlap of the peptides with the much more concentrated (nondeuterated) detergents PC-8, PC10, PC-14, and PC-16 (for the structure see Figure 1), it was not possible to obtain a useful number of NOEs for a complete structure determination of CM15 and TM7 in these micelles. However, the dispersion found in the amide-, and aromaticproton region is indicative of structured conformations for CM15, TM7, and LdrD in all detergents tested (Figure 2). The differences are usually most pronounced for PC-8 and in the case of LdrD larger differences are also found in the spectrum obtained in PC-16 solution. Information about the secondary structure content can also be obtained from CD measurements. The CD spectra (Figure 3) show varying degrees of R-helical structure formation of the three peptides in the series of detergent solutions. While there is no standard method to calculate the exact secondary structure content of short peptides containing aromatic groups29 the normalized CD spectra clearly show that the smallest peptide CM15 exhibits a high degree of R-helical structure in PC-8, PC-12, and PC-16 micelles. R-helical peptide and protein conformations are characterized by minima at 208 and 222 nm and a maximum at 190 nm. Because of the small size of CM15, even the relatively small PC-8 provides enough hydrophobic space to accommodate the helix of approximately 16 Å. TM7 shows similarly high helical content but the most pronounced R-helix is found in PC-12. This can be explained by the ideal radius of PC-12 micelles for the transmicellar orientation of TM7.27 The structured R-helical backbone is around 23 Å long. The terminal polar lysine residues are near the surface of the micelle and stabilize the position of the peptide. A larger or smaller micelle radius poses problems in binding the helix in the same orientation and thus obviously disturbs its secondary structure to some extent. The R-helical structure of LdrD which is indicated by NMR data is cor-
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Figure 2. Low field regions of 1H spectra showing the aromatic and amide region of CM15, TM7, and LdrD.
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TABLE 2: Self-Diffusion Coefficients in m2/s × 1010 PC-8 PC-10 PC-12 PC-14 PC-16
CM15
TM7
LDR
1.78 ( 0.06 1.36 ( 0.05 1.12 ( 0.04 0.92 ( 0.02 0.82 ( 0.02
1.77 ( 0.05 1.21 ( 0.20 1.15 ( 0.01 0.99 ( 0.02 0.74 ( 0.17
1.76 ( 0.16 1.45 ( 0.10 1.18 ( 0.09 0.97 ( 0.04 0.87 ( 0.05
TABLE 3: Hydrodynamic Radii (in Å) Obtained from NMR Self-Diffusion Coefficients and the Radii of the Two Step Models from the SAXS Measurements PC-8a PC-10 PC-12 PC-14 PC-16
CM15
TM7
LdrD
MIC only
SAXS
14.3 ( 1.0 19.2 ( 0.9 23.1 ( 1.0 28.2 ( 0.9 32.5 ( 1.1
14.3 ( 0.9 20.9 ( 3.6 22.6 ( 0.4 26.2 ( 0.8 36.6 ( 3.0
14.5 ( 2.5 18.0 ( 1.2 22.0 ( 1.3 26.8 ( 1.4 29.9 ( 1.9
13.2 ( 2.2 22.6 ( 2.2 23.3 ( 1.4 29.6 ( 1.4 33.3 ( 1.5
22.5 27.0 32.0 34.0
a Spherical approximation used despite no SAXS data providing shape information.
the nondeuterated detergent and therefore the latter were used for obtaining the diffusion parameters. The validity of this approach was checked for PC-12 where the deuterated form is commercially available. The self-diffusion coefficient obtained on the peptide signals of CM15 and TM7 in DPC-d38 are within experimental error identical to the ones obtained on nondeuterated PC-12 after correction according to eq 2. The hydrodynamic radius can be obtained from the diffusion coefficient using the Einstein-Stokes equation
RH )
Figure 3. Far UV CD spectra of LdrD (top), TM7 (center), and CM15 (bottom) in PC-8 (dashed line), PC-12 (solid line), and PC-16 (dotted line) micelles. The estimated error of the molar ellipticity is around 10%.
roborated by the negative band at 208 nm in the CD spectra. For this larger peptide there is a clear tendency of increasing secondary structure by going to longer chain detergents. However, compared to CM15 and TM7 the molar ellipticity per residue even in PC-16 is rather low which points to less well-defined helical structures. To gain information about the micelle sizes we first determined NMR self-diffusion coefficients (see Table 2). Because of the much lower concentration of the peptides (typically ∼1 mM) compared to the detergents (200 mM for PC-8 and 100 mM for all others) the peptide signals were overwhelmed by
kT 6πηDeff
(7)
where RH represents the hydrodynamic radius, k the Boltzmann constant, T the temperature, η the dynamic viscosity, and Deff the effective diffusion coefficient. A value of 0.851 mPa · s was found for the viscosity of the buffer solution. The hydrodynamic radii of the micelles (Table 3) increases steadily by going to longer chain length detergents and the binding of the peptides has basically no influence on the micelle radius within experimental error. Thus the peptides seem to replace detergent molecules and the hydrodynamic radius of the overall complex stays approximately the same. Similar observations have been made for melittin in DPC9 and an amphipathic peptide bound to various phospholipids.16,30 However, it contradicts an analysis of a 21-residue peptide bound to DPC micelles,30 where volumes were obtained from an analysis of T1 and T2 relaxation times. In this case, we assume an altered flexibility of the detergent to be the reason for the differences in relaxation times rather than a change of the correlation time. Surprisingly even the larger peptides TM7 and LdrD can bind to smaller micelles. Therefore, the micelle size is determined mainly by the detergent used and only the amount of secondary structure decreases if a peptide binds to a micelle whose size is not optimal. For LdrD, we also determined the hydrodynamic radius in 100 mM PC-8 (under its cmc of 114 mM), and got 14.5 ( 1.4 Å, which is very similar to the value presented in Table 3 for 200 mM PC-8. This indicates that for this larger peptide the dissolved, “detergentcoated” form has a size similar to PC-8 micelles themselves, a situation similar to proteins bound to PC-12. For the determination of the micelle radius from NMR selfdiffusion coefficients the shape of the micelle is important. The information from the literature about the shapes of small
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Figure 4. PDDF (open squares), the fit corresponding to a two step core shell spherical model (full line), and the fit corresponding to a continuous core shell model using splines (dashed line) of PC-10 (a), PC-12 (b), PC14 (c), and PC-16 (d). Inset: electron density difference profiles corresponding to the fits
micelles, in particular phosphocholine micelles, is inconsistent. While small micelles are often referred to as spherical without experimental proof some molecular modeling studies indicated nonspherical shapes for phosphocholine micelles. This was corroborated by a recent experimental study2 that also found ellipsoidal shapes for phosphocholine micelles. However, Tieleman et al.17 found spherical shapes in their modeling studies and relaxation enhancements induced by a paramagnetic environment could also be well interpreted by assuming spherical micelle-peptide assemblies. The small-angle X-ray scattering curves of phosphocholines PC-8, PC-10, PC-12, PC-14, and PC16 were evaluated using the GIFT method. As mentioned above, the structure factor model used was the one for hard-sphere interaction, but this was appropriate for this study because the charges of the micelles were effectively screened by the salt present in the solution. Furthermore, we were only interested in the form factor of the micelles that could be determined in all cases. The PDDFs of all samples show the typical characteristics of an inhomogeneous globular aggregate with opposite sign in the contrast for the core and the shell. The core of micelles in aqueous systems has a negative contrast with respect to the solvent. For the micelles without any polypeptides added, the PDDFs and the corresponding spherical fit from a two step model as well as from a continuous core shell model using splines for the radial difference electron density distribution are in very good agree-
ment (Figure 4). Only for the smallest of the phosphocholines (PC-8) we were not able to fit the PDDF with a spherical model. But this is not surprising as the aggregation number can be considered to be very low in this case. This may give rise to a nonspherical but still globular object. But apart from that the PDDF of the PC-8 micelles also show the same characteristics as those formed by the other phosphocholines. To see if and to what extent the shape and size of the micelles are influenced by the addition and incorporation of polypeptides, two phosphocholines (PC-8 and PC-12) were investigated in the presence of polypeptides. In case of PC-8, the polypeptides CM15 and TM7 were investigated. The PDDFs of all three systems, PC-8, PC-8 + CM15, and PC-8 + TM7 are shown in Figure 5a. In this special representation, the height of all three functions was normalized with respect to their maximum value. It can be seen immediately that the maximum dimension in all three cases is nearly the same and also the positions of the maxima and minima on the abscissa are only slightly shifted. This is a strong indication that the shape and size of the micelles is not changed considerably by addition of the polypeptides. It can also be seen in Figure 5a that the height decreased, that is, the amplitude of the first maximum and minimum in case of added polypeptides is more pronounced in case of TM7 as it is the longer one. This means that the polypeptides are incorporated into the micelles and therefore the contrast of the core relative to the solvent is decreased as the polypeptides have a higher
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Figure 6. Scattering curve of PC10 (a) and PC12 (b) on absolute scale (open squares), the fit from GIFT corresponding to the respective PDDF in Figure 4 (full line) and the fit to the spherical model (dashed line).
Figure 5. The PDDFs of PC 8 (red dots), PC-8 + CM15 (green stars), and PC-8 + TM7 (blue triangles) is shown in (a) and the PDDFs of PC 12 (red dots), PC-12 + CM15 (blue triangles) in (b), all are normalized with respect to the second maximum. The PDDF of PC-12 + CM15 (open squares), the fit corresponding to a two step core shell spherical model (full line), and the fit corresponding to a continuous core shell model using splines (dashed line) is given in (c). Inset: electron density difference profiles corresponding to the fits.
electron density than the alkyl chains. For PC-12 only the effects of addition of CM15 were investigated. Here a very similar behavior as for the PC-8 + CM15 case can be seen (Figure 5b). The first maximum as well as the minimum have decreased in amplitude but to a much smaller extent. This is to be expected as the aggregation number of PC-12 is much higher than that
of PC-8. In addition, it was also possible to fit the PDDF of PC-12 + CM15 with a spherical two step model (Figure 5c) that strengthens the conclusion that upon addition of a polypeptide no major size and or shape changes in the micelles are induced. Our results contradict a recent report2 on the size and shape of two of the surfactants, PC-10 and PC-12. Therefore, we also tried a very similar approach and fitted the scattering curves directly. But also these results (Figure 6) show spherical symmetry of the micelles. Furthermore the length of the head groups from the direct fit was very similar to those that were obtained by deconvolution and also to the findings of Yaseen et. al31 obtained from neutron reflectivity measurements of PC12 on the water air interface. Information related to the rotational correlation time (the size) of the peptide-micelle complex can also be obtained from NMR transverse relaxation time (T2) measurements. Because of the strong signals from the detergents we used well separated, lowfield aromatic resonances for obtaining averaged T2 times of nonexchanging signals (Figure 7). A comparison between the different peptides is not meaningful as the individual T2 values depend on the specific aromatic signals that were used for the analysis and their dynamical behavior. However, it can be easily seen that for all peptides the T2 times continuously decrease by using longer chain length detergents. This can be explained by
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Go¨bl et al. in the micellar shape and size was found upon the addition of the polypeptides. The transverse relaxation slows down and the NMR signal dispersion is almost unchanged by using smaller chain length detergents. Thus, for small- to medium-sized peptides the use of smaller detergents, which are currently unfortunately not commercially available, would be warranted for solution NMR studies. For larger peptides longer hydrophobic chain detergents would be necessary for structural studies. Acknowledgment. Funding by the Austrian Science Foundation (FWF) under project numbers 20020 to K.Z. and through the Doktoratskolleg “Molecular Enzymology” is gratefully acknowledged. C.G. wants to express his gratitude to the ¨ AW) for a DOC scholarship. Austrian Academy of Sciences (O References and Notes
Figure 7. Transverse (T2) relaxation times of aromatic region signals of CM15 (white), TM7 (hatched), and LdrD (black).
the higher tumbling rates of smaller micelles and is also manifested by the narrower lines found in the NMR spectra in PC-8 and PC-10 (see Figure 2). Thus there is a clear advantage of using shorter chain length detergents as long as the peptide still has enough space for the formation of its 3D structure. Besides sharper spectral lines, the longer transverse relaxation times found in shorter detergents are even more important for the acquisition of two- and multidimensional NMR experiments where any loss of magnetization during the pulse-sequence translates directly into lower intensity of peaks in the resulting spectra. Considering the high degree of structure in the CD spectra and the much longer T2 times for small- to mediumsized peptides like CM15 and TM7, the use of PC-8 or PC-10 should be advantageous for NMR studies. Therefore, the commercial availability of deuterated phosphocholines of varying chain lengths would be desirable for NMR studies. On the other hand for LdrD, which could not be structurally characterized in PC-12 due to extensive signal overlap and fast relaxation the use of short phosphocholines would likely not yield a stable structure and one would rather need deuterated longer chain detergents and use isotopically labeled peptide. The peptides used for this study all form helical structures and although CM15 is amphipathic, they are all well embedded in the micelles. However, it has been recently shown that the cyclic disulfide forming peptide Kalata B7 binds to the surface of DPC micelles and increases its radius by ∼3 Å.11 Such a change in the micelle radius likely also has an influence on choosing the optimal membrane-mimetic. Conclusions We have determined the sizes, shapes, influences on the structure, and NMR-relevant parameters of phosphocholines with alkyl chain lengths between 8 and 16 carbons on three membrane-binding peptides with 15, 25, and 35 residues. For all peptides tested, the size of the micelles is only determined by the hydrophobic chain length of the detergent but the size of the peptide determines how well its secondary structure can form. Therefore, we also determined the shapes as well as radii of the micelles with and without peptides by SAXS. The radii correlate very well with the radii obtained by the NMR measurements and there is basically no change by adding the peptides. The shapes of all but one of the investigated phosphocholine micelles were found to be spherical and no change
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