J. Phys. Chem. B 2008, 112, 1251-1261
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Interaction of a Tripeptide with Cesium Perfluorooctanoate Micelles Silvia Pizzanelli,*,† Claudia Forte,† Susanna Monti,† and Reinhard Schweitzer-Stenner‡ Istituto per i Processi Chimico-Fisici (IPCF-CNR), Area della Ricerca, Via G. Moruzzi 1, I-56124 Pisa, Italy, and Department of Chemistry, Drexel UniVersity, Philadelphia, PennsylVania 19104 ReceiVed: May 22, 2007; In Final Form: October 31, 2007
The interaction of alanyl-phenylalanyl-alanine (Ala-Phe-Ala) with the micelles formed by cesium perfluorooctanoate (CsPFO) in water was studied in the isotropic phase by means of 1H NMR and by molecular dynamics (MD) simulations. Information on the location of the peptide was experimentally obtained from selective variations in Ala-Phe-Ala chemical shifts and from differential line broadening in the presence of the paramagnetic ion Mn2+. The peptide-micelle association constant was estimated analyzing the chemical shift variations of the most sensitive Ala-Phe-Ala resonances with the peptide concentration. MD simulations of Ala-Phe-Ala in the micellar environment confirmed the experimental observations, identifying the hydrogen bonding interactions of the different peptide moieties with the micelle, yielding a binding constant close to the experimental one. NOESY experiments suggest that the peptide in the micellar environment does not adopt a preferred conformation but is mainly unstructured. Details on the conformational behavior of the peptide in the micellar solution observed through MD were consistent with a different conformational equilibrium in the proximity of the micelle. Information on Ala-Phe-Ala dynamics was obtained from 1H T1 data and compared to MD simulation results on the overall tumbling motion.
1. Introduction
SCHEME 1. Ala-Phe-Ala Peptide
The interaction of peptides with micelles and bicelles is important in a variety of fields, including biological processes, separation techniques, and drug delivery. Considerable effort has been made to give a rationale of the partitioning of short peptides at membrane interfaces,1,2 to collect evidence on peptide location, and to collect evidence on its conformational and motional changes induced by the presence of micelles. In particular, among the short peptides, great attention has been focused on the conformation of enkephalins in different micelles, such as those formed in water by sodium dodecyl sulfate, cetyltrimethylammonium bromide, dimyristoylphosphatidylcholine, ganglioside GM1 (see, for example, refs 3-6). Limiting ourselves to the case of oligopeptides with less than five amino acids, studies concerning the location of Ala-Trp-Ala-O-tertbutyl in dioleylphosphocholine by means of X-rays and neutron diffraction7 and of Ala-Phe-Ala-O-tert-butyl in unilamellar phosphatidylcholine vesicles by 1H nuclear magnetic resonance (NMR)8 and molecular dynamics (MD) simulations9 have been reported. Diffusion-based studies of binding of very short peptides have also been performed.3,10 We chose to investigate the interaction of the tripeptide AlaPhe-Ala (Scheme 1) with the bilayer-like disk-shaped micelles formed by cesium perfluorooctanoate (CsPFO) in water,11 providing the association constant of the peptide to the micelle and information on its location and dynamics. The collection of data regarding this interaction is of great interest from a fundamental point of view, since this type of micelle is used in its liquid crystal phase as an orienting medium for studying the conformation of small molecules.12-14 However, the interaction of peptides with fluorinated micelles is of interest also from an * Author to whom correspondence should be addressed. Phone: +39050-3152518. FAX +39-050-3152442. E-mail:
[email protected]. † Istituto per i Processi Chimico-Fisici (IPCF-CNR). ‡ Drexel University.
applicative point of view. For example, in micellar chromatography, the use of fluorocarbon surfactants allows the separation of small peptides with high structural similarities.15-17 To the best of our knowledge, the interaction of small peptides with fluorinated micelles has been studied only indirectly by observing the efficiency of chromatographic separation of mixtures of peptides differing in size, hydrophobicity, and hydrogen bonding ability; however, direct evidence on molecular details of binding was never reported. Our study is meant to ultimately contribute in the effort to provide a rationale to the behavior of small peptides in the presence of fluorinated micelles and possibly suggest solutions to separation problems. The system was studied in the isotropic micellar phase using 1H NMR and exploiting observables such as chemical shift and relaxation times, which are informative on the location, structure, and dynamics of small peptides interacting with micelles or bilayers.18,19 NMR was combined with MD simulations, which, over the past few decades, has proved to be one of the most direct methods to theoretically investigate molecular behavior of complex systems, complementing the experimental data of micellar systems by leading to the understanding of the morphologies and dynamics of such aggregates.20-24 Regarding our system, recently, Balasubramanian and Bagchi25,26 studied characteristic features of an aggregate of 62 CsPFO molecules in water, focusing their analysis on the diffusion of water
10.1021/jp073947x CCC: $40.75 © 2008 American Chemical Society Published on Web 01/04/2008
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molecules and Cs+ ions at the micellar surface. Here, a series of MD computer simulations of Ala-Phe-Ala in a mixed solvent composed of water and CsPFO was carried out to explore the details of Ala-Phe-Ala/CsPFO micelle interactions.
TABLE 1: 1H Chemical Shift and T1 Values of Ala-Phe-Ala Signals in Water and When Bound to CsPFO Micelles at 310 K chemical shift (ppm) proton
2. Materials and Methods
Ala1-R
2.1. Preparation of the Samples. Ala-Phe-Ala (95%; customsynthesized by Peptide International), CsOH‚H2O (Aldrich, 99.97%), CF3(CF2)6COOH (Aldrich, 96%), MnSO4‚H2O (99%; Carlo Erba, Milano, Italy), D2O (Aldrich, 99.9%) were used without further purification. CsPFO was prepared according to the procedure reported in the literature.13 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 chemical shifts of Ala-Phe-Ala in the presence of the micelles were measured on 5-90 mM CsPFO/ D2O solutions of Ala-Phe-Ala at 310 K in order to estimate the association constant. Such samples were obtained introducing successive weighed amounts of Ala-Phe-Ala into the least concentrated sample (sample 1). In the NMR experiments involving Mn2+, 0.7 mg of Ala-Phe-Ala was dissolved in a CsPFO/D2O solution containing 300 µL of D2O, resulting in an Ala-Phe-Ala concentration of 7.6 mM. Successive aliquots of a MnSO4 solution in D2O were added to the sample in the NMR tube. In this series of experiments, the highest Mn2+ concentration reached was 0.1 mM, and the water/CsPFO molar ratio ranged between 40 and 47. The DQF-COSY experiment was performed on a 43 mM CsPFO/water (90 v% H2O/10 v% D2O) solution of Ala-Phe-Ala, while the NOESY experiments and T1 measurements were performed on a 15 mM solution of Ala-Phe-Ala in CsPFO/water (90 v% H2O/10 v% D2O) solution. The 1H experiments of Ala-Phe-Ala in D2O without CsPFO were conducted on a sample characterized by a 0.9 mM AlaPhe-Ala concentration. In all the samples studied, the pH was approximately 7; at this pH, Ala-Phe-Ala is zwitterionic. 2.2. NMR Experiments. All the NMR experiments were carried out on a Bruker AMX-300 WB, 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) variabletemperature unit, with a temperature stability of (0.1 K. 1H chemical shifts are given in parts per million after external calibration using a solution of acetone in D2O. For 1H T1 experiments, the standard inversion-recovery pulse sequence was used. 1H NOESY and DQF-COSY experiments were performed using standard Bruker pulse programs. NOESY experiments with mixing times ranging from 100 to 300 ms were acquired. DQF-COSY spectra were used for the 1H peak assignment. Where required, the linewidths were determined with the SPORT-NMR spectral analysis package.27 The chemical shifts of the Phe2-β protons, together with the J coupling values involving these protons, were obtained through a fitting procedure implemented in the program SpinWorks, written and made available by Dr. Kirk Marat.28 2.3. Determination of Binding Constant. The binding of Ala-Phe-Ala to CsPFO micelles has been monitored by measuring the 1H chemical shifts in isotropic micellar solution as a function of Ala-Phe-Ala concentration. The following equilibrium between the peptide P and the surfactant S establishes
Sn + P h SnP
(1)
Ala1-β Phe2-aromaticd Phe2-βe Ala3-R Ala3-β
T1 (s)
sample 1a
sample 2b
sample 1c
sample 2b
3.66 1.32 7.48 3.43/3.19 4.35 1.55
4.14 1.65 7.51 3.39/3.21 4.28 1.49
2.50 0.77 1.38 0.38/0.41 2.97 0.80
2.69 0.96 2.18 0.57/0.52 3.00 0.99
a Measured in a CsPFO/D O solution characterized by an Ala-Phe2 Ala concentration of 5 mM. b Measured in a 0.9 mM Ala-Phe-Ala solution in D2O. c Measured in a CsPFO/D2O solution characterized by an Ala-Phe-Ala concentration of 15 mM. d Center of gravity of the multiplet. e The two different values represent the two magnetically nonequivalent Phe2-β protons.
with n being equal to the number of surfactant molecules representing a binding site for the peptide.29 The equilibrium constant is assumed to be 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.30 The peptide is assumed to be monomeric, which is valid for the concentrations considered.35 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 by31
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 peptides, respectively. The overall association constant, Ka, of the peptide with the surfactant is given by the following expression:29
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. A global fit of the chemical shift values of the R and β protons of residue Ala1 observed at different Pt values was performed through eqs 2 and 3, using a home-written Microsoft Visual Fortran program; only these chemical shifts were chosen since they are the most sensitive to the presence of the micelles. Ka, n, and δbound were variable parameters of the fitting, while δfree values were fixed to the chemical shifts observed in D2O solution. In the fitting procedure, the value of n was constrained in the range of 1-150, with the upper limit given by the micelle aggregation number estimated by interpolating literature data reported for CsPFO/ D2O solutions characterized by different CsPFO weight fractions and at different temperatures.32 2.4. Construction of the Starting Model and MD Simulation Details. A stable conformation of the micelle, sampled by Balasubramanian and Bagchi during their MD production runs, was kindly provided by Prof. Balasubramanian and was used as the starting structure for our calculations. This configuration
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Figure 1. Regions of the 1H spectra due to R and β protons of residues Ala1 and Ala3 in an isotropic CsPFO/D2O solution (spectra a and c, respectively, sample 1) and in D2O (spectra b and d, respectively, sample 2).
Figure 2. Comparison of the calculated (lines) and experimental 1H chemical shift values of Ala1-R (filled circles, left vertical axis scale) and Ala1-β (open circles, right vertical axis scale) of Ala-Phe-Ala in CsPFO/D2O at different Ala-Phe-Ala concentrations (Pt).
comprised a micelle made of 62 PFO molecules, 62 Cs+ counterions, and 10 652 water molecules. In order to define initial starting models for our MD simulations, the original structure was manipulated using the programs Sybyl7.233 and xleap (AMBER934). First, water molecules were removed from the snapshot, and a minimum energy conformation of the AlaPhe-Ala peptide adopting an inverse γ-turn, with dihedral angles ψ(Ala1) ) 142.5°, φ(Phe2) ) -88.8°, ψ(Phe2) ) 68.2°, φ(Ala3) ) -142.4°, ψ(Ala3) ) 146.7°, χ1(Phe2) ) -59.4°, and χ2(Phe2) ) 100.8°,35 was located in the bulk region of the solvent at a distance of about 43 Å from the micelle core, with random orientation. The peptide sequence was designed as a zwitterion according to the experimental conditions (pH ) 7), and a Mn2+ ion with the corresponding SO42- counterion were added to the system to reproduce environmental effects. The positions of the 62 Cs+ counterions present in the provided configuration were maintained unchanged. The model was then immersed in a periodic octahedral box of TIP3P36 water molecules of dimension 78 × 78 × 78 Å3. The total number of solvent molecules was about 12 000. The built configuration was used as the starting structure for a series of MD simulations. All simulations were performed using the AMBER9 suite of programs. Partial charges for molecular fragments not present in the standard amber force fields (gaff and ff03)37,38 were obtained from DFT-B3LYP/6-31G* calculations and the RESP39,40 procedure. The preliminary preparation of the system, geared toward the relief of bad solute-solvent interactions, consisted of relaxation by energy minimization and a short
constant volume MD run (150 ps) during which the temperature was gradually increased to 310 K while the micelle, the peptide, and Cs+ counterions were kept fixed. The resulting structure was then equilibrated at T ) 310 K through an additional 150 ps of MD simulation in the NVT ensemble, allowing the peptide, but not the micelle with its counterions, to move freely along with the solvent. At this point, the simulation conditions were changed from constant volume and temperature to constant pressure (1 atm) and temperature (NPT) and, starting from this configuration, NPT-MD calculations were conducted for 7 ns, with the first nanosecond considered as equilibration and the last 6 ns used for analysis. During NPT runs, in order to keep the integrity of the micelle, harmonic position restraints with a force constant of 25 kcal/(mol Å2) were applied to the fluorocarbon tails of PFO, whereas carboxyl headgroups and the first two CF2 groups were not constrained. During the simulations, all bond lengths were fixed at their equilibrium values by using the SHAKE41 algorithm with a tolerance of 0.0004 Å, which allowed a time step of 2 fs. A nonbonded pair list was used to accelerate calculations and was updated every 25 steps. All calculations used periodic boundary conditions to avoid edge effects. Nonbonded cutoff was fixed at 10 Å, and Coulomb interactions were treated using the particle mesh Ewald method included in AMBER9. Constant temperature was maintained with the Andersen coupling scheme,42,43 whereas pressure was preserved using a Berendsen’s barostat.44 Coordinates were saved every 10 ps and the simulation trajectory was either analyzed with tools from the AMBER9 package or coded in-house. 2.5. Analysis of Molecular Tumbling. The correlation time for the overall molecular tumbling was estimated by calculating the reorientational correlation function defined as
C(t) ) 〈P2(µ b(0)‚µ b(t))〉
(4)
where P2 is the second-order Legendre polynomial and b µ(t) is the unit vector along the N-terminus-C-terminus direction at time t. According to the procedures and formalism reported in the literature,45-51 C(t) may be factored as
C(t) ) CO(t)CI(t)
(5)
where CO(t) and CI(t) describe the global and internal reorientations, respectively. Since the internal correlation for the N-terminus-C-terminus vector was almost equal to unity, CO(t) had the same correlation time as the correlation function for overall motion. The description of rotational relaxation was obtained by assuming a double exponential decay due to the
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Figure 3. Expansions of selected regions of the 1H spectra of Ala-Phe-Ala in CsPFO/D2O in the presence of 0.1 mM Mn2+ (upper trace) are compared to the corresponding regions of spectra recorded without Mn2+.
contribution of faster and slower tumbling of the peptide far from and close to the micelle, respectively. The global correlation function can thus be written as
CO(t) ) Aslowe-t/τslow + Afaste-t/τfast
(6)
where Aslow + Afast ) 1, and τslow and τfast are the correlation times of the overall reorientational motion in the two different situations mentioned above. The average correlation time, τ, is
τ ) Aslowτslow + Afastτfast
(7)
2.6. Determination of Ala-Phe-Ala/Micelle Adsorption Free Energy. The Ala-Phe-Ala/micelle adsorption free energy was calculated using the probability ratio method.52,53 This technique has been successfully used on similar systems and described in detail.54 The overall free energy of adsorption, ∆Gads, for the peptide was calculated as
∆Gads ≈ -RT
() Pi
∑i Pi ln P
∆di
(8)
0
where R is the ideal gas constant, T is the absolute temperature, Pi and P0 represent the probability densities of the peptide being in two particular distance intervals from the micelle center of mass, and ∆di is the width of the interval, chosen to be equal to 0.2 Å. P0 is the normalized probability density of a reference state defined considering positions far away from the micelle where interactions were negligible; P0 was calculated by averaging the Pi value between a cutoff distance of 40 Å and the maximum distance found. 3. Results 3.1. 1H NMR. Spectra of Ala-Phe-Ala in CsPFO Micellar Solutions. The assignment of the 1H signals of Ala-Phe-Ala observed in a CsPFO/D2O solution containing 0.1 mg of AlaPhe-Ala and in aqueous solution without the micelles was made on the basis of DQF-COSY spectra and is reported in Table 1. Phe2-R is not given because this signal is overlapped to that due to HOD. The chemical shifts due to protons on residue Ala1 are particularly sensitive to the presence of the micelles. In Figure 1, the regions of the 1H spectra due to R and β protons of residues Ala1 and Ala3 in an isotropic micellar solution (a and c, respectively) and in D2O (b and d, respectively) are shown as an example.
NOESY spectra of Ala-Phe-Ala in CsPFO show weak intraresidue cross-peaks for all residues, but no interresidue signals. Binding Constant. A global fit of the chemical shifts of the R and β protons of residue Ala1, the most sensitive to the presence of the micelles, was performed according to the model described in the Materials and Methods section. In Figure 2 the best-fit chemical shift values are compared to the experimental ones. A Ka value on the order of 250 ( 50 M-1 was found, which implies that ∆G0 ≈ -3.8 kcal/mol. The best fitting n value was 75, and δbound values of 3.5 and 1.2 ppm were determined for the R and β protons, respectively. Spectra of Ala-Phe-Ala in CsPFO/D2O Solutions in the Presence of Mn2+. The addition of Mn2+ to Ala-Phe-Ala in the isotropic micellar solution causes differential line broadening of the peptide 1H signals, indicating different average distances between different peptide moieties and the paramagnetic ion Mn2+. In Figure 3 the regions of the 1H spectra of Ala-Phe-Ala due to the proton groups of interest in the presence of Mn2+ are compared to the corresponding regions without Mn2+. The observed changes in line width are summarized in Table 2. The resonances due to residue Ala1 are broadened more extensively than those of Phe2 and Ala3. Significant broadening is induced at a 0.1 mM Mn2+ concentration. On the basis of studies reported in the literature for analogous systems,19,55 we hypothesize that Mn2+ ions are located preferentially close to the surface of the micelles, electrostatically interacting with CsPFO carboxylate groups. In fact, in the presence of Mn2+, the resonance of CsPFO carboxylic carbon is broadened, while that due to the CF3 carbon, located inside the micelle and far from water, is not affected (data not shown). This indicates that the Mn2+ ion does not deeply penetrate the micelle. In addition, in a 0.1 mM Mn2+ solution, Ala-Phe-Ala signals are broader when micelles are present, rather than when they are not (data not shown). This can be interpreted as being due to a confinement effect of Ala-Phe-Ala and Mn2+ in the proximity of the micelles. 1H Spin-Lattice Relaxation Times. 1H T experiments were 1 performed both on Ala-Phe-Ala in an isotropic micellar solution and in D2O at 310 K. The Ala-Phe-Ala concentration used ensured that the T1 measured was primarily that of the bound peptide. For all peptide NMR signals observed, the integrals as a function of the delay after the π pulse showed a single exponential behavior characterized by a time constant T1 in both water and micellar solution. The values found are given in Table 1. All the T1 values of Ala-Phe-Ala protons when the peptide is bound to the micelle are slightly reduced with respect to those
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TABLE 2: Line Width of Ala-Phe-Ala Resonances before (A) and after (B) the Addition of Mn2+ (0.1 mM) to the Solution of the Peptide in CsPFO/D2O at 310 K line width (Hz) proton group 1
Ala -R Ala1-β Phe2-aromatica Phe2-β Ala3-R Ala3-β a
A
B
2.0 2.9 29 3.3 2.9 3.8
15.0 6.9 29 6.5 4.3 5.3
Width at half height of the whole multiplet.
observed when the peptide is free, with the largest reduction being shown by the Phe2-aromatic protons. 3.2. MD Simulations. Ala-Phe-Ala Location and Dynamics. Examination of Ala-Phe-Ala individual structures revealed that, as expected, the molecule approached the micelle orienting its charged N-terminus group toward the micelle surface in such a way to form hydrogen bonds with the carboxyl headgroups of the PFO molecules, thus suggesting that the driving force behind Ala-Phe-Ala diffusion is of electrostatic type and mainly due to the positive charge of the N-terminus. Five representative snapshots illustrating a possible approaching path and characteristic features of plausible peptide-micelle complexes are shown in Figures 4 and 5. To elucidate further Ala-Phe-Ala motion, the distances of four different peptide points from the center of mass of the micelle, that is, the peptide center of mass, the benzene ring center, the nitrogen on NH3+, and the center of mass of the two oxygens in the COO- groups, were monitored as a function of the simulation time. As it appears from the evolution of distances (Figure 6), during the first 800 ps of the simulation, Ala-Phe-Ala diffused from the bulk region of the solvent, where it was located at the end of the equilibration phase and where all its groups had similar distances from the micelle core, to the interfacial region of the micelle. Then, a time interval of about 1600 ps was necessary for the molecule to accommodate close enough to the surface in order to engage direct interactions with the various groups of the micelle. During this period, both the location and conformation of the molecule changed frequently, and contacts were repeatedly broken and reformed. After this transient phase, a stable configuration was reached, and the peptide remained bound to the surface for about 1400 ps (t ) 3800 ps). MD data indicate that Ala-Phe-Ala approached the PFO aggregate, reorienting the NH3+ group toward the micelle surface, adopting, at first, a nearly perpendicular alignment with respect to the micelle-water interface, turning away the C-terminus group in order to minimize C-terminus-COO- (PFO) repulsion (Figure 5). When the NH3+ moiety was close enough to the surface acceptor headgroups, quite strong hydrogen bonds were formed and maintained, allowing the peptide to rearrange its conformation again and favorably interact with the micelle also through the backbone and ring atoms. However, the N-terminus hydrogen bonds were extremely intermittent, frequently breaking and reforming. Indeed, the N-terminus group could form at least three unique hydrogen bonds at any given time because of its three donor atoms, which competed for acceptors. All donors were attracted electrostatically to different acceptor sites simultaneously, because the headgroups were abundant, densely packed, and able to move to some extent in response to the environment. As a consequence, competition, and hence intermittency in hydrogen bond formation, was observed. Competition with water molecules was also noticed; in fact, favorable interactions of
Figure 4. Ala-Phe-Ala/micelle approaching path. All atoms are shown in stick mode. Water molecules and ions are not shown for clarity. Color codes: hydrogen ) white, oxygen ) red, carbon ) gray, nitrogen ) blue, and fluorine ) green.
the micelle head groups with water molecules perturbed peptide adsorption to some extent, and, after about 900 ps (t ) 4700 ps), the molecule moved away at a distance of about 12 Å from the interface (35 Å from the micelle core) in an apparently random manner. A relatively stable water layer was formed and maintained for about 250 ps (t ) 4950 ps), then again the highly mobile and flexible Ala-Phe-Ala molecule approached the surface, and the temporary hydrogen bonds formed between water molecules and carboxyl groups of the PFO chains were promptly displaced by the positively charged and polar functional groups of the peptide, which again formed close contacts with the micellar surface.
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Figure 5. Possible Ala-Phe-Ala/micelle complexes. All atoms are shown in stick mode. Hydrogen bonds are displayed as dotted black lines. Water molecules are not shown for clarity. Color codes: hydrogen ) white, oxygen ) red, carbon ) gray, nitrogen ) blue, fluorine ) green, Cs+ ) violet, and Mn2+ ) magenta.
Figure 6. Distance of the Ala-Phe-Ala groups from the center of mass of the micelle as a function of the simulation time.
Intermolecular Hydrogen Bonds. Intermolecular hydrogen bonds were identified when donor-acceptor distances were smaller than 3.5 Å, and the angle formed by hydrogen-donoracceptor atoms were smaller than 40°. Intermolecular NH‚‚‚ F(PFO) (nhf) and NH‚‚‚O(PFO) (nho) hydrogen bonds were observed, and both of them were equally populated. In both types, the majority of H-bonds involved the NH3+ (Ala1) group, representing about 92% of the nhf type and 75% of the nho type, respectively. H-bonds with NH(Phe2) were less populated (8% of nhf and 17% of nho), and for NH(Ala3), only nho type bonds were observed (8%). All these hydrogen bonding interactions were established with, at most, five different PFO molecules of the micelle, in the whole simulation time, suggesting that the Ala1-Phe2 portion of the peptide has the tendency to move on the interface and, at the same time, insert
its functional groups into accessible cavities of complementary potential. The total micelle surface area (ASA) available for interaction with the surrounding environment was quantified through the method of Lee and Richards,56 and the section of the surface in contact with Ala-Phe-Ala was evaluated. Smooth oscillations around an ASA of 7220 ( 50 Å2 were observed for the micelle, and the peptide-micelle contact region was about 15% of the total surface area. Residence Times. A series of Ala-Phe-Ala residence times on the micelle surface were calculated by using the whole trajectory and averaged out to obtain a mean residence time (MRT). Such values were determined for various moieties of the peptide, namely, NH3+(Ala1), NH(Phe2), ring(Phe2), NH(Ala3), and COO-(Ala3), to give a description of Ala-Phe-Ala mobility and the strength of the intermolecular interactions (Table 3). Residence times of a given PFO molecule around the aforementioned groups were calculated as follows. At a given configuration, only those PFO molecules within a distance less than 3.5 Å (corresponding to the distance chosen for hydrogen bond identification) were considered to be bound to the group of interest. Any PFO molecule that returned to this coordination shell after escaping for less than 10 ps was considered to be continuously bound to the selected group. However, when any of the PFO molecules was out of the coordination shell of the chosen Ala-Phe-Ala group for longer than 10 ps, it was considered to be a free molecule (i.e., not bound to that group). A variation from bound to free and vice versa is here defined as a transition between two different states. Thus, residence time is the duration of time for which a PFO molecule was bound to an Ala-Phe-Ala group. Considering that more than one PFO molecule can be present in the coordination sphere, different residence times, corresponding to the presence of zero (0 PFO), one (1 PFO), and two (2 PFOs) PFO molecules can be calculated.57,58 In Table 3, the MRTs calculated over all the different states observed in the
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TABLE 3: Residence Times (ps) for a Particular Number of PFO Molecules around NH3+(Ala1), NH(Phe2), Ring(Phe2), NH(Ala3), COO-(Ala3) Groups in Ala-Phe-Ala Tripeptidea 0 PFO group
mean
σ
1 PFO max mean
79 124 550 NH3+(Ala1) NH(Phe2) 197 323 1100 Ring(Phe2) 133 273 1290 NH(Ala3) 641 1069 2460 3 COO (Ala ) 2395 290 2600
57 59 14 53 10
σ
2 PFOs max mean σ max
68 260 120 460 6 30 61 150 0 10
15 0 10 0 0
7 0 0 0 0
20 0 10 0 0
a Three different cases are reported, namely the occurrence of zero (0 PFO), one (1 PFO) and two (2 PFOs) PFO molecules in proximity to the group of interest. For each case, mean and maximum (max) values and the standard deviation (σ) are reported.
sampling time as well as the maximum values and standard deviations are reported. Taking into account that the first contact was observed at t ) 930 ps (according to the aforementioned definition), residence times were evaluated starting from that time until the end of the simulation run. As can be expected, the 0-PFO MRT for NH3+(Ala1) was the shortest, whereas the 2-PFO MRT was the longest. 1-PFO MRT was comparable with the ones displayed by both NH(Phe2) and NH(Ala3) groups. On the other hand, the 0-PFO MRTs of these moieties were much longer than the one observed for NH3+(Ala1), supporting the picture that contacts with the NH groups of Phe2 and Ala3 residues were established only after the NH3+(Ala1)‚‚‚PFO salt bridge was formed. Binding of the NH3+(Ala1) group was highly intermittent, as confirmed by a greater number of transitions (71) of one PFO molecule between the first and second (distance > 3.5 Å) coordination shell, observed in about 5 ns, with respect to the other moieties where this number was 37, 55, 13, and 3 for NH(Phe2), ring(Phe2), NH(Ala3), and COO-(Ala3), respectively. The total number of transitions in the case of 2 PFO molecules is equal to 2 for both the ring and the NH3+ group. From the data reported in Table 3, it is evident that most of the time the benzene ring was found relatively far from the micelle surface, even though a few short-lived contacts with one or two PFO molecules were observed. A very long 0-PFO MRT, an almost zero 1-PFO MRT, and a zero 2-PFO MRT for COO-(Ala3) indicate that this moiety during the simulation time only very rarely reached the micelle surface and preferred to remain oriented toward the solvent favorably interacting with the surrounding water molecules. Molecular Motions. A rough account of the molecular motions can be gained from time-correlation functions even at fairly short simulation times.59 Figure 7a displays the timecorrelation functions of the interatomic N-H vectors for Phe2 and Ala3, the HR-Cβ vectors for Ala1 and Ala3, the CR-Cγ vector for Phe2, and the N(Ala1)-COO(Ala3) vectors. In principle, these functions depend on both internal motions and overall rotational tumbling. Figure 7b illustrates the timecorrelation functions for the same internuclear vectors after removal of the overall peptide rotation by superimposing each MD snapshot onto the starting conformation (backbone atoms). All the vectors except the one connecting N(Ala1) with the carbon of the carboxylic group of Ala3 show the typical behavior of a highly flexible system with remarkable internal motions during the whole simulation time, and the correlation functions do not reach plateau values. On the other hand, the timecorrelation function for the N-terminus-C-terminus vector has the characteristic trend of a rigid structure, being substantially flat and deviating only slightly from unity.
Figure 7. Reorientational correlation functions for Ala-Phe-Ala before (a) and after (b) removing overall rotation. Best fit of a double exponential function for the N-terminus-C-terminus vector correlation function is shown as an orange line (c).
The correlation time for the overall molecular tumbling was estimated according to eqs 4 and 5, considering CI(t) ≈ 1, as observed above. The fitting of the global correlation function CO(t), assuming a double exponential decay, as outlined in the Materials and Methods section, gave the following best-fit parameters: Afast ) 0.65 ( 0.02, Aslow ) 0.35 ( 0.02, τfast ) 55 ( 3 ps, and τslow ) 450 ( 17 ps, with R2 ) 0.995, and the fitting curve is shown in Figure 7c. The average correlation time was about 193 ps. Results of analogous MD simulations of Ala-Phe-Ala molecules in pure water (data not shown) gave A ) 0.99 ( 0.01 and a correlation time of 49.5 ( 0.5 ps (R2 ) 0.999). Ala-Phe-Ala Conformational Characteristics. The conformational behavior of Ala-Phe-Ala was analyzed considering its backbone and side chain dihedral angles, focusing our attention on the important regions of the Ramachandran maps corresponding to the β-strand (β) (-180° < φ < -100°, 90° < ψ < 180° and -180° < φ < -100°, -180° < ψ < -120°), polyproline II (PPII) (-100° < φ < -50°, 100° < ψ < 180°), right-handed R-helix (RR) (-180° < φ < 0°, -120° < ψ
