Study of the Interaction of GFG Tripeptide with Cesium

Langmuir 2008, 24, 5809-5815

5809

Study of the Interaction of GFG Tripeptide with Cesium Perfluorooctanoate Micelles by Means of NMR Spectroscopy and MD Simulations Silvia Pizzanelli, Claudia Forte,* and Susanna Monti Istituto per i Processi Chimico-Fisici, CNR, Via G. Moruzzi 1, I-56124 Pisa, Italy ReceiVed NoVember 30, 2007. ReVised Manuscript ReceiVed March 10, 2008 The interaction of glycyl-phenylalanyl-glycine (GFG) with bilayers formed by cesium perfluorooctanoate (CsPFO) in water was investigated in the isotropic phase by means of 1H NMR and molecular dynamics (MD) simulations. Details on the preferential location of the different residues of GFG were obtained from selective variations of chemical shift with peptide concentration and of line width in the presence of the paramagnetic ion Mn2+. The analysis of 1H NMR spectra recorded at different concentrations and temperatures allowed the association constant and the enthalpy change upon binding to be evaluated. MD simulations highlighted the hydrogen bonds formed between the different GFG functional groups and the micelle. Both NMR and MD gave indications of high affinity of GFG with the micelle, with the N-terminal residue anchoring on the surface via hydrogen bonds with the micelle COO- groups.

1. Introduction The binding of peptides to lipid bilayers is an important issue in biochemistry, and a detailed comprehension of their interaction mechanisms is of fundamental importance for a variety of cellular processes (i.e., action of toxins, antimicrobial and channel-forming peptides). Among other techniques, NMR has been extensively used to study the mutual interactions between proteins and bilayers.1,2 Considerable interest has been given also to short peptides (see, for example, refs 3–8): in fact, the binding of small peptides to bilayers, aside from being involved in a variety of fields, such as chromatography and drug delivery, may aid in identifying the contributions of the different peptide functional groups to the binding process of polypeptides. In particular, in the case of lipid bilayers, polarity scales of amino acids have been proposed concerning hydrophobic binding.9,10 In the present work, the interaction of the tripeptide glycylphenylalanyl-glycine (GFG) with bilayers formed by cesium perfluorooctanoate (CsPFO) in water11 is investigated. Fluorinated bilayers of this type have been used in conformational studies of small molecules12–14 and in micellar chromatography for separating mixtures of small peptides;15 in addition, fluorinesurfactant-based drug delivery systems, including micelles, have been engineered for delivering peptides.16,17 Therefore, the collection of details on GFG binding to fluorinated micelles is important both from a fundamental and an applicative point of view. In a previous study, we investigated the binding of alanylphenylalanyl-alanine (AFA) with CsPFO micelles18 and found that the charged NH3+ terminus preferentially interacts with the * To whom correspondence should be addressed. E-mail: [email protected]. (1) Arora, A.; Tamm, L. K. Curr. Opin. Struct. Biol. 2001, 11, 540–547. (2) Marcotte, I.; Auger, M. Concepts Magn. Res. A 2005, 24A, 17–37. (3) Chandrasekhar, I.; van Gunsteren, W. F.; Zandomeneghi, G.; Williamson, P. T. F.; Meier, B. H. J. Am. Chem. Soc. 2006, 128, 159–170. (4) Hicks, R. P.; Beard, D. J.; Young, J. K. Biopolymers 1992, 32, 85–96. (5) Palian, M. M.; Boguslavsky, V. I.; O’Brien, D. F.; Polt, R. J. Am. Chem. Soc. 2003, 125, 5823–5831. (6) Chatterjee, C.; Majumder, B.; Mukhopadhyay, C. J. Phys. Chem. B 2004, 108, 7430–7436. (7) Brown, J. W.; Huestis, W. H. J. Phys. Chem. 1993, 97, 2967–2973. (8) Fielding, L. Tetrahedron 2000, 56, 6151–6170. (9) White, S. H.; Wimley, W. C. Biochim. Biophys. Acta 1998, 1376, 339–352. (10) Mayer, P. T.; Xiang, T. X.; Niemi, R.; Anderson, B. D. Biochemistry 2003, 42, 1624–1636.

negatively charged micelle surface, with the phenyl ring tending to remain outside of the micelle. Here, GFG was chosen with the aim of understanding the role played by the methyl groups on the N and C termini. Conformational studies of GFG19,20 and its complexes with Pd(II),21 Ni(II),22 and Cu(II)23 as well as investigation on its dynamics in water24 and adsorption on colloidal silver25 are reported in the literature, but, to the best of our knowledge, no studies concern the behavior of this peptide in the presence of micelles. GFG was here studied in the isotropic micellar phase using 1H NMR, exploiting the sensitivity of the chemical shift of selected peptide hydrogens to the interaction with the micelle. The NMR experimental evidence was combined with molecular dynamics (MD) simulations, an approach which was revealed to be extremely powerful in similar studies. Molecular dynamics simulations of perfluorinated micellar surfaces26 and internal structures27 have been proposed, but recent experimental findings suggest that the models only partially agree with experimental data.28 On the other hand, a molecular-thermodynamic theory recently developed for these systems,29 although correctly reproducing the experimental observables,28 requires complex and demanding computational procedures. In the present work, considering that the simulation is only meant to support and possibly explain the experimental findings, the model by Balasubramanian and Bagchi26 was chosen for the starting micellar assembly because it is not highly demanding from a computational point of view, although it is not suitable to describe the micellar interior. This given, the MD calculations were limited to the description of the approach path of the peptide to the (11) Boden, N.; Corne, S. A.; Jolley, K. W. J. Phys. Chem. 1987, 91, 4092– 4105. (12) Poon, C. D.; Samulski, E. T.; Weise, C. F.; Weisshaar, J. C. J. Am. Chem. Soc. 2000, 122, 5642–5643. (13) Kimura, A.; Kuni, N.; Fujiwara, H. J. Am. Chem. Soc. 1997, 119, 4719– 4725. (14) Kimura, A.; Takamoto, K.; Fujiwara, H. J. Am. Chem. Soc. 1998, 120, 9656–9661. (15) Ye, B.; Hadjmohammadi, M.; Khaledi, M. G. J. Chromatogr., A 1995, 692, 291–300. (16) Krafft, M. P.; Riess, J. G. Biochimie 1998, 80, 489–514. (17) Krafft, M. P.; Riess, J. G. J. Polym. Sci., Part A: Polym. Chem. 2007, 45, 1185–1198.

10.1021/la703756u CCC: $40.75  2008 American Chemical Society Published on Web 05/02/2008

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micelle surface identifying the driving force of binding, and to the estimate of the binding free energy.

temperatures by measuring the 1H chemical shifts in isotropic micellar solutions as a function of GFG concentration. The following equilibrium between the peptide P and the surfactant S establishes:

2. Materials and Methods

Sn + P T SnP

2.1. Preparation of the Samples. GFG (custom-synthesized by Peptide International), CsOH · H2O (Aldrich, 99.97%), CF3(CF2)6COOH (Aldrich, 96%), MnSO4 · H2O (Carlo Erba, Milano, Italy, 99%), and D2O (Aldrich, 99.9%) were used without further purification. CsPFO was prepared according to the procedure reported in the literature.30 The 1H NMR experiments of GFG in D2O without CsPFO were conducted on a sample characterized by a 0.9 mM GFG concentration. All the NMR samples containing CsPFO were prepared by weighing CsPFO directly into an NMR tube and adding water with a micropipet, reaching a water/CsPFO molar ratio of 40; the micropipet was provided with a disposable glass capillary. Homogeneous solutions were obtained simply by shaking the samples while heating to the isotropic phase. 1H NMR chemical shifts of GFG in the presence of the micelles were measured on 11 1.2-69.3 mM CsPFO/D2O solutions of GFG at 310, 325, and 340 K in order to estimate the association constant at these temperatures. The samples containing the different amounts of GFG were obtained by introducing successive weighed amounts of the peptide into the least concentrated sample; the pH of these solutions was 7. On the basis of the GFG isoelectric point, at this pH, GFG is mainly zwitterionic, with a small fraction of the anionic form also occurring. In the NMR experiments involving Mn2+, successive aliquots of a MnSO4 solution in D2O were added to a 1.2 mM GFG solution in CsPFO/D2O contained in an NMR tube. In this series of experiments, the highest Mn2+ concentration reached was 268 µM, and the water/CsPFO molar ratio ranged between 40 and 46. After each addition of Mn2+, the phase was checked to be isotropic at 310 K by recording 2H NMR spectra. The GFG concentration was 6 mM in the micellar sample at pH 5.5 and 0.9 mM in the water sample at pH 10; both samples were prepared using commercial aqueous buffers (Hanna Instruments): a potassium hydrogen phthalate and a sodium hydrogen carbonate/sodium carbonate buffer, respectively. The pH was measured with a Hamilton Biotrode electrode for microsamples (Hamilton, Switzerland) attached to a pHmeter Eutech CyberScan pH1100 instrument. 2.2. NMR Experiments. All the NMR experiments were carried out on a Bruker AMX-300 WB instrument, equipped with a 5 mm probe. The π/2 pulse was 11.5, 10.5, and 6.9 µs on the 1H, 2H, and 13C channels, respectively. The sample temperature was controlled employing a BVT 1000 (Eurotherm) variable temperature unit, with a temperature stability of (0.1 K. For all the experiments, a 4 s relaxation delay was used. 1H chemical shifts are given in ppm after external calibration using a solution of acetone in D2O. 13C, 1H Heteronuclear multiple quantum coherence (HMQC) experiments on the 69.3 mM GFG sample in CsPFO/D2O at 325 K and the 0.9 mM GFG sample in D2O at 310 K were performed to aid in the 1H spectral assignment. To this end, the 13C assignment reported for similar systems31 was exploited. In the experiments, the GARP sequence was applied for decoupling during acquisition and the BIRD sandwich was used in order to suppress the signals from protons not coupled with 13C; the BIRD delay was 550 ms, while the 1/(2J) delay was set to 3.5 ms for both samples.32 The line widths of the signals in the 1H spectra were determined with the SPORT-NMR spectral analysis package.33 The chemical shifts of the R and β protons, together with the J coupling values involved, were obtained through a fitting procedure implemented in the program SpinWorks, written and made available by Dr. Kirk Marat.34 2.3. Determination of the Binding Constant. The binding of zwitterionic GFG to CsPFO micelles has been monitored at different (18) Pizzanelli, S.; Forte, C.; Monti, S.; Schweitzer-Stenner, R. J. Phys. Chem. B 2008, 112, 1251–1261. (19) Newmark, R. A.; Miller, M. A. J. Phys. Chem. 1971, 75, 505–508. (20) van der Spoel, D. Biochem. Cell Biol. 1998, 76, 164–170. (21) Vestues, P. I.; Martin, R. B. J. Am. Chem. Soc. 1980, 102, 7906–7909. (22) Tsangaris, J. M.; Chang, J. W.; Martin, R. B. J. Am. Chem. Soc. 1969, 91, 726–731.

(1)

with n being equal to the surfactant molecules representing a binding site for the peptide.35 The equilibrium constant is assumed independent of the number of peptide molecules bound to the same micelle, as long as a free binding site is available for the binding of an additional peptide molecule, in agreement with the model of random distribution of solubilizates among micelles.36 Moreover, the peptide is assumed to be monomeric, which is reasonably valid for the concentrations considered.24 An estimate of the association constant was performed assuming that the exchange of the peptide between the two sites is fast in the NMR time scale. In this case, the mole fraction of the peptide bound, xSnP, is given by37

xSnP )

δobs - δfree δbound - δfree

(2)

where δobs is the observed chemical shift of any peptide resonance sensitive to the binding at a given peptide concentration, and δfree and δbound are the chemical shifts of the same resonance in the free and fully bound peptide, respectively. The overall association constant of the peptide with the surfactant, Ka, is given by the following expression35

Ka )

(

(1 - xSnP)

xSnP

)

[St] - xSnP[Pt] n

(3)

where [St] is the total concentration of surfactant and [Pt] is the total concentration of the peptide. For each temperature, a global fit of the chemical shift values of the R protons of residue G1 observed at different Pt values was performed using eqs 2 and 3 by means of a home-written program in the Mathematica 5 environment (Trademark of Wolfram Research Inc.) with Ka, n, δbound, and δfree being the variable parameters of the fitting. Only these R proton chemical shifts were chosen, since they are the most sensitive to the presence of the micelles. 2.4. Molecular Dynamics Simulations. The creation of the initial structure, equilibration, and dynamics was performed as described in ref 18. The xleap module of AMBER938 was used to build all the starting configurations for the simulations. The structure of the CsPFO micelle was kindly provided by Balasubramanian who extracted a stable conformation from 3 ns atomistic molecular dynamics simulations of the CsPFO micelle in water.26,39 The snapshot consisted of 62 perfluorooctanoate (PFO) surfactant molecules, 62 cesium ions, and 10 562 water molecules. All water molecules and cesium ions far from the micelle, that is, farther than 10 Å from any PFO head group, were removed. Fifty neutralizing Cs+ counterions were placed at the carboxyl groups of the micelle, and four GFG molecules (hereafter called GFG(n) with n ) 1-4) with minimum energy conformation, in random orientation, were placed in close proximity to the micelle surface, with particular attention to avoid any bad van der Waals contact. Four GFG molecules were considered in order to obtain more significant data from a statistical point of view. GFG was in the zwitterionic form, and a Mn2+ ion with the corresponding SO42-counterion was added to the system to reproduce environmental effects. The general Amber (23) Tsangaris, J. M.; Martin, R. B. J. Am. Chem. Soc. 1970, 92, 4255–4260. (24) Mikhailov, D. V.; Washington, L.; Voloshin, A. M.; Daragan, V. A.; Mayo, K. H. Biopolymers 1999, 49, 373–383. (25) Herne, T. M.; Ahern, A. M.; Garrell, R. L. J. Am. Chem. Soc. 1991, 113, 846–854. (26) Balasubramanian, S.; Bagchi, B. J. Phys. Chem. B 2001, 105, 12529– 12533. (27) Mei, D.; O’Connell, J. P. Langmuir 2002, 18, 9067–9079.

Interaction of GFG with CsPFO Micelles (GAFF) and the ff03 force fields40,41 were employed to represent the molecular mechanical potentials of the micelle and peptide molecules. This choice was dictated by the fact that the Duan et al. force field6 has a considerable balance between R-helix and β-strand secondary structure elements, and thus, it can be appropriate for extended-time simulations of peptides whose structures can contain both types of motifs. The surfactant was modeled with explicit fluorine atoms, and interactions between the fluorocarbon tails were described using the parameters reported by Sprik et al.42 Partial charges for nonstandard molecular fragments were obtained from DFT-B3LYP/ 6-31G* calculations and following the RESP43,44 procedure. The micelle, the four GFG molecules, and the ions were surrounded by a periodic octahedral box of TIP3P water molecules45 (box size ) 80 × 80 × 80 Å3, number of water molecules ) 12 000). All simulations were performed using the Sander module of AMBER9 with SHAKE46 (tolerance ) 0.0004 Å) on hydrogen atoms in the NpT ensemble with a time step of 2 fs. Constant temperature (T ) 310 K) was maintained with Andersen coupling47,48 and a time constant of 0.2 ps. Berendsen’s barostat49 with isotropic molecule based scaling and a time constant of 2 ps was employed to keep the pressure constant (1 bar). All Lennard-Jones interactions were cut off at 12 Å, and the electrostatic interactions were handled by the particle mesh Ewald method (PME). Equilibration was performed by first holding the positions of the micelle and the peptides fixed and running 1000 steps of minimization followed by a short 50 ps run in the NVT ensemble to remove unphysical voids due to structural modifications. Equilibration was continued for 100 ps applying harmonic position restraints, with a force constant of 25 kcal/(mol Å2), on the fluorocarbon tails located inside the micelle; the carboxyl head groups and the first four CF2 groups were not constrained. This equilibration was followed by 50 ps NpT dynamics. Finally, the production run in the NpT ensemble was initiated. The equilibration protocol was necessary to let water molecules and ions equilibrate and then let the four molecules and the micelle slowly relax from their starting geometries, to avoid bad contacts and to relieve poor bond angle and dihedral deviations in the model structures. To determine if the total equilibration time was enough to relax the system, pressure, volume, and density were monitored. Although these quantities were equilibrated, the first nanosecond of the production run was considered part of the equilibration phase. The system resulting from equilibration was simulated for 13 ns, and the last 12 ns data, sampled every 10 ps, were used for analysis. In order to investigate the effect due to the presence of the micelle, a shorter simulation (about 6 ns) of four GFG molecules in pure water was also performed. 2.5. Analysis Procedure and Definition of the Examined Quantities. A GFG molecule was considered bound to the micelle when the distance between the center of mass of the micelle and any one among the GFG center of mass, the NH3+ group, the phenyl ring, and the COO- group was less than 25 Å. Pair radial distribution functions (RDFs) between both the N- and C-terminus groups of each GFG molecule and a given atom of the solvent, with their coordination numbers (CNs), were calculated in pure water and in the presence of the micelle in order to monitor any change in the solvation properties of these two groups induced by the micelle. In order to identify the preferred location of both the NH3+ and ring groups of each GFG molecule with respect to the micelle and to provide a general picture of the peptide binding, the distribution of these moieties in the space around the micelle was calculated by binning the atom position from root mean square (rms) coordinate (28) Nordstierna, L.; Pavel, V.; Furo´, I. J. Phys. Chem. B 2006, 110, 25775– 25781. (29) Srinivasan, V.; Blankschtein, D. Langmuir 2005, 21, 1647–1660. (30) Weise, C. F.; Weisshaar, J. C. J. Phys. Chem. B 2003, 107, 3265–3277. (31) Spectral Data Base System (SDBS), catalogue number 4334, 4336, 4373. (32) Garbow, J. R.; Weitekamp, D. P.; Pines, A. Chem. Phys. Lett. 1982, 93, 504–509. (33) Geppi, M.; Forte, C. J. Magn. Reson. 1999, 137, 177–185. (34) Marat, K. Spin Works; University of Manitoba: Winnipeg, Manitoba, Canada; http://www.umanitoba.ca/chemistry/nmr/spinworks/ (35) Vogel, H. FEBS Lett. 1981, 134, 37–42.

Langmuir, Vol. 24, No. 11, 2008 5811 Table 1. 1H NMR Chemical Shifts (ppm) of Zwitterionic GFG signals in Water and in CsPFO Micellar Solution at 310 K G1 R F2 aromaticd F2 βc G3 Rc c

in CsPFO micellar solutiona

in waterb

3.49/3.55 7.54 3.18/3.42 3.92/4.02

3.79/3.88 7.54 3.18/3.41 3.85/3.97

a Measured in a 1.2 mM GFG solution. b Measured in a 0.9 mM GFG solution. c The two different values correspond to the diastereotopic methylene protons. d Center of gravity of the multiplet.

fit frames over all micelle atoms at 10 ps intervals into 1 Å3 grids over the whole trajectory. The value found in each grid element represents the number of times the coordinates of the center of mass of the selected group were within the grid element. These grid elements were contoured using the density tool of Chimera.50 The contouring of the NH3+ and ring densities were performed at 2 and 6 hits per Å3, that is, 2 and 6 visits to each grid element from all the frames of the trajectory. Hydrogen bonds between the peptide groups and the micelle were analyzed considering donor-acceptor distances smaller than 3.5 Å and hydrogen donor-acceptor angles smaller than 40°. The peptide-micelle adsorption free energy was calculated using the probability ratio method.18,51–53 The overall free energy of adsorption, ∆Gads, for the peptide was calculated as follows:

∆Gads ) -RT

() P

∑ Pi ln P0i ∆di i

(4)

where R is the ideal gas constant, T is the absolute temperature, Pi and P0 are the probability densities of the peptide being in two particular distance intervals from the center of mass of the micelle, and ∆di is the width of the interval, which was chosen as equal to 0.2 Å. P0, which is the normalized probability density of a reference state far away from the micelle, was calculated by averaging the Pi value between a cutoff distance of 35 Å and the maximum distance found.

3. Results and Discussion 1H NMR. Spectra of GFG in CsPFO Micellar Solutions.

3.1. NMR spectra of GFG in CsPFO/D2O micellar system were recorded at 310, 325, and 340 K with GFG concentrations ranging between 1 and 70 mM. Figure 1 shows the spectra of GFG in water and in the micellar environment at 310 K. The spectra are characterized by four distinct sets of signals, one for each glycine methylene group, one for the phenylalanine aromatic protons, and one for the β protons, with the R proton peak overlapped to the strong signal of residual water. The F2 β protons together with the R proton give rise to an ABC type spectrum, while the G1 and G3 diastereotopic protons, characterized by different chemical shifts, give rise to two AB type spectra. The assignment of the 1H signals of GFG in CsPFO/D2O isotropic solution and in aqueous solution without the micelles was made by correlating 1H/13C signals by means of a [13C,1H]-HMQC experiment and exploiting the 13C assignment reported for similar systems.31 This assignment is given in Table 1. 1H

(36) Moroi, Y. J. Phys. Chem. 1980, 84, 2186–2190. (37) Deber, C. M.; Behnam, B. A. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 61–65. (38) Case, D. A.; Darden, T. A.; Cheatham, T. E., III; Simmerling, C. L.; Wang, J.; Duke, R. E.; Luo, R.; Merz, K. M.; Pearlman, D. A.; Crowley, M.; Walker, R. C.; Zhang, W.; Wang, B.; Hayik, S.; Roitberg, A.; Seabra, G.; Wong, K. F.; Paesani, F.; Wu, X.; Brozell, S.; Tsui, V.; Gohlke, H.; Yang, L.; Tan, C.; Mongan, J.; Hornak, V.; Cui, G.; Beroza, P.; Mathews, D. H.; Schafmeister, C.; Ross, W. S.; Kollman, P. A. AMBER 9; University of California: San Francisco, 2006. (39) Balasubramanian, S.; Bagchi, B. J. Phys. Chem. B 2002, 106, 3668–3672. (40) Duan, Y.; Wu, C.; Chowdhury, S.; Lee, M. C.; Xiong, G.; Zhang, W.; Yang, R.; Cieplak, P.; Luo, R.; Lee, T. J. Comput. Chem. 2003, 24, 1999–2012.

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Figure 2. Chemical shift of selected GFG protons in CsPFO/D2O in the zwitterionic form as a function of GFG concentration at 310 K.

Figure 1. From left to right, spectral regions of the phenyl F2 protons, of R G3 and G1 protons, and of β F2 protons in the micellar environment (upper trace, [GFG] ) 6.0 mM) and in D2O (lower trace, [GFG] ) 0.9 mM) at 310 K.

The spectra for the micellar solution clearly show a minor set of both F2 β and aromatic protons and a peak in the G3 region. Whereas for the aromatic signals a detailed analysis is prevented by the strong overlap with the major component and by signal complexity, in the case of β protons, which are only partially overlapped to the signals of the major set, it can be observed that the chemical shifts are slightly downfield shifted with respect to those observed in water and completely insensitive to different GFG concentrations. Also the singlet resonating in the G3 region is insensitive to GFG concentration. The presence of these additional resonances can be justified by only assuming the occurrence of two populations, with the minor one decreasing with peptide concentration in the temperature range studied, reaching a plateau for GFG concentration larger than 30 mM. This minor component has been ascribed to GFG in the anionic form on the basis of spectra recorded at different pH values. In fact, the second set of resonances is not observed in a micellar sample characterized by pH 5.5, when the peptide is supposed to be completely zwitterionic, but occurs in an aqueous sample at pH 10, when the peptide is completely anionic. It should be noted that the singlet in the G3 region is ascribable to G1 protons which, although diastereotopic, resonate at the same chemical shift, as observed in many glycyl peptides in water in their cationic or anionic form.54,55 As far as the major zwitterionic form is concerned, the chemical shifts of the different resonances have a different dependence on GFG concentration in CsPFO/D2O, as shown in Figure 2. In particular, for the F2 residue, the β protons are only slightly sensitive to different GFG concentrations, resonating at about 3.2 and 3.4 ppm, values close to those in water. G3 protons, characterized by a chemical shift of about 4 ppm, close to the value observed in water, are insensitive to GFG concentration variations. On the contrary, G1 proton resonance frequencies show a considerable dependence on GFG concentration and are (41) Lee, M. C.; Duan, Y. Proteins 2004, 55, 620–634. (42) Sprik, M.; Ro¨thlinsberger, U.; Klein, M. L. Mol. Phys. 1999, 97, 355– 373.

Figure 3. Temperature dependence of the chemical shift difference (∆δ) between the diastereotopic protons of residues G1 and G3 of zwitterionic GFG in a micellar solution characterized by [GFG] ) 4.8 mM (empty symbols) and in water (filled symbols).

significantly upfield shifted by the presence of the micelles with respect to the corresponding signal in water. This indicates that the peptide interacts with the micelle preferentially through the G1 residue rather than with the G3 one. Such preferential interaction is confirmed by the temperature dependence of the chemical shift difference between the diastereotopic protons of G1 and G3 in micellar solution and in water shown in Figure 3. In fact, the temperature dependence for G3 protons is comparable in water and in the micellar solution, while it is much more pronounced for G1 protons in the micelles rather than in water, with the chemical shift difference sensibly decreasing with increasing temperature. In addition, for the G3 residue, the chemical shift difference is larger than that for G1; this has been observed in glycyl dipeptides in their zwitterionic form in water,54–56 where the large difference in chemical shift in C-terminal glycyl dipeptides has been interpreted to indicate that selective population of only a few rotamers occurs, contrary to N-terminal glycyl dipeptides, where a large number of rotamers is significantly populated. In our case, this seems to occur also in the micellar environment, where, moreover, the number of populated rotamers sensibly increases with temperature for G1. Thus, the micelles seem not to significantly modify the rotameric preferences of G3 with respect to water but to sensibly affect those of G1. This interpretation agrees with the preferential interaction of the G1 residue with the micelle in comparison with G3. It should also be pointed out that the insensitivity of the resonances of anionic GFG to GFG concentration indicates a (43) Cieplak, P.; Cornell, W. D.; Bayly, C. I.; Kollman, P. A. J. Comput. Chem. 1995, 16, 1357–1377.

Interaction of GFG with CsPFO Micelles

Langmuir, Vol. 24, No. 11, 2008 5813

temperature from 310 to 340 K; these values are higher than the micelle aggregation numbers reported for CsPFO/D2O solutions characterized by similar CsPFO weight fractions and at temperatures close to those imposed by us, but the trend with the temperature is the same.57 Considering that

ln Ka ) -

Figure 4. Expansions of selected regions of the 1H NMR spectra of GFG in CsPFO/D2O at 310 K in the presence of different amounts of Mn2+.

low affinity of GFG in this form to the micelle, as expected on the basis of the fact that the peptide anion and the micellar surface are characterized by the same charge. Spectra of GFG in CsPFO/D2O Solution in the Presence of Mn2+ The addition of Mn2+ to GFG in the isotropic micellar solution causes differential line broadening of the peptide 1H NMR signals, indicating different average distances between different peptide moieties and the paramagnetic ion Mn2+. In Figure 4, the regions of the 1H spectra of GFG due to the proton groups of interest in the presence of different Mn2+ concentrations are compared to the corresponding regions without Mn2+. It can be observed that, for zwitterionic GFG, the resonances due to residue G1 are broadened more extensively than those of F2 and G3. In fact, significant broadening is induced on the resonances of G1 protons already at a 23 µM Mn2+ concentration, while a solution concentrated more than 10 times is necessary to induce broadening on G3 protons. Since Mn2+ ions are located preferentially close to the surface of the micelles,18 electrostatically interacting with CsPFO carboxylate groups, these data indicate a preferential affinity to the micelle surface of the peptide N-terminus, in agreement with the chemical shift data. Moreover, a close inspection of Figure 4 reveals that the signal due to F2 β protons of the anionic form is less broadened than that of the corresponding protons of the zwitterion. This indicates that F2 β protons of the anion experience a larger average distance from the Mn2+ ion than those of the zwitterion, in agreement with the chemical shift data, again revealing a lower affinity to the micellar surface for the negatively charged peptide. 3.2. Determination of the Binding Constant. The GFG/ micelle binding constant was determined from the G1 R proton chemical shifts at different GFG concentrations, according to the procedure described in the Materials and Methods section. The experimental chemical shift values are shown in Figure 5 together with the curves calculated using the best fitting values reported in Table 2. All parameter values were well determined except for Ka, which was obtained with a large standard deviation. The best fitting δfree values were reasonably close to the corresponding values independently measured in aqueous solution. Upon increasing the temperature, a decrease was observed for Ka and also for n. The latter decreased from 237 to 167 with increasing

∆G0 ∆H0 ∆S0 )+ RT RT R

(5)

and assuming that ∆H0 and ∆S0are independent of temperature in the range investigated, a rough estimate of -12 ( 4 kcal/ mol was obtained for ∆H0; given the uncertainty on the equilibrium constant values, the value of ∆S0 was illdetermined, ranging between -35 and -6 cal/mol. The nonnegligible negative enthalpy change upon binding of GFG to the micelle suggests that the interaction is essentially ascribable to a “nonclassical” hydrophobic effect58 arising from van der Waals interactions between the nonpolar part of the peptide and the micellar fluorocarbon core. This effect has been observed, for example, in the case of some peptides with phospholipid vesicles.59 In the present case, however, the micelle interior is fluorinated, and therefore, it is not only hydrophobic but also lipophobic. Nonetheless, solubility enhancement measurements on aromatic hydrocarbons in fluorinated surfactant solutions60 highlighted favorable hydrophobic interactions between the solutes and the micelles, although smaller with respect to those observed when hydrocarbon surfactants are used because of the mutual phobicity between hydrocarbons and fluorocarbons. Moreover, the retention factors of a variety of solutes in micellar electrokinetic chromatography experiments using lithium perfluorooctanesulfonate micelles have been justified on the basis of either favorable dipole-dipole and dipole-induced dipole interactions61 or relative ease of cavity formation.62 The ∆G0 value of -5.1 kcal/mol obtained for GFG at 310 K is higher in absolute value than that obtained for AFA (-3.8 kcal/mol) in the same conditions;18 this indicates a more favorable binding of GFG to CsPFO micelles, as discussed in the following sections. 3.3. Dynamics of GFG in Aqueous and Micellar Media from Molecular Dynamics Simulations. In the molecular dynamics simulation, the four GFG molecules considered were (44) Bayly, C. I.; Cieplak, P.; Cornell, W. D.; Kollman, P. A. J. Phys. Chem. 1993, 97, 10269–10280. (45) Jorgensen, W. L. J. Am. Chem. Soc. 1981, 103, 335–350. (46) Ryckaert, J. P.; Ciccotti, G.; Berendsen, H. J. C. J. Comput. Phys. 1977, 23, 327–341. (47) Andrea, T. A.; Swope, W. C.; Andersen, H. C. J. Chem. Phys. 1983, 79, 4576–4584. (48) Andersen, H. C. J. Chem. Phys. 1980, 72, 2384–2393. (49) Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; DiNola, A.; Haak, J. R. J. Chem. Phys. 1984, 81, 3684–3690. (50) UCSF Chimera, version 1; University of California: San Francisco, 2006. (51) Mezei, M. Mol. Simul. 1989, 3, 301–313. (52) van Gunsteren, W. F.; Weiner, P. K. Computer Simulation of Biomolecular Systems: Theoretical and Experimental Applications; ESCOM Science: Leiden, The Netherlands, 1989. (53) Monti, S. J. Phys. Chem. C 2007, 111, 6086–6094. (54) Morlino, V. J.; Martin, R. B. J. Am. Chem. Soc. 1967, 89, 3107–3111. (55) Nakamura, A.; Jardetzky, O. Proc. Natl. Acad. Sci. U.S.A. 1967, 58, 2212–2219. (56) Beeson, C.; Dix, T. A. J. Chem. Soc., Perkin Trans. 2 1991, 1913–1918. (57) Holmes, M. C.; Reynolds, D. J.; Boden, N. J. Phys. Chem. 1987, 91, 5257–5262. (58) Seelig, J.; Ganz, P. Biochemistry 1991, 30, 9354–9359. (59) Seelig, J. Biochim. Biophys. Acta 1997, 1331, 103–116. (60) An, Y. J.; Jeong, S. W. J. Colloid Interface Sci. 2001, 242, 419–424. (61) Poole, C. F.; Poole, S. K.; Abraham, M. H. J. Chromatogr., A 1998, 798, 207–222. (62) Fuguet, E.; Ra`fols, C.; Bosch, E.; Rose´s, M.; Abraham, M. H. J. Chromatogr., A 2001, 907, 257–265.

5814 Langmuir, Vol. 24, No. 11, 2008

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Figure 5. Experimental chemical shift values of the diastereotopic R protons of residue G1 (9, 0) at 310 (a), 325 (b), and 340 K (c). The curves calculated using the best fitting values reported in Table 2 are also shown.

Figure 6. Distance of the center of mass (CM) of the NH3+(G1), ring(F), and COO-(G3) groups of each GFG molecule [GFG(1) (a), GFG(2) (b), GFG(3) (c), and GFG(4) (d)] from the center of mass of the micelle as a function of the simulation time.

driven toward the micelle by the electrostatic interactions between the positively charged N-terminus groups of the peptides and the negatively charged head groups of the PFO molecules. These interactions were responsible for the increase in the concentration of the peptide in close proximity to the micelle surface and played a key role in both the initiation and duration of the binding process. The time evolution of the distances between selected peptide groups and the center of mass of the micelle are shown in Figure 6 It can be observed that the initially randomly placed peptides reached, within 1 ns, the surface of the micelle orienting their NH3+ group in such a way to form quite strong hydrogen bonds

with the surface anionic head groups. A further indication of the role played by the NH3+ group in the binding is given by the coordination number of water molecules around this moiety, which decreases from 9 to 5 when the micelle is added in the calculation, indicating desolvation. On the other hand, for the COO- terminus, this number decreases only from 9 to 7. This mechanism of attraction and first anchoring of the peptide to the micelle surface is very similar to that observed for AFA.18 In addition, as already observed in the case of AFA, all the GFG molecules were not rigidly fixed on the micelle surface throughout the whole simulation time, but contacts were broken and reformed several times, and in two cases the molecule left

Interaction of GFG with CsPFO Micelles

Langmuir, Vol. 24, No. 11, 2008 5815

Table 2. Best Fitting Parameters Used in the Calculation of Chemical Shift Trends Reported in Figure 5 for the Two r Protons of G1 in GFG R1 R2 R1 R2 R1 R2 a

Ka (M-1)

n

δbound (ppm)

δfree (ppm)

δfree,exp (ppm)a

T (K)

3600 ( 1000

237 ( 10 204 ( 10

700 ( 250

167 ( 10

3.93 ( 0.01 3.71 ( 0.01 3.93 ( 0.01 3.73 ( 0.01 3.95 ( 0.01 3.77 ( 0.01

3.90 3.80 3.91 3.81 3.91 3.81

310 ( 0.1

2700 ( 700

3.53 ( 0.01 3.48 ( 0.01 3.50 ( 0.01 3.47 ( 0.01 3.41 ( 0.01 3.42 ( 0.01

325 ( 0.1 340 ( 0.1

Values measured on a 0.9 mM GFG solution in D2O.

the micellar surface in the course of the simulation (Figure 6a and d). However, a difference is observed for the phenylalanine ring between AFA and GFG; in fact, in two cases, after the first anchoring of the molecule to the micelle surface through the positively charged N-terminus, further interactions involve the aromatic ring, as highlighted by the shorter distance from the micellar center of mass (Figure 6a and c). The majority of H-bonds involved the NH3+ group, representing about 64% of the total, whereas H-bonds with NH(F) were less populated (36%). Only a few involved fluorine atoms (5%), while the majority (95%) involved the oxygens of PFO. Hydrogen bonding interactions were established simultaneously with at most five different PFO molecules, suggesting that the G1-F portion of the peptide has indeed the tendency to insert its functional groups into accessible cavities of complementary potential. As far as peptide-micelle adsorption free energies are concerned, ∆Gi values were negative for all peptide molecules for distances shorter than 35 Å, confirming the tendency of GFG to favorably associate with the micelle. As expected, the most favorable adsorption was observed for GFG(3) (∆Gads ) -2.5 kcal/mol), while for GFG(4) it was the least favorable (∆Gads ) -1.8 kcal/mol); on average, the adsorption free energy was about -2.2 kcal/mol. Although this quantity should be considered an upper limit to the real adsorption free energy, considering that the MD sampling was not exhaustive, it is higher in absolute value than that estimated for AFA, which was ∼ -1.4 kcal/ mol,18 as already observed for the values determined experi-

mentally. The stronger association of GFG molecules with the micelle is possibly due to the greater flexibility of GFG peptides.

4. Conclusions 1H NMR spectra of zwitterionic GFG in CsPFO/D O at 2 different peptide concentrations clearly revealed the presence of fast exchange between micelle bound and free GFG. In particular, the stronger concentration dependence was observed for protons of the N-terminal residue, confirming that this residue is preferentially bound to the micelle surface via hydrogen bonds between the peptide NH3+ and the micelle COO- group, as indicated by the MD simulations and by GFG differential line broadening in the presence of Mn2+. Analysis of the chemical shift variations with concentration allowed the association constant of GFG to the micelle to be estimated using a two-site exchange model. The measurements performed at different temperatures yielded a non-negligible negative enthalpy change upon binding, indicating that the interaction is essentially ascribable to a nonclassical hydrophobic effect arising from van der Waals interactions between the nonpolar part of the peptide and the micelle fluorocarbon core.

Acknowledgment. The authors thank Prof. Reinhard Schweitzer-Stenner for providing the GFG sample and for critical and precious comments. The authors also thank Dr. E. Pitzalis for help in the pH measurements. LA703756U