Positioning of Micelle-Bound Peptides by Paramagnetic Relaxation

Mar 3, 2009 - Positioning of Micelle-Bound Peptides by Paramagnetic Relaxation ... Physical and Theoretical Chemistry, Graz UniVersity of Technology, ...
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J. Phys. Chem. B 2009, 113, 4400–4406

Positioning of Micelle-Bound Peptides by Paramagnetic Relaxation Enhancements Klaus Zangger,*,† Michal Respondek,† Christoph Go¨bl,† Walter Hohlweg,† Kenneth Rasmussen,‡ Gu¨nter Grampp,‡ and Tobias Madl†,§ Institute of Chemistry/Organic and Bioorganic Chemistry, UniVersity of Graz, Graz, Austria and Institute of Physical and Theoretical Chemistry, Graz UniVersity of Technology, Graz, Austria ReceiVed: September 25, 2008; ReVised Manuscript ReceiVed: December 18, 2008

Many peptides, proteins, and drugs interact with biological membranes, and knowing the mode of binding is essential to understanding their biological functions. To obtain the complete orientation and immersion depth of such a compound, the membrane-mimetic system (micelle) is placed in an aqueous buffer containing the soluble and inert paramagnetic contrast agent Gd(DTPA-BMA). Paramagnetic relaxation enhancements (PREs) of a specific nucleus then depend only on its distance from the surface. The positioning of a structurally characterized compound can be obtained by least-squares fitting of experimental PREs to the micelle center position. This liquid-state NMR approach, which does not rely on isotopic labeling or chemical modification, has been applied to determine the location of the presumed transmembrane region 7 of yeast V-ATPase (TM7) and the membrane-bound antimicrobial peptide CM15 in micelles. TM7 binds in a trans-micelle orientation with the N-terminus being slightly closer to the surface than the C-terminus. CM15 is immersed unexpectedly deep into the micelle with the more hydrophilic side of the helix being closer to the surface than the hydrophobic one. Introduction Interactions with biological membranes is an intrinsic property of many peptides, proteins, natural compounds, and drugs.1-3 The orientation and location of such compounds in the membrane are key in determining their function and activity. Due to the flexibility of small membrane-bound peptides, their atomic resolution three-dimensional (3D) structure can essentially only be determined by solution NMR spectroscopy. To keep the dimension of the peptide-membrane-mimetic assembly below the NMR size limit, micelles are typically used for such studies.4,5 Although the 3D atomic resolution structure of small micelle-bound ligands can be obtained routinely by high-resolution NMR spectroscopy, investigating the orientation, and especially immersion depth, within the micelle presents a bigger challenge. Methods are available for probing either the immersion depth6-9 or the orientation10,11 of preferably helical peptides bound to micelles. However, most of them depend on isotopic labeling of the ligand and/or chemical modifications of the micelle-forming detergents or lipids and often provide only a rough estimate of the immersion depth. Here we present a quantitative solution-state NMR method for obtaining both the complete orientation and immersion depth of structurally characterized molecules in a micelle without any chemical perturbation through the use of relaxation enhancements exerted by a soluble paramagnetic agent. Instead of using a localized (and covalently attached) paramagnetic center as typically employed for obtaining structurally relevant information, we use the freely soluble gadolinium-diethylenetriamine pentaacetic acid-bismethylamide Gd(DTPA-BMA). Other known water* To whom correspondence should be addressed. E-mail: klaus. [email protected]; phone: ++43 316 380-8673; fax: ++43 316 3809840. † University of Graz. ‡ Graz University of Technology. § Current address: Department Chemie, Technische Universita¨t Mu¨nchen, Germany.

soluble paramagnetic agents, for example Ni2+ or Mn2+ ions and nitroxide spin-labels like TEMPOL, can interact with certain groups on proteins.12,13 In contrast, Gd(DTPA-BMA) was shown to be inert in aqueous solution,14,15 and we did not find any indications of specific binding to micelles or peptides. In addition, the stronger paramagnetism of gadolinium versus nitroxide15 allows the use of lower concentrations to achieve the same relaxation enhancements. If the paramagnetic compound does not penetrate into the micelle, then one can describe this situation as making the solvent around the micelle paramagnetic. Experimental relaxation enhancements, which are proportional to the third power of the immersion depth, can be least-squares fitted to obtain the proportionality constant and the location and orientation within the micelle, or in other words, the position of the micelle center relative to the peptide. In cases where the number of PREs does not enable a stable least-squares fitting, the proportionality constant obtained on a larger system can be used. The positions of two membrane-bound peptides in DPC micelles were determined with this approach: the transmembrane helical peptide TM7 of yeast V-ATPase and the antimicrobial peptide CM15. Thus, a few fast experiments enable the determination of the location within the micelle without the need for covalent modification or isotopic labeling. Experimental Methods Materials. CM15 (KWKLFKKIGAVLKVL) and TM7 (KKSHTASYLRLWALSLAHAQLSSKK)weremadebyFMOCbased solid-phase peptide synthesis and were purchased from Peptide Specialty Laboratories GmbH (Heidelberg, Germany). DPC-d38 (98%) was from Cambridge Isotope Laboratories Inc. (Andover, MA). Gd(DTPA-BMA) was purified from the commercially available MRI contrast agent Omniscan (Nycomed, Oslo, Norway) as described in ref 11. All other chemicals were bought from Sigma-Aldrich (St. Louis, MO) in the highest purity available.

10.1021/jp808501x CCC: $40.75  2009 American Chemical Society Published on Web 03/03/2009

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NMR Spectroscopy. All 2D NMR spectra (NOESY and TOCSY for determining the structure of TM7 and the PRE values) were acquired on a Bruker Avance DRX 500 MHz spectrometer using a TXI triple-resonance probe with z-axis gradients at 304 K. Samples of 1.2 mM TM7 dissolved in 100 mM DPC-d38, 50 mM phosphate buffer (pH ) 5.0) including 10% D2O, and 0.02% sodium azide were used. To obtain paramagnetic relaxation enhancements, the samples were titrated with Gd(DTPA-BMA) (60 mM) to final concentrations of 0.5 1, 1.5, 2, 3, 4, and 5 mM. Proton T1 relaxation times were obtained from a series of 2D NOESY or TOCSY spectra with a saturation recovery sequence at the beginning as described for CM15.11 For each series the delays between the saturation and start of the actual 2D sequence were 100, 300, 500, 700, 1000, 1500, 2000, and 3000 ms. PREs of samples containing a doxyl derivative were obtained by dissolving 16-doxyl-stearic acid at a concentration of 90 mM in methanol. Twenty microliters of this solution were evaporated to dryness and then dissolved in 600 µL of the NMR sample, giving a final concentration of about 3 mM 16-doxyl-stearic acid in the presence of 100 mM DPC. The relaxation rates of this sample were obtained as for Gd(DTPA-BMA) and were compared to the sample without any paramagnetic compound to obtain the doxyl-derivative relaxation enhancements. For the assignment and solution structure determination of TM7 we used TOCSY and NOESY spectra with mixing times of 40 and 150 ms, respectively. The solvent signal was suppressed using two excitation sculpting blocks before the start of the acquisition. Partial assignment of 13C chemical shifts was accomplished with a 1H-13C HSQC on unlabeled peptide for which 352 scans were acquired for each of the 256 increments. All spectra were processed using nmrPipe16 and were analyzed by NMRViewJ.17 The hydrodynamic radii of the micelles were calculated from self-diffusion coefficients. The latter were obtained by measuring a series of stimulated-spin-echo pulse sequences with variations of gradient-strengths. The signal intensity decay was leastsquares fitted to the following equation

ln(I) ) a - bDG2

(1)

where I is the signal intensity, G is the gradient strength, D is the self-diffusion coefficient, and the constant b is obtained through a calibration on a system where D is known. For this calibration we used a sample containing 90% H2O/10% D2O and the temperature-corrected H2O diffusion coefficient of 2.1 × 10-9 m2/s.18,19 To measure the diffusion coefficient of only the micelles that contain a peptide molecule we used intensities of peptide signals rather than of DPC itself. Structure Calculation of TM7. NOESY cross peaks of TM7 were assigned manually; the peak volumes were integrated with the program NMRViewJ and translated into distance restraints using the built-in median method. Additionally, φ and ψ dihedral angle restraints were obtained using the program TALOS,20 based on HR proton as well as CR and Cβ carbon chemical shifts. A total of 397 NOEs and 20 dihedral angle restraints were used for the structure determination. The structure calculation was carried out with the program CNS21 using the full simulated annealing method. ESR Spectroscopy. ESR measurements were performed on a Bruker ELEXSYS 500 continuous wave spectrometer working at 9.6 GHz (X band). The modulation frequency and amplitude were 100 kHz and 0.05-0.2 mT, respectively, and the applied microwave power was 2 mW. Samples of 10 mM Gd(DTPABMA) in the presence and absence of 100 mM DPC in 50 mM

KPi (potassium phosphate) pH 6.5 were sealed off under vacuum before ESR spectra were acquired. Theory The paramagnetic relaxation enhancements are exerted by an inert and freely soluble paramagnetic agent, which does not penetrate into the micelle.11 For this system the “second sphere interaction” model22 best describes the paramagnetic contribution to the longitudinal relaxation rates, which are given by:

R1M )

( )

(

2 2 7τc 3τc 2 µ0 2 γI (geµB) J(J + 1) + 2 2 6 15 4π r 1 + ωHτc 1 + ωS2τ2c

)

(2)

Here, R1M is the contribution of a single paramagnetic center to R1. The other parameters are: µ0, magnetic permeability of the vacuum; γI, gyromagnetic ratio of the proton; ge, free electron g value; µB, electron Bohr magneton; J, total angular momentum quantum number (due to the strong spin-orbit interaction in Gd, the spin quantum operator S has to be replaced by the total angular momentum operator J); τc, correlation time; ωH, proton Larmor frequency; and ωS, electron frequency. The correlation time τc is given by15

1 1 1 1 ) + + τc T1e τM τr

(3)

where T1e is being the electron relaxation time (10 ns), τM is the lifetime of an intermolecular adduct between Gd(DTPABMA) and the micelle, and τr is the rotational correlation time. Because we will be only interested in the 1/r6-dependence, all other parameters are combined into a single constant. To get the overall relaxation enhancement induced by the combined effect of all paramagnetic centers we then use the integrated form of eq 211 and include the concentration into the proportionality constant giving

PRE )

z d3

(4)

where d is the distance (in Å) between a specific nucleus and the closest paramagnetic center (i.e., the immersion depth within the micelle plus the radius of the paramagnetic probe). This equation is the exact solution for a planar surface. However, it also provides a good approximation for all immersion depths in micelles with radii typically used for solution NMR studies.11 For the positioning of micelle-bound molecules we need to know the proportionality constant “z”. For its calculation through eq 2 we would need the correlation time τc, which depends on the electron relaxation time of gadolinium, the rotational correlation time of the micelle, and the lifetime of the weak intermolecular adduct between the micelle and the paramagnetic probe. Although a value for τc of 0.5 ns was found by Pintacuda and Otting15 for proteins, it cannot be assumed a priori that the correlation time of the micelle-Gd(DTPA-BMA) system is the same as between a protein and Gd(DTPA-BMA). Due to the mobility of the micelle-forming detergent molecules, z cannot be obtained by calibration on detergent signals either. To obtain z together with the location in a micelle, we used the 25-residue structurally characterized transmembrane helix 7 of yeast V-ATPase (TM7).23 PREs of this helix show large variations

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along the peptide chain, having high values at the termini and much smaller ones in the middle. The immersion depth d of each peptide nucleus is given by d ) R - r, where R is the effective radius of the micelle (including the solvent layer and the radius of the paramagnetic probe) and r is the distance between the center of the micelle and the individual peptide proton. The radius of the micelle was obtained by NMR selfdiffusion measurements. We then carried out a least-squares fit of eq 4 for all possible micelle center coordinates of the transmembrane peptide. Due to the large variations in PREs for this peptide, the least-squares fitting procedure was very stable and yielded the coordinates of the micelle-center relative to the peptide as well as the z value. Once z and the radius of the micelle is known, the orientation and immersion depth of any structurally characterized peptide can be obtained by looking for the position of the peptide in the micelle that gives the lowest deviation of calculated versus measured PREs. For compounds showing smaller variations of PREs, the knowledge of z makes the least-squares fitting procedure much more stable. To find the center of the micelle, a grid is placed around the peptide. Each grid point is then assumed to be the center of a micelle. Theoretical PREs are calculated (PREcalc), and the sum of squared deviations between calculated and experimental PREs (eq 5) is used as a criterion for the quality of the fit.

χ)

∑ (PREcalc - PREexp)2

(5)

Figure 1. Stereo diagrams of least-squares fitted bundle of the 17 lowest energy structures of TM7 bound to DPC micelles. The backbone is shown in panel a, and side-chain nuclei are included in panel b.

The index i runs over all protons for which PREs could be determined. The grid point that gives the lowest χ value is the center of the micelle. A standalone computer program called “Parapos” (for paramagnetic positioning) was written that carries out a least-squares fit of eq 4 for each grid point in a cube with side lengths of 50 Å around the peptide. Parapos can be downloaded at www.uni-graz.at/nmr.

specific interactions with the peptide or the micelle. Due to faster relaxation upon the addition of the paramagnetic agent, the signal intensities decayed, but the resolution was still good enough to allow the extraction of relaxation enhancements (see Figure 2). At each step of the titration a set of eight saturation-recovery 2D-NOESY and TOCSY spectra with differing delays between the saturation and start of the TOCSY or NOESY sequence were recorded. Only clearly separated peaks in these 2D data sets were used for the extraction of PREs. Exchangeable protons (NHs) were not used for the PRE analysis due to the unknown contribution of relaxation enhancements from already relaxed water protons. The PREs of TM7 (see the Supporting Information) are high at the termini and are much lower in the middle of the peptide, as expected for a transmicellar orientation. Beside the size, the shape of the micelles also determines the PREs. Molecular dynamics simulations pointed to spherical shapes,24 whereas other studies reported elliptical micelles.25 Small angle X-ray scattering and residual dipolar coupling NMR measurements carried out by us on DPC micelles containing a series of small membrane-bound peptides (up to 35 residues, including CM15 and TM7) show that these micelles are spherical.26 The radius of the micelle containing TM7 was determined by NMR self-diffusion measurements to be 22.6 ( 0.4 Å. This value is in good agreement with the hydrodynamic radii of typical DPC micelles found in the literature.27,28 The radius of Gd(DTPA-BMA) of ∼3.5 Å (measured from its structure with the program HyperChem) has to be added for the least-squares fitting. Due to the rather large variations in PREs, the micelle center position can be obtained together with the proportionality constant z by least-squares fitting of eq 4. To evaluate the stability of this fitting procedure for TM7, we randomly deleted individual PREs from the initial list of 50 PREs and subsequently calculated the changes in the obtained z-value, the micelle center coordinate, and the sum of squared deviations χ (eq 5). As shown in Figure 3, the standard deviation

i

Results and Discussion Upon dissolving TM7 in an aqueous buffer it showed poor chemical shift dispersions typical of random coil peptides. Addition of DPC-d38 led to large shift changes up to concentrations of around 70 mM DPC. To ensure that all of the peptide is bound to micelles, a concentration of 100 mM DPC was subsequently used. Proton signals of TM7 were assigned using TOCSY, NOESY, and 1H-13C-HSQC spectra. The solution structure was determined using 397 NOEs together with 20 dihedral angle restraints that were obtained using the program TALOS with chemical shifts of HR, CR, and Cβ nuclei. During a later stage in the structure refinement C′O to NH hydrogen bond restraints were introduced for residues being involved in the R-helix based on their typical NOE pattern, chemical shifts, and TALOS-derived φ and ψ angles. A total of 50 structures were calculated, and the 17 lowest energy structures of TM7 are shown in Figure 1 as a least-squares fit bundle showing only the backbone (Figure 1a) or also the side-chain nuclei (Figure 1b). An R-helix is formed for the most part with the first and last residues being less well-defined. The rmsd between residues 2-24 is 0.28 Å and 1.03 Å for the backbone and side chain atoms, respectively. The structure has been deposited in the PDB database under accession number 2JTW. To obtain PRE values of TM7, a titration of the peptide-DPC micelle complex with Gd(DTPA-BMA) was performed. Upon addition of Gd(DTPA-BMA) to final concentrations of up to 5 mM, no shifts of resonances were detected, indicating the absence of

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Figure 3. Changes in the z-value (black circles) and the sum of leastsquares deviations χ (gray triangles) during the least-squares fitting by randomly deleting individual PREs of TM7.

Figure 4. Orientation and location of TM7 in DPC micelles. Protons for which PREs could be unambiguously determined are drawn colorcoded with PREs going from blue (low values) to red (high PREs). The center of the micelle is indicated by a large green sphere. The radius of the micelle is drawn to scale, and the radius of the closest approaching paramagnetic centers is depicted by a dotted line. The micelle is indicated by blue DPC molecules with the phosphate groups colored red.

Figure 2. Expansions from TOCSY spectra of TM7 for various concentrations of Gd(DTPA-BMA). The spectra are drawn using the same vertical scale.

of z increases steadily upon reducing the numbers of PREs but yields consistent z values around 2600 Å3 s-1 mM-1 for the most part. Accordingly, the squared deviations are almost unchanged by the random deletion of up to ∼15 PREs. In addition, the micelle-center position remains within a space element of 1 Å3 by reducing the PREs down to 37 and only moves more than 2 Å when the number of PREs is reduced to less than 27. It should be noted that, because of the transmicellar orientation, there are large variations in PREs along the peptide

chain, which makes the least-squares fitting more robust compared to an orientation parallel to the surface. The lowest square deviations were obtained if the center of the micelle is placed close to residue 12, giving a z ) 2647 Å3 s-1 mM-1. The resulting positioning of TM7 is shown in Figure 4, where the micelle center is indicated by a large green sphere. The N-terminus is slightly closer to the surface of the micelle compared to the C-terminus. The micelle is drawn to scale, and the position of the closest approaching paramagnetic centers (radius of the micelle plus the radius of the paramagnetic molecule) is depicted by a dotted line. The structure and the location of TM7 gives clear experimental evidence that this peptide stretch corresponding to residues starting at V727 in the full membrane-bound subunit a of yeast V-ATPase forms a transmembrane helix, which is in contrast to an older model29 suggesting that R735 in the V-ATPase a subunit (R10 of TM7) is close to the lumen. However, it perfectly correlates with a newer model30 suggesting that R735 is right within a transmembrane helix. Arg-735 is essential for the proton transport process of yeast V-ATPase.30,31

4404 J. Phys. Chem. B, Vol. 113, No. 13, 2009 Rather low PREs were found for protons of R10 of TM7 (PREs: 0.39 for R10-HR, 0.60 for R10-Hδ and 0.64 for R10-Hε). In the presence of 16-doxyl stearic acid R10-Hδ has the highest PRE (21.9), thus confirming its deep immersion in the micelle. They indicate that even the side chain of this arginine residue is close to the center of the micelle and does not snorkel to the surface as previously hypothesized.23 However, the determination of pKa values of membrane-immersed amino acids is a considerable problem; two independent computational studies32,33 recently found that the side-chain pKa values of arginine residues decrease with increasing immersion depth in a membrane. For the core of a bilayer, consistent pKa values for the side chain guanidino group around neutral have been found, whereas this value is 12.48 in an aqueous environment. The location of R10 of TM7 as found with PREs thus points to a pKa value of this residue being close to neutral. This drastically reduces the energy needed for proton translocation in V-ATPase, which involves frequent protonation transfers to and from R735. In this process it is assumed that protons can move from the cytoplasmic space into hemichannels formed in the a subunit to carboxyl groups (glutamate residues) in the c, c′, and c′′ subunits.31 After rotation of the c subunit ring, the acquired proton is released into the other hemichannel (in contact with the lumen) through interaction of the carboxyl groups with R735. Thus, the location of R735 close to the center of the membrane is important for the catalytic reaction for two reasons: (1) to be able to interact with glutamic acids on the c subunit, which are also close to the bilayer center30,31 and (2) to lower the pKa value in order to allow faster and less energy-consuming protonation and deprotonation during catalytic turnover. The z value obtained on TM7 can then be used together with the micelle radius for the least-squares fitting of the position and orientation of other compounds, like smaller peptides or ones that are bound closer to the surface. They are characterized by smaller variations of PREs and therefore give a less stable least-squares fit if both the coordinates of the micelle center and z were unknown. If we use the z-value determined for TM7 on another system we need to confirm that the influence of Gd(DTPA-BMA) on relaxation rates is the same. For this reason we measured PREs of the micelle-forming DPC protons for a free DPC micelle, a DPC micelle with TM7 bound, and one containing the peptide CM15. For all systems we measured basically identical DPC relaxation enhancements (see the Supporting Information). As an example of determining the orientation of a smaller peptide within a micelle we used the antimicrobial peptide CM15. CM15 (KWKLFKKIGAVLKVL) is a fifteen-residue hybrid peptide of cecropin A (residues 1-7) and mellitin (residues 2-9), which was recognized as the minimal sequence that displays antimicrobial activity.34 Although unstructured in aqueous solution, CM15 adopts an amphipathic helical structure upon addition of lipids. It is bound parallel to the surface between residues 4 and 14 and displays increased flexibility toward the N-terminus.11 The solution structure and PREs of CM15 are available from previous work11 (for PREs see the Supporting Information), and the latter show variations between the two sides of the helix with a periodicity of 3.6 residues. All measured PREs (backbone and side chain) of nonexchangeable protons of CM15 were used as input for the least-squares fitting. The effective micelle hydrodynamic radius as obtained by NMR self-diffusion measurements is again 22.6 ( 0.8 Å. The position of the center of the micelle relative to the peptide determines

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Figure 5. Orientation and location of CM15 in DPC micelles obtained from all measured PRE values using the program Parapos as explained in the text. The peptide is drawn as ball-and-stick models color-coded with PREs going from blue (low values) to red (high PREs). The center of the micelle is shown as a green sphere, and the radius of the closest approaching paramagnetic centers is depicted by a dotted line. The micelle is indicated by blue DPC molecules with the phosphate groups colored red.

the relative orientation and immersion depth. Figure 5 shows the micelle center obtained for CM15 using Parapos as a green sphere. CM15 is immersed quite deeply into the micelle, with the hydrophobic side of the helix being placed in the hydrophobic core of the micelle. This immersion of the majority of the peptide in the hydrophobic part of the micelle explains why hydrophobic amino-acid side-chains (residues Ala10 and Val14) can be on the “hydrophilic” side of an amphipathic helix. Furthermore, the polar sidechains on the hydrophilic side of the helix are all lysine residues. Due to their lengths, the charged amino groups can “snorkel” to the polar regions close to the surface even when the peptide is immersed in the micelle. Such an orientation of lysine chains does not lead to nontrivial NOEs and is therefore not represented in the NMR structure. The orientation and immersion depth of CM15 is in agreement with the rather small micelle radius, which can accommodate a straight R-helical peptide of 15 residues in an orientation parallel to the micelle surface only when it is bound not too close to the surface. The deep immersion of CM15 in the micelle seems to contradict the orientation (parallel to the surface) found using paramagnetic relaxation waves.11 However, the latter only provide the orientation, and as long as the PREs differ between the two sides of the helix, it is recognized as being parallel to the surface. The actual distance from the surface can only be reliably obtained from PREs using the complete fitting procedure described here. The very short helix of CM15 is also characterized by small differences in PREs along the helix. For longer helices, an orientation similar to CM15 with the center of the micelle being on the edge of the peptide backbone would lead to a paramagnetic relaxation wave having a parabolic shape with higher values near the termini. For such a longer helix, the closeto-transmembrane orientation should thus be found also using a paramagnetic relaxation wave.11 The orientation of CM15 with the hydrophilic side (also containing Ala10 and Val14) being

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TABLE 1: PREs of CM15 in the Presence of 16-Doxylstearic Acid proton

PRE (s-1 mM-1)

1.HR 7.HR 8.HR 10.HR 12.HR 13.HR 13.Hε1 14.HR 14.Hβ

3.2 9.6 24.4 5.4 7.8 6.9 8.4 6.3 9.0

TABLE 2: PREs of TM7 in the Presence of 16-Doxylstearic Acid proton

PRE (s-1 mM-1)

2.HR 3.HR 8.Hδ 8.Hε 10.Hδ 12.Hε3 12.Hξ3 13.HR 16.HR 17.HR 18.HR 19.HR 20.HR 22.Hβ 23.HR 25.Hβ 25.Hγ

1.3 3.5 12.0 14.8 21.9 12.1 8.9 9.6 15.5 7.6 2.8 10.0 7.2 2.0 3.6 2.4 1.7

closer to the surface of the micelle is in agreement with an EPR investigation observing oxygen accessibility on six individually introduced spin labels in CM156 and with the orientation found using paramagnetic relaxation waves.11 However, these two studies were not able to provide the immersion depth. To confirm the unexpected mode of binding of CM15 rather deep in the micelle, we also determined relaxation enhancements in the presence of small amounts of 16-doxyl-stearic acid. The doxyl radical at the end of the hydrophobic chain is immersed deep in the micelle and should therefore lead to larger PREs for nuclei close to the center of the micelle. The results for TM7 and CM15 are shown in Tables 1 and 2. Only well-resolved proton signals were used for the analysis. For both peptides, clearly enhanced PREs are found for residues that are also found close to the center of the micelle with our Gd(DTPA-BMA) approach. Only a rather small number of protons gave useful PREs in the presence of the doxyl radical due to strong linebroadening of nuclei near the intrinsic paramagnetic probe. On the contrary, the paramagnetic environment of Gd(DTPA-BMA) outside the micelle gives less-severe broadening even for the nuclei closest to the surface since each paramagnetic center is still at least 4 Å away. Therefore, a more uniform distribution of PREs along the peptide chain can be observed. A further difficulty in using PREs of a doxyl radical attached to a lipid molecule is the less well-defined position within the micelle due to the mobility of micelle forming molecules, whereas the distance to the Gd(DTPA-BMA) layer is determined by the micelle radius. In addition to paramagnetic relaxation enhancements, one could also use NOEs between the peptide and micelle for obtaining information about the mode of binding. However, we could not detect any NOEs between DPC and the peptide, probably due to fast mobility of the detergent molecules. Once the positioning of a peptide in a micelle is known, one can easily

Figure 6. Comparison of experimental (gray squares) and backcalculated (dark triangles) PREs of (a) TM7 and (b) CM15. PREs are given as a function of atom number, indicating the number of the atom for which a PRE could be determined. Therefore, these atom numbers can not be translated directly into the residue number but indicated the change of PRE by going along the peptide chain.

back-calculate PREs. A comparison of such calculated with experimental PREs for TM7 and CM15 is shown Figure 6, panels a and b, respectively. Larger variations are found near the termini of the peptides. These likely stem from the increased mobility of the peptides there. In regions of structural flexibility, NOEs are averaged differently to PREs. Although NOEs exaggerate a “closed” confirmation, PREs emphasize close encounters with the paramagnetic environment and therefore tend to point to a more open peptide structure. It should be noted that the proposed approach only works for compounds that do not change the spherical micelle shape. For larger proteins the a priori unknown shape of the protein-lipid complex prevents a direct translation of PREs into immersion depth. It is also advantageous for the interpretation of relaxation data when the paramagnetic compound has no specific interaction with the micelle. It has been shown previously by ESR spectroscopy that 3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(4-ethoxybenzyl)undecandicarboxylicacidGd(EOBDTPA) and other polar contrast agents do not penetrate or interact with membrane-forming lipids.35,36 A possible specific interaction of Gd(DTPA-BMA) with the micelles would be expected to lead to much larger relaxation enhancements of protons located near the surface of the micelle than the ones observed. To get additional confirmation about the absence of any such interaction we acquired ESR spectra of Gd(DTPABMA) in the presence and absence of 100 mM DPC and found no differences (see the Supporting Information). The z-value found for TM7 (2647 Å3 s-1 mM-1) can be used to calculate the correlation time τc of eq 2. Using literature values for all constants in eq 2, a correlation time τc of 1.62 ns

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can be obtained. For a micelle of radius 22.6 Å, the rotational correlation time τr is 10.4 ns as obtained through eq 6,24

τr )

4πr3η 3kT

(6)

where r is the radius, η is the solvent viscosity (8.9 × 10-4 Pas), k is the Boltzmann constant, and T is the temperature. Using eq 3 thus yields τM ) 2.4 ns. For a protein-Gd(DTPABMA) system a value of 0.5 ns has been found,15 indicating that the interaction complex is slightly stronger between the micelle and Gd(DTPA-BMA). Conclusion We have shown that the orientation and immersion depth of a structurally characterized micelle-bound peptide can be obtained by using relaxation enhancements induced by a paramagnetic agent that is freely soluble in an aqueous buffer and inert toward peptides and the micelle-forming detergent. Because this method does not imply any covalent modification of the peptide or micelle, there is no perturbation of the peptide-lipid interaction. In addition, isotopic labeling of the peptide is not required, as long as it can be assigned using homonuclear NMR techniques. The applicability of the presented method is, of course, not restricted to peptides bound to micelles but should prove useful whenever a structurally characterized compound is positioned in any large molecular complex of defined spherical shape. Acknowledgment. Funding by the Austrian Science Foundation (FWF) under project Nos. 19902 and 20020 to K.Z. is gratefully acknowledged. M.R. thanks the Heinrich-Jo¨rg Stiftung for financial support, and T.M. wants to express his gratitude ¨ AW) for a DOC to the Austrian Academy of Sciences (O scholarship. Supporting Information Available: Two tables (Tables S1 and S2) showing the experimental PREs of TM7 and CM15 in PDB format. A figure (Figure S1) depicting the structure of DPC with PREs measured in the presence of CM15 and TM7. EPR spectra (Figure S2) of Gd(DTPA-BMA) in the presence and absence of DPC micelles. This information is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Brogden, K. A. Nat. ReV. Microbiol. 2005, 3, 238. (2) Epand, R. M.; Epand, R. F.; McKenzie, R. C. J. Biol. Chem. 1987, 262, 1526.

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