NMR Investigation of the Electrostatic Effect in Binding of a

Mar 16, 2007 - Location, Structure, and Dynamics of the Synthetic Cannabinoid Ligand CP-55,940 in Lipid Bilayers. Tomohiro Kimura , Kejun Cheng , Kenn...
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J. Phys. Chem. B 2007, 111, 3831-3838

3831

NMR Investigation of the Electrostatic Effect in Binding of a Neuropeptide, Achatin-I, to Phosphatidylcholine Bilayers Tomohiro Kimura,*,† Keiko Ninomiya, and Shiroh Futaki Institute for Chemical Research, Kyoto UniVersity, Uji, Kyoto 611-0011, Japan ReceiVed: October 29, 2006; In Final Form: February 5, 2007

Achatin-I (Gly1-D-Phe2-Ala3-Asp4), known as a neuropeptide containing a D-amino acid, binds to the surface of a zwitterionic phosphatidylcholine (PC) membrane only when the peptide N-terminal amino group is in the ionized state, NH3+ (Kimura, T.; Okamura, E.; Matubayasi, N.; Asami, K.; Nakahara, M. Biophys. J. 2004, 87, 375-385). To gain mechanistic insights into how the binding equilibrium is delicately controlled by the ionization state of the N-terminal amino group, peptide-lipid binding interactions are investigated by selectively enriched 15N (at the N-terminus) and natural-abundance 13C NMR spectroscopy. Upon binding to the PC membrane, the 15N NMR of the N-terminal NH3+ shifts upfield. This observation supports a mechanism that the role of the N-terminal NH3+ in stabilizing the binding state is through electrostatic attraction with a headgroup negative charge, i.e., PO4-. Interestingly, when the side chain β-carboxyl group in Asp4 is deionized at acidic pH, the 15N signal of the N-terminal NH3+ exhibits no significant chemical-shift change upon membrane binding of achatin-I. The Asp4 side chain thus regulates efficiency of the electrostatic binding between the peptide N-terminal NH3+ and the lipid headgroup PO4-. 13C chemical shifts in the hydrophobic D-Phe2 residue are largely perturbed upon membrane binding, in the case where the side chain β-CO2- in Asp4 is deionized; the deionization of Asp4 β-CO2- increases the net hydrophobicity of achatin-I with a reduction of both the electrostatic hydration and the electrostatic attraction with the headgroup N(CH3)3+ in the most superficial region of the PC membrane, resulting in deeper anchoring of the phenyl ring. Hence, the electrostatic effect of the side chain β-CO2- in Asp4 floats achatin-I on the PC membrane surface, and the binding equilibrium is sensitively controlled by the ionization state of the N-terminal NH3+.

1. Introduction Elucidation of microscopic peptide-lipid interactions controlling the binding state and equilibrium of bioactive peptides on the phospholipid membrane surface is a challenging key subject to understand the sophisticated physiological functions performed through the membrane-binding phenomena.1-3 In a previous work, we aimed at solving issues on the binding equilibrium, location, and side chain conformations of a neuropeptide achatin-I (Gly1-D-Phe2-Ala3-Asp4) in phosphatidylcholine (PC) bilayers by using natural-abundance 13C and high-resolution (on the order of 0.01 Hz) 1H NMR spectroscopy; egg-yolk PC was used for the model PC bilayers.4 Within the short primary amino acid sequence, achatin-I contains ionic, hydrophobic, and stereoisomeric (D-configuration) residues. The peptide therefore can provide a strategic model system to gain basic concepts on peptide-lipid interactions.5-12 As one of the notable features, achatin-I binds to the zwitterionic PC membrane surface only when the peptide N-terminal amino group is in the ionized state NH3+.4 To have mechanistic insights into the notable role played by the N-terminal NH3+ in determining the binding equilibrium, here we selectively enriched the 15N isotope at the NH3+ group (Figure 1), and interactions with the PC lipids have been probed by the 15N NMR. In scrutinizing the electrostatic effect of the N-terminal NH3+, we pay special attention to the presence of another ionic group, CO2-, in the * To whom correspondence should be addressed. Phone: (301) 4356727. Fax: (301) 594-0035. E-mail: [email protected]. † Present address: Laboratory of Membrane Biochemistry and Biophysics, NIAAA, National Institutes of Health, Bethesda, MD 20892.

Figure 1. Chemical structures of an amphipathic neuropeptide achatin-I and phosphatidylcholine at a membrane surface. The N-terminal NH3+ of achatin-I marked with asterisk has been selectively enriched with the 15N isotope.

C-terminal Asp4, because the electrostatic effect of CO2- in free energy (kJ/mol) is considered to be as large as the electrostatic effect of NH3+.13-15 Achatin-I is widely known as a neuropeptide containing a D-amino acid.16 In the central nervous system of an African giant snail, Achatina fulica Fe´russac, the peptide excites heart regulatory neurons by inducing inward transmembrane current

10.1021/jp067100x CCC: $37.00 © 2007 American Chemical Society Published on Web 03/16/2007

3832 J. Phys. Chem. B, Vol. 111, No. 14, 2007 due to Na+ ions16 and controls cardiac activities.17 A reason why this species is of significant interest for neuroscientists is the availability of huge neurons to study electrophysiological activities.18 Besides the excitatory function, modulatory functions have been discovered on achatin-I against transmembrane current induced by other neurotransmitters, such as oxytocin and acetylcholine.19 In elucidating the molecular mechanism of such diverse physiological roles of achatin-I in the microscopic membrane environment,16,17,19-23 it is important to have fundamental knowledge on binding interactions with phospholipids as an essential component of the membrane embracing receptor proteins and ion channels. Previously, we studied binding of achatin-I to PC membranes, because PC is a major phospholipid in diverse neural membranes.24 We reported that achatin-I binds to PC membranes at neutral pH and the binding equilibrium sensitively depends on the peptide ionization state; the electrostatic effect in determining the equilibrium at physiological neutral pH can be examined by monitoring how the binding equilibrium is affected by changes in the peptide ionization state through pH variation.4 The observation that achatin-I binds to the membrane tells the significance of peptide-lipid interactions in regulating the thermodynamics and kinetics of physiological events at the synaptic membranes. What then is the underlying molecular mechanism of the peptide ionization-state dependence of the binding equilibrium? The analysis of phospholipid 13C NMR has shown an outline view of the location of achatin-I in the PC membrane; the large part of the peptide structure lies around the glycerol to the ester carbonyl groups (Figure 1) at physiological pH.4 The 31P signal of the headgroup PO4- in PC shifts downfield upon peptide binding to the membrane surface.4 Those observations, together with the binding equilibrium showing the notable ionizationstate dependence of the peptide N-terminal NH3+, imply electrostatic binding between the peptide N-terminal NH3+ and the lipid headgroup PO4-. Does an ammonium ion bind at the phospholipid PO4-? As for free NH4+ ions there is a longstanding interest in binding interactions with phospholipid membranes, in relation to mechanistic interpretations of the lyotropic series known as the Hofmeister effect.25-28 Molecular dynamics simulations of the PC membrane successfully probed the preferential binding between NH4+ ions and the headgroup PO4-.29 This computational result also leads us to consider that the N-terminal NH3+ of achatin-I has a propensity to bind to the headgroup PO4-. Recently one of us (T.K.) reported that the N-terminal NH3+ of a human neuropeptide, Met-enkephalin (Tyr1-Gly2-Gly3-Phe4-Met5), electrostatically binds at the headgroup PO4- in the PC membrane.30 The headgroup PO4of phospholipids is known to attract a positively charged guanidinium group of arginine in cationic peptides;31,32 cf., for example, the binding of antimicrobial peptide tachyplesin I to membranes containing phosphatidylglycerol.31 To gain a direct evidence of the binding between the N-terminal NH3+ of achatin-I to the headgroup PO4- of PC, however, spectroscopic probing of the membrane-binding effect on 15N NMR at the N-terminal NH3+ of achatin-I is necessary. The 15N resonance within the functional group of interest, i.e., NH3+, is expected to reflect sensitively the peptide-lipid binding interaction in spite of the significant hydration of achatin-I on the PC membrane surface.4 While the unbinding of achatin-I in response to the deionization of the peptide N-terminal NH3+ indicates a significant electrostatic effect of the NH3+ in stabilizing the binding state,4 it should be emphasized that this unbinding phenomenon does not mean that the other structural parts of achatin-I do not control

Kimura et al. the binding state. The observed electrostatic effect of the NH3+ on the binding equilibrium is most likely only an outcome in a good counterbalance between stabilizing and destabilizing factors of the binding state in the other structural parts of achatin-I. The peptide contains negatively charged R- and β-CO2- in the C-terminal Asp4 and a hydrophobic phenyl ring in the D-Phe2, as well as the positively charged N-terminal NH3+. In stabilizing the binding state, the CO2- groups can contribute through the electrostatic attraction with the lipid headgroup N(CH3)3+ and the phenyl ring through the hydrophobic interactions. In stabilizing the free state in bulk water, the intense electrostatic hydration of the CO2- groups plays a key role.4,14,15 What if we then perturb the counterbalancing factors by deionizing the side chain β-CO2- in Asp4 by decreasing pH? As the electrostatic effect of ionic CO2- is larger than that of nonionic CO2H, the deionization of the side chain β-CO2- is expected to induce significant changes of the binding state; changes of the binding state on the membrane surface can be sensitively probed by natural-abundance 13C NMR of the peptide.4,30 Therefore, it is of significance to examine the efficiency of electrostatic attraction of interest between the peptide N-terminal NH3+ and the lipid headgroup PO4- by changing the ionization state of the side chain β-carboxyl group in Asp4. 2. Experimental Section 2.1. Materials. Achatin-I (Gly1-D-Phe2-Ala3-Asp4) selectively 15N-labeled at the N-terminal NH3+, [15NH3+]-achatin-I, was prepared by the Fmoc solid-phase synthesis.33 Fmoc-amino acid derivatives for D-Phe, Ala, and Asp, and Wang resin were purchased from Merck Japan (Tokyo, Japan). A t-Boc derivative of 15N-glycine (Boc-[15N]-Gly) was from ISOTEC (Kawasaki, Japan). Achatin-I monoammonium salt without the 15N-labeling, used for 13C NMR measurements, was purchased from Sigma Chemical (St. Louis, MO). Egg-yolk phosphatidylcholine (PC) was from NOF (Tokyo, Japan). The egg-yolk PC membrane is in the liquid-crystalline phase at the investigated temperature of 30.0 ( 0.1 °C, as the transition temperature between the gel and liquid-crystalline phases is at -15 to -7 °C.34 Solvent heavy water (2H2O; 99.90% 2H) was from Euriso-top (Saint Aubin, France). Solvent light water (H2O) used for 15N NMR measurements was purified by a WEX-10 water-purification system from Millipore (Billerica, MA). The 2H2O solution of 2HCl (18 wt %) or NaO2H (40 wt %) used to adjust the solution pH (p2H) was from Aldrich Chemical (Milwaukee, WI). 2.2. Synthesis of [15NH3+]-Achatin-I. [15NH3+]-achatin-I was prepared by Fmoc solid-phase peptide synthesis.33 Wang resin (0.61 g, substitution level 0.55 mmol/g) was treated with FmocAsp(OtBu)-OH (0.41 g, 1 mmol) in the presence of diisopropylcarbodiimide (DICDI) (157 µL, 1 mmol), HOBt (135 mg, 1 mmol), and dimethylaminopyridine (12.3 mg, 0.1 mmol) in dimethylformamide (DMF) at 20 °C for 1.5 h. After deprotection of the Fmoc group by treating with 20% piperidine in DMF for 20 min, Fmoc-Ala-OH (311 mg, 1 mmol) was introduced in the reaction with DICDI (157 µL, 1 mmol) and HOBt (135 mg, 1 mmol) for 2 h. Repetitive removal of the Fmoc groups followed by introduction of amino acid derivatives gave the protected peptide resin (Boc-[15N]-Gly-Phe-Ala-Asp(OtBu)resin) (810 mg). Then, the whole amount of protected peptide resin was treated with trifluoroacetic acid (TFA) (9.5 mL) in the presence of 1,2-ethanedithiol (0.5 mL) at 20 °C for 3 h. After the resin was removed by filtration, the filtrate was evaporated under N2 atmosphere. Ether (40 mL) was added to the resulting oil to give a powder, which was collected by

Membrane Binding of Achatin-I centrifugation. The powder was dissolved in 10% acetic acid and subjected to lyophilization. The resultant powder (110 mg) was then further purified by high-performance liquid chromatography (HPLC) with a YMC-Pack Pro C18 column (20 mm × 150 mm) [eluent, A ) H2O containing 0.1% TFA, B ) CH3CN containing 0.1% TFA; gradient, 10-25% B in A over 40 min; flow rate, 4 mL/min; detection, 215 nm]. The eluate corresponding to the main peak was collected and lyophilized to afford a white fluffy powder (43 mg, 32% yield from the initial introduction of Asp on the resin). Fast atom bombardment mass spectrometry (FAB-MS): m/z ) 410 (M + H)+ (calcd 410). After the purification by HPLC, impurity was confirmed to be negligible based on 1H NMR. 2.3. Sample Preparation. A mixture of achatin-I and the LUV of egg-yolk PC was prepared at the concentrations of 5 and 50 mM for the peptide and lipid, respectively. The LUV were prepared by the extrusion method as follows. A required amount of egg-yolk PC was dissolved in chloroform (Wako Pure Chemical; Osaka, Japan), and the chloroform solution was evaporated by a rotary vacuum evaporator. The lipid film thus formed on the flask wall was dried overnight under vacuum. The dried lipid film was vortex mixed in water for ∼5 min to prepare a homogeneous suspension of multilamellar vesicles (MLV). To obtain the LUV with a diameter of 100 nm, 10 cycles of freezing and thawing were carried out for the suspension, and the resultant was extruded 21 times through a polycarbonate filter with the pore diameter of 100 nm. Then solid achatin-I was dissolved in the LUV dispersion. Achatin-I in aqueous solution was prepared at 5 mM corresponding to the concentration in the peptide-LUV mixture. For the 15N NMR measurements, the samples were prepared with 9:1 mixture in volume of H2O and 2H2O solvents. For the 13C NMR measurements, 2H2O solvent was used. The samples were prepared at three different pH values, i.e., 3.20, 7.00, and 11.60, to change the ionization states of the amino and carboxyl groups in achatin-I. The R-amino group at the N-terminus, the R-carboxyl group at the C-terminus, and the β-carboxyl group in the C-terminal Asp4 side chain are in the dominant ionization states of (R-NH3+, R-CO2-, β-CO2H) at acidic pH ) 3.20, (R-NH3+, R-CO2-, β-CO2-) at neutral pH ) 7.00, and (R-NH2, R-CO2-, β-CO2-) at basic pH ) 11.60. Those ionization states correspond to the ones in the previous work,4 where we investigated the ionization-state dependence of the equilibrium of binding between achatin-I and the LUV of egg-yolk PC. The LUV were confirmed to be stable in the examined pH range by the 1H NMR spectrum. It should be noted that the acidification to pH ) 3.20 deionizes the side chain β-CO2- in the C-terminal Asp4, corresponding to the pKa of 3.9.35 The pKa of the Asp R-carboxyl group is ∼2 units smaller than that of the β-site.35 We did not decrease the pH below 3.2 to deionize the Asp R-CO2-, because it does not ensure the full ionization of the PO4- in PC according to the intrinsic pKa ) 0.8.36 The ionization state of the PC headgroup was thus fixed throughout the examined pH range in the zwitterionic PO4- and N(CH3)3+ as confirmed by the choline methylene CH2OP signal in 13C NMR. At each of the acidic, neutral, and basic conditions, the difference in pH values between the samples of (1) achatin-I in aqueous solution and (2) the mixture of achatin-I and the LUV was controlled within 0.01 at the resolution of the pH meter (D-21 model, HORIBA; Kyoto Japan), so we can neglect uncertainties of the ionization state due to pH adjustment. On samples prepared with 2H2O solvent for 13C NMR measurements, the pH values shown are corrected for the isotope effect

J. Phys. Chem. B, Vol. 111, No. 14, 2007 3833 by adding 0.4 unit to the reading of the pH meter with a glass electrode.37 On samples prepared with the 9:1 mixture in volume of H2O and 2H2O solvents for the 15N NMR measurements, the pH values shown are the direct readings of the pH meter. 2.4. 15N NMR Measurements. The 15N signal of the labeled achatin-I at the N-terminus was measured on a JEOL (Tokyo, Japan) 600 MHz spectrometer (JNM-ECA 600 model with a superconducting magnet of 14.10 T) operating at the 15N frequency of 61 MHz. The spectrum was recorded at three different pH values of 3.20, 7.00, and 11.60 for both (1) achatin-I (5 mM) in aqueous solution and (2) the mixture of achatin-I (5 mM) and the PC(50 mM)-LUV. We equipped the magnet with a newly developed high-power tunable probe (T10A model, JEOL) for a large volume tube of 10 mm o.d. The temperature was controlled at 30.0 ( 0.1 °C. Measurements were carried out with the irradiation of 1H signals. The 32 768 data points were sampled over the spectrum range of 100 ppm, corresponding to a digital frequency resolution of 0.003 ppm (0.2 Hz). A total of 500-8000 scans were acquired for the free induction decay (FID) signals. The 15N chemical shifts (δ) relative to free 15NH + in aqueous solution (observed for 5 mM 15NH NO 4 4 3 (g98% 15N; CIL, MA) in aqueous solution) were obtained by referring to the locking frequency of the solvent deuteron. The spectra are shown without phase correction regarding the negative nuclear Overhauser enhancement factor for the 15N1H dipolar interactions.38 2.5. 13C NMR Measurements. The binding state of achatin-I on the PC membrane surface was probed by natural-abundance 13C NMR of the peptide. Probing of the peptide 13C NMR is advantageous over probing of the lipid 13C NMR in determining details of the binding state of the peptide on the membrane surface. Note that, when the membrane-bound fraction of achatin-I is, e.g., 10% at pH ) 3.20 at the peptide-to-lipid molar ratio of 1/10, the perturbation on the peptide 13C signals upon membrane binding stems from 10% of the bound peptide, while the perturbation on the lipid 13C signals is less efficient due to the 10 times larger total number of lipid molecules. Measurements were performed on the 600 MHz spectrometer operating at the 13C frequency of 151 MHz. The spectrum was recorded for both (1) achatin-I (5 mM) in aqueous solution and (2) the mixture of achatin-I (5 mM) and the PC(50 mM)-LUV at acidic pH ) 3.20, and the results can be compared with our previous report for the neutral pH; note that achatin-I does not bind to the PC membrane at basic pH ) 11.60.4 The magnet was equipped with the high-power tunable probe (T10A model, JEOL) for the sample tube of 10 mm o.d. The temperature was controlled at 30.0 ( 0.1 °C. The 32 768 data points were sampled over the spectrum range of 250 ppm to give a digital resolution of 0.0076 ppm (1.2 Hz). A total of 10 000 scans were acquired for the FID signals. 13C chemical shifts (δ) relative to the DSS (sodium 2,2-dimethyl-2-silapentane-5sulfonate) methyl carbons were obtained by referring to the absorption frequency of the solvent deuteron monitored as the lock signal. Assignment of the 13C signals was made according to the previous report.4 3. Results and Discussion 3.1. 15N NMR of Selectively Labeled Achatin-I at the N-Terminal NH3+. Achatin-I (Gly1-D-Phe2-Ala3-Asp4) binds to the surface of the zwitterionic PC membrane only when the peptide N-terminal amino group is in the ionized state, NH3+; the membrane-bound fraction, which is in rapid exchange with the free fraction in bulk water in the NMR time scale, decreases from ∼15% to nearly none upon deionization of the NH3+

3834 J. Phys. Chem. B, Vol. 111, No. 14, 2007 according to an ultracentrifugation experiment.4 To gain mechanistic insights into the notable electrostatic effect of the N-terminal NH3+, we designed to probe the peptide-lipid electrostatic interactions responsible for stabilizing the binding state. The N-terminal NH3+ was selectively enriched with the 15N isotope by solid-state peptide synthesis, and perturbation on the 15N NMR upon membrane binding of achatin-I was examined. 3.1.1. Electrostatic Effect of the N-Terminal NH3+. 15N NMR spectra recorded at neutral pH ) 7.00 for [15NH3+]-achatin-I (5 mM) (1) in bulk water and (2) in the binding equilibrium with the LUV of egg-yolk PC (50 mM) are shown in Figure 2a. In bulk water, the N-terminal NH3+ gives the 15N resonance at 5.67 ppm. On the other hand, in the case where the peptide is in binding equilibrium with the PC membrane, the signal appears in the upper field by 0.13 ppm at 5.54 ppm. The upfield chemical-shift change indicates the electrostatic attraction with PO4- in the PC headgroup; the 15N signal of NH3+ shifts upfield upon deprotonation in pH titration,38 and the deprotonation of NH3+ can take place as a result of a salt bridge with a basic group in the phospholipid headgroup as observed notably in the case of a human opioid peptide Met-Enkephalin (Tyr1-Gly2Gly3-Phe4-Met5) even through changes in 13C resonances at carbons close to the N-terminal amino group.30 Thus, it is concluded that the notable electrostatic effect of the N-terminal NH3+ in controlling the binding equilibrium is through the attraction with the headgroup PO4-. Electrostatic binding between those groups agrees with the location of achatin-I around the glycerol to the ester carbonyl groups and with the downfield shift in 31P NMR of PO4- upon binding of achatinI.4 When achatin-I approaches from bulk water to the PC membrane surface, the positively charged N-terminal NH3+ encounters a repulsive positively charged surface layer due to the terminal trimethylammonium of the zwitterionic PC headgroup. The observed binding between the peptide N-terminal NH3+ and the lipid PO4- indicates that the potential barrier due to the charge distribution along the bilayer normal on the membrane surface is low enough to reach thermodynamic equilibrium between the bound and free states of those oppositely charged groups within the NMR time scale. The current observation thus supports the importance of charge distribution along the bilayer normal in determining the binding state and equilibrium of peptides on the membrane surface; cf., Metenkephalin anchors the negatively charged C-terminal CO2- at the lipid headgroup NH3+ on the phosphatidylserine (PS) membrane surface despite the net negative charge of the PS headgroup.30 As seen in Figure 2a, the insignificant change in the 15N signal line width upon membrane binding indicates minor changes in dynamics of the molecular environment around the N-terminal NH3+ in achatin-I. This is obviously due to the presence of the hydration effect on the peptide on the membrane surface.4 At basic pH ) 11.60, where the N-terminal amino group is in the deionized form NH2, the 15N signal shows no significant chemical-shift difference between that in aqueous solution and in the mixture with membrane (Figure 2b). This is obviously because of the negligible fraction of membrane-bound achatin-I. 3.1.2. Electrostatic Effect of the CO2- in the C-Terminal Asp4. As pointed out in the Introduction, while the electrostatic binding with the lipid headgroup PO4- is responsible for the notable electrostatic effect of the peptide N-terminal NH3+ in determining the binding equilibrium, it is important to emphasize that this notable effect of the N-terminal NH3+ is most likely only an outcome of a good counterbalance between the stabilizing

Kimura et al.

Figure 2. 15N NMR spectra of achatin-I (5 mM), selectively enriched with the 15N isotope at the N-terminal amino group, in bulk water and in the mixture with the LUV of egg-yolk PC (50 mM): (a) at neutral pH ) 7.00, (b) at basic pH ) 11.60, and (c) at acidic pH ) 3.20. The dominant ionization states of the ionizable functional groups in achatin-I are as shown.

and destabilizing factors of the binding state in the other structural parts of achatin-I. In this section, we pay particular attention to a role of the electrostatic effect played by another ionic group, i.e., CO2-. A perturbation on the counterbalancing factors was made by deionizing the side chain β-CO2- in the C-terminal Asp4. Then, the influence on the electrostatic binding between the peptide N-terminal NH3+ and the lipid headgroup PO4- was examined. The pH was adjusted to acidic 3.20 to deionize the side chain β-CO2-; it was not decreased further to deionize the C-terminal R-CO2-. This is to avoid a change in the ionization state of the zwitterionic PC headgroup N(CH3)3+

Membrane Binding of Achatin-I

J. Phys. Chem. B, Vol. 111, No. 14, 2007 3835

Figure 3. (a) Natural-abundance 13C NMR spectrum of the mixture of achatin-I (5 mM) and the LUV of egg-yolk PC (50 mM) at p2H ) 3.20. Signals from achatin-I are marked with asterisks with the carbon assignment as shown. (b) 13C chemical-shift changes ∆δ (ppm) of achatin-I upon binding to the LUV of egg-yolk PC. The ∆δ values were estimated according to the observed chemical-shift differences ∆δ between those measured in bulk water and those in the binding equilibrium with the LUV, by taking account of the membrane-bound fraction at 10% (ref 4).

and PO4-.35 At the pH ) 3.20, the fraction of membrane-bound achatin-I is 10%.4 The 15N NMR spectra of [15NH3+]-achatin-I (5 mM) recorded at acidic pH ) 3.20 (1) in bulk water and (2) in the binding equilibrium with the LUV of egg-yolk PC (50 mM) are shown in Figure 2c. In spite of the membrane-bound fraction at 10%, the 15N signal shows no significant chemical-shift difference from the one observed in bulk water. The absence of a chemicalshift difference indicates that achatin-I with the deionized side chain β-CO2H in Asp4 binds to the membrane without efficient electrostatic binding between the peptide N-terminal NH3+ and the lipid headgroup PO4-. In other words, the electrostatic effect of the Asp4 side chain β-CO2- plays a key role in controlling the binding state such that the electrostatic effect of the N-terminal NH3+ is in the efficient manner. Let us see then how the binding state is perturbed by deionizing the side chain β-CO2- in Asp4. Natural-abundance 13C NMR of achatin-I provides detailed information on the binding state according to its high atomic-site distinguishability and sensitivity to the molecular environment.

3.2. Changes in the Binding State Induced by Deionizing the Side Chain β-CO2- in the C-Terminal Asp4. 3.2.1. 13C NMR of Achatin-I. The binding state of achatin-I on the PC membrane surface can be sensitively inspected by monitoring changes of the peptide 13C NMR upon transfer from bulk water to the membrane surface.4,30 Natural-abundance 13C NMR spectrum of achatin-I (5 mM) in the mixture with the LUV of egg-yolk PC (50 mM) at pH ) 3.20 is presented in Figure 3a. The peptide signals, observed as the average for the membranebound and free states, are marked with asterisks. In Table 1 we summarize the chemical shifts (δ) together with those observed in bulk water at pH ) 3.20. Chemical-shift changes ∆δ upon transfer from bulk water to the PC membrane surface can be estimated by the observed chemical-shift differences ∆δ between those measured in bulk water and those in the mixture with the LUV, by taking account of the molar fraction of the membranebound peptide at 10%. The obtained chemical-shift changes ∆δ show us a marked carbon-site dependence. The carbon-site dependence enables us to gain insight into the site-dependent peptide-lipid binding interactions as discussed below.

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TABLE 1: 13C Chemical Shifts (δ, ppm) of Achatin-I (Gly1-D-Phe2-Ala3-Asp4) (5 mM) Observed at p2H ) 3.20 in Bulk Water and in the Binding Equilibrium with the LUV of Egg-Yolk PC (50 mM)a,b amino acid residue Gly1 D-Phe2

Ala3 Asp4

carbon site

(a) in bulk water (observed (ppm))

(b) in the mixture with the PC-LUV (observed (ppm))

chemical-shift difference ∆δ (b - a) (observed (ppm))

chemical-shift change ∆δ upon binding to the PC-LUV (estimated (ppm))

R-C R-CO R-C β-C ring-C(1) ring-C(2,6) ring-C(3,5) ring-C(4) R-CO R-C β-C R-CO R-C β-C β-CO2H R-CO2-

40.44 166.96 55.59 36.19 136.12 129.35 128.94 127.48 174.32 49.55 16.28 172.76 49.92 37.18 174.93 174.79

40.46 166.96 55.61 36.19 136.17 129.38 128.92 127.42 174.23 49.51 16.30 172.72 49.89 37.20 174.89 174.73

+0.02 0.00 +0.02 0.00 +0.05 +0.03 -0.02 -0.06 -0.09 -0.04 +0.02 -0.04 -0.03 +0.02 -0.04 -0.06

+0.19 0.00 +0.19 0.00 +0.49 +0.29 -0.19 -0.58 -0.87 -0.39 +0.19 -0.39 -0.29 +0.19 -0.39 -0.58

a Free induction decay signals were accumulated until it was confirmed that the signal-to-noise ratios were large enough to analyze the chemical shifts (δ) at the signal peaks within errors due to the digital frequency resolution at 0.0076 ppm. b Chemical-shift changes ∆δ (ppm) upon binding to the membrane are estimated from the observed chemical-shift differences ∆δ (ppm) between those measured in bulk water and those in the LUV mixture, by taking account of the fraction of bound peptide at 10% (ref 4).

The chemical-shift changes ∆δ upon membrane binding are illustrated as a bar graph in Figure 3b. Compare with the corresponding ∆δ values at neutral pH for achatin-I with the ionic β-CO2- in the Asp4 side chain.4 At any amino acid residue, the carbon-site dependence of ∆δ is obviously different between the acidic and the neutral pH’s. At neutral pH, prominent upfield shifts (∆δ < 0) are observed at Asp4 more than 5 times as large as the shifts ∆δ at the other residues. In contrast, such prominent upfield shifts at Asp4 are no longer present at acidic pH. The chemical-shift changes ∆δ in the other residues, i.e., Gly1-D-Phe2-Ala3, are more drastic on the contrary at acidic than at neutral pH. This observation is evidence that the binding state is perturbed substantially by deionizing the side chain β-CO2- in Asp4. Before examining in detail the ∆δ values at acidic pH, let us review the binding state of achatin-I at neutral pH determined by the previous natural-abundance 13C and high-resolution (on the order of 0.01 Hz) 1H NMR results4 and the present selectively labeled 15N NMR result. At neutral pH, the peptide N-terminal NH3+ binds to PO4- in the lipid headgroup, the C-terminal Asp4 R- and β-CO2- bind to N(CH3)3+ at the edge of the headgroup, and the phenyl ring of D-Phe2 locates around the ester groups. With those site-specific interactions, the peptide structure mainly lies on the membrane surface in the range from the glycerol to the ester carbonyl groups. Here we should note that the 13C chemical-shift changes ∆δ of achatin-I upon membrane binding are prominent at the C-terminal Asp4 which locates at the headgroup N(CH3)3+ in the most superficial region of the PC membrane. The prominent upfield shifts ∆δ at Asp4 result from extensive dehydration of the R- and β-CO2groups.39-43 That is, the C-terminal Asp4 electrostatically binds to the headgroup N(CH3)3+ on the membrane surface at the large expense of dehydration. At acidic pH ) 3.20, where the side chain β-carboxyl group in the C-terminal Asp4 is deionized, the 13C chemical-shift changes ∆δ in Asp4 are comparable in magnitude with those in the other residues. The absence of prominent upfield changes ∆δ at Asp4, in contrast to neutral pH, is because of the lesser dehydration of Asp4 upon membrane binding; the electrostatic hydration of Asp4 in bulk water is weaker with the deionized β-CO2H than with the ionic β-CO2-. Note that the deionization

of the side chain β-CO2- in Asp4 reduces the electrostatic hydration not only of the functional group itself but also of the overall Asp4, through a reduced degree of charge separation represented such as by the dipole moment in the Asp4 residue.14,15 As a notable trend observed in the structure aside from the C-terminal Asp4, i.e., Gly1-D-Phe2-Ala3, 13C signals of the backbone carbonyl groups in D-Phe2 and Ala3 shift upfield. These upfield shifts ∆δ, much larger than the corresponding ones at neutral pH, tell notable changes in the hydration state of the backbone upon membrane binding. The perturbation on the chemical shifts at the other carbon sites in the residues D-Phe2 and Ala3 also indicates significant changes in the hydration state at those sites most likely due to deep anchoring of the side chains compared to neutral pH. How then can the deep anchoring be induced by deionizing the side chain β-CO2in Asp4? Deionization of the side chain β-CO2- in Asp4 weakens the electrostatic hydration and the electrostatic attraction with the headgroup N(CH3)3+ in the most superficial region of the PC membrane. The weakened electrostatic effect of Asp4 increases the net hydrophobicity of achatin-I. Hence it is considered that deeper binding of the peptide on the membrane surface is favored as a result of the deionization. Here we conclude that the less efficient binding between the peptide N-terminal NH3+ and the lipid headgroup PO4- caused by deionizing the side chain β-CO2- in Asp4 is related to the deeper binding state. The absence of an 15N chemical-shift difference at the N-terminal NH3+ between in bulk water and on the membrane surface shows, at the same time, that the NH3+ group is not buried so deep as to change its electronic structure because of dehydration. Changes ∆ν1/2 (Hz) in half-widths of the peptide 13C signals in response to the membrane binding provide us with supportive information on the binding state from the viewpoint of dynamics. Observed ∆ν1/2 values at pH ) 3.20 show a carbon-site dependence in the range from ∼0 to +6 Hz. Broadening of the 13C-signal line width (∆ν 1/2 > 0) reflects in general both (1) slowdown of the rate of exchange between the membrane-bound and free states regarding the entire peptide molecule, and (2) local slowdown of motion at a given carbon nucleus due to membrane binding. Here we attempt to look into the latter

Membrane Binding of Achatin-I contribution by examining the carbon-site dependence of ∆ν1/2. The largest broadening (+6 Hz) is observed at the ring-C(4) carbon in D-Phe2, and moderate broadening (+3 Hz) is observed at the carbons, R-C and ring-C(1) in D-Phe2, R-CO in Ala3, and β-C in Asp4. The largest broadening at the edge of the hydrophobic phenyl ring (ring-C(4) carbon) explains the anchoring of the ring on the membrane surface; the broadening at this site is prominent probably because of an insertion depth below the ester carbonyl groups, where the probability of water molecules decreases drastically.44,45 It should be noted, on the other hand, that at many carbon sites the line broadening upon membrane binding is small, which is close to the detectable limit at the digital frequency resolution (1.2 Hz). This is in marked contrast to the observed chemical-shift changes as discussed above. The small changes in the line widths indicate minor changes in dynamics of the molecular environment around many of the carbon sites in achatin-I. The importance of hydration effect15 is emphasized in this regard of soft peptide binding on the membrane surface.4 3.2.2. Hydrophobic Anchoring of the Phenyl Ring and the Side Chain Conformation. In this section, we discuss the deeper binding state of achatin-I on the PC membrane surface induced by deionizing the Asp4 side chain β-CO2-, by referring to the previous observation on the side chain conformational equilibrium.4 Let us see if there is some correlation between anchoring of the phenyl ring in D-Phe2 residue and the side chain conformation; deeper anchoring of the hydrophobic ring is expected to be accompanied by larger perturbation on the conformational equilibrium. According to the vicinal 1H-1H couplings JRβ1 and JRβ2 in the side chains, we investigated changes (∆P) in side chain conformer probabilities of the D-Phe2 and Asp4 residues upon binding to the PC membrane surface. ∆P values in response to a transfer of achatin-I from water to methanol solutions were also investigated to see the effect of reduced electric polarity in the surrounding environment; the ionization state of achatin-I in methanol has been identified to be identical to the one in water at neutral pH.4 At neutral pH, membrane binding results in conformational changes ∆P in D-Phe2 in almost the same trend with the ∆P for a transfer from bulk water to bulk methanol. ∆P due to the membrane binding is -0.09 for the trans conformer, +0.03 and -0.06 for the gauche conformers with respect to the side chain β-phenyl and the backbone R-carbonyl groups. The corresponding change ∆P due to a transfer from water to methanol is -0.10 for the trans, +0.04 and -0.06 for the gauche conformers. The side chain conformational change upon membrane binding can therefore be interpreted by the reduced polarity of the surrounding molecular environment. This observation implies that the binding of achatin-I on the PC membrane surface is not tight to force the hydrophobic side chain into dominantly taking a particular conformer because of the steric effect of membrane lipids. In contrast, at acidic pH, where the side chain β-carboxyl group in Asp4 is in the deionized state, membrane binding induces side chain conformational changes ∆P in D-Phe2 in a drastic manner. ∆P is +0.02 for the trans, -0.43 and +0.41 for the gauche conformers. The almost exclusive probability exchange between the two conformers may be explained by the steric effect of lipids. The previous 1H NMR observations on the side chain conformational equilibrium thus support the current conclusion that the deionization of the side chain β-CO2- in Asp4 induces a deeper anchoring of the phenyl ring of the D-Phe2 residue.

J. Phys. Chem. B, Vol. 111, No. 14, 2007 3837 The change in the geminal 1H-1H coupling Jβ1β2 between the side chain β-protons of the D-Phe2 residue is also large at acidic pH upon membrane binding (+1.45 Hz, 13.50 f 14.95 Hz).4 Compare with an order of magnitude smaller change at neutral pH (+0.23 Hz, 13.20 f 13.43 Hz). In the C-terminal Asp4, which is considered to be more exposed to a water-rich region than the hydrophobic D-Phe2 residue, the corresponding change of Jβ1β2 is smaller both at acidic pH (-0.41 Hz, 16.81 f 16.40 Hz) and at neutral pH (-0.07 Hz, 15.88 f 15.81 Hz). Hence, deionizing the side chain β-CO2- in the C-terminal Asp4 induces deeper anchoring of the hydrophobic phenyl ring in D-Phe2, being accompanied by a large perturbation on the side chain conformation as probed by JRβ1 and JRβ2 and on the electronic structure as probed by Jβ1β2. 4. Conclusions Achatin-I (Gly1-D-Phe2-Ala3-Asp4), known as a neuropeptide containing a D-amino acid, binds to the surface of a zwitterionic phosphatidylcholine (PC) membrane only when the peptide N-terminal amino group is in the ionized state, NH3+.4 In this study, the molecular mechanism of this notable electrostatic effect of the N-terminal NH3+ in determining the binding equilibrium was scrutinized. To examine if the peptide N-terminal NH3+ electrostatically binds to the lipid headgroup PO4-, we selectively enriched the NH3+ with the 15N isotope and probed the membrane-binding effect on the 15N NMR. An upfield shift of the 15N NMR of the N-terminal NH3+ was observed upon membrane binding of achatin-I at neutral pH, where all the ionizable groups in the peptide are in the ionized states: the N-terminal NH3+ and the C-terminal Asp4 R- and β-CO2-. The upfield shift ∆δ indicates electrostatic binding with the headgroup negative charge, i.e., PO4-. In contrast, at acidic pH, where the side chain β-carboxyl group in Asp4 is deionized, the 15N signal exhibits no significant chemical-shift change upon membrane binding. Deionization of the Asp4 β-CO2- thus alters the binding state such that efficient electrostatic binding between the peptide N-terminal NH3+ and the lipid headgroup PO4- no longer takes place. In other words, the electrostatic effect of the N-terminal NH3+ is efficient in the presence of the ionic side chain β-CO2- in the C-terminal Asp4. The overall binding state was inspected through examining changes ∆δ in 13C chemical shifts of achatin-I upon membrane binding. Deionization of the side chain β-CO2- in Asp4 diminishes prominent upfield changes ∆δ at Asp4 observed when it is ionized.4 Achatin-I with the deionized β-CO2H in Asp4 binds at a lesser expense of dehydration of Asp4. In contrast, the magnitude and carbon-site dependence of ∆δ in the other residues, i.e., Gly1-D-Phe2-Ala3, are enhanced by deionizing the side chain β-CO2- in Asp4. The enhanced perturbation ∆δ on the chemical shifts indicates deeper binding induced by the deionization. Deionization of the side chain β-CO2- in Asp4 weakens the electrostatic hydration and the electrostatic attraction with the lipid headgroup N(CH3)3+ in the most superficial region of the PC membrane. The weakened electrostatic effect of Asp4 increases the net hydrophobicity of achatin-I. Hence, deeper binding on the membrane surface becomes favored. The ionic side chain β-CO2- in Asp4 plays a key role in floating the amphipathic neuropeptide achatin-I on the PC membrane surface such that the electrostatic binding between the peptide N-terminal NH3+ the lipid headgroup PO4- plays the notable determining role of the binding equilibrium.

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