Interaction of Alcohols with [Val5] angiotensin in Alcohol− Water Mixtures

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Interaction of Alcohols with [Val5]angiotensin in Alcohol-Water Mixtures R. C. Neuman, Jr. and J. T. Gerig* Department of Chemistry & Biochemistry, UniVersity of California, Santa Barbara, California 93106 ReceiVed: February 10, 2010; ReVised Manuscript ReceiVed: April 7, 2010

Intermolecular solvent-solute NOE experiments have been used to probe interactions of various alcohols with the peptide hormone [val5]angiotensin II at 0 °C. It is found that these NOEs are detectable but dependent on the kind of alcohol present and the conformation of the peptide. Solvent-solute NOEs in 100% methanol and 89% methanol-water are basically those predicted by a hard sphere model for intermolecular spin dipole interactions. NOEs at the peptide backbone (N-H, CR-H) protons in 25% methanol-water and 36% ethylene glycol-water mixtures indicate that alcohol interactions near these groups are also adequately described by this model. However, in 35% ethanol-water, interactions of alcohol methyl protons with the peptide result in unexpectedly negative NOEs, probably signaling that peptide-alcohol interactions in this solvent take place on a significantly slower time scale than that defined by mutual diffusion of these species. Some side chain-alcohol interactions result in NOEs up to 8 times larger than expected. Possible reasons for these enhanced effects are discussed. Introduction Aliphatic alcohols are added to solutions of peptides and proteins in water for many reasons.1 The cosolvent may change the relative amounts of conformation(s) of a polypeptide present or alter solubility.2–4 Alcohol may be needed to help solubilize small molecules for studies of interactions of these molecules with the biomolecule. Alcohol cosolvents can play a role in the formation of nanostructures from peptides and proteins.5–7 The strength and stability of fibers formed from proteins such as collagen or silk fibroin can depend on the alcohol used in their preparation.8–11 Alcohols, particularly polyhydric alcohols, may be used to alter the viscosity of a solution as an aid to examining the motions of a protein or the dynamics of its interactions with other molecules.12–14 Alcohol-water mixtures provide media that remain fluid at very low temperatures, facilitating studies of biochemical processes that are too rapid to be studied at more normal temperatures.15 Chemical joining of alcohols, particularly polyethylene glycol, to peptides and proteins is a standard method for altering stability and pharmacological properties.16,17 In all of these situations, the alcohol is often regarded as a bystander to the process of interest and specific interactions of an alcohol with peptides and proteins have generally been disregarded. Fluorinated alcohols, particularly trifluoroethanol, can have strikingeffectsontherelativestabilitiesofpeptideconformations.18–22 The origins of these effects have been highly investigated and may reside in the physicochemical properties of aqueous fluoroalcohol,23–25 site-specific interactions with a polypeptide,26–28 or alteration of the orientations of amino acid side chains.29 Identification of interaction sites for alcohols and other organic cosolvents on protein structures by NMR methods can provide valuable indications of possible targets for pharmaceutical agents.30,31 Liepinsh and Otting have examined binding of methanol, 2-propanol and tert-butanol to specific sites on the surface of hen egg-white lysozyme.32 NMR studies of a tetrapeptide in water-alcohol mixtures indicated site-specific * Corresponding author. Phone: 805-893-2113. Fax: 805-893-4120. E-mail: [email protected].

selective solvation of the peptide by the alcohol components.33 Byerly et al. have used perturbations of 1H-15N HSQC spectra to identify binding sites for several small organic solvents on a peptide deformylating enzyme.34 Dalvit employed NMR spectroscopy to probe interactions of dimethyl sulfoxide (DMSO) and water with a small peptide.35 His results suggested exclusion of DMSO from the vicinity of the peptide N-H protons, possibly as a result of preferential solvation of these by water molecules. Intermolecular NOE experiments have suggested that, even when a definite, long-lived complex between an alcohol and a biomolecule is not formed, there can be associations or interactions with dissolved peptides in alcohol-water solvents that persist for times that are long relative to the time characteristic of diffusive encounters.28,36,37 It was the purpose of the present work to explore the dependence of such weak interactions on the chemical nature of the alcohol. To this end, intermolecular NOEs resulting from spin dipolar interactions between protons of the alcohol and those of the peptide were used to probe interactions of methanol, ethanol and ethylene glycol with the octapeptide [val5]angiotensin II in alcohol/water mixtures.

H3N+-Asp1-Arg2-Val3-Tyr4-Val5-His6-Pro7-Phe8-COO[val5]angiotensin II Angiotensin II is a peptide hormone that exerts a variety of physiological effects by interacting with a number of specific receptors.38,39 The amino acid residue at position 5 is key to the activity of the peptide; in some species this is replaced by an Ile residue. The peptide likely has a flexible structure in aqueous solution but takes up a specific conformation upon receptor binding.40,41 Solvation-desolvation effects may have a significant role in such binding.42 Experimental Section Sample Preparation. [Val5]angiotensin II was obtained in >95% purity from Sigma-Aldrich or as a > 98% purity synthetic

10.1021/jp101305u  2010 American Chemical Society Published on Web 04/27/2010

Alcohol Interaction with [Val5]angiotensin product from GenWay (San Diego, CA). Mass spectra of all samples were consistent with the expected structure. Both materials were used as received. A trace impurity with a proton chemical shift of 2.05 ppm (assumed to be acetate ion) was present in the Sigma-Aldrich product. 3-(Trimethylsilyl)propionic acid-d4 sodium salt (TSP) was obtained from Stohler Isotope Chemicals. Deuterium oxide (100 atom %), anhydrous methanol (99.8%), methanol-d3 (CD3OH), ethanol-1,1-d2 (98 atom %), anhydrous ethylene glycol (99.8%), and tetramethylsilane (TMS) were used as received from Sigma-Aldrich. Distilled deionized water was used in sample preparation. Solvent mixtures containing water were prepared on a v/v basis with the water portion being a mixture of 85% H2O/15% D2O for the methanol/water and ethyleneglycol/water samples. Pure H2O was used for the ethanol-d2/water samples. The solvent mixture referred to in the text as “100% methanol” was a mixture of 88.6% CH3OH/11.4% CD3OH (v/v). The mixture called “89% methanol-water” contained 10.9% water, 78.9% CH3OH, and 10.2% CD3OH by volume. For water-containing solutions, the apparent sample pH (3-4) was determined by a Model IQ150 pH meter (IQ Instruments, San Diego, CA) equipped with a 4 mm o.d. stainless steel electrode. The reported pH is the meter reading and was not corrected for the presence of alcohol or isotopic composition. Deuterium in the solvent provided the spectrometer lock signal for all samples examined. Samples were ∼10 mM in peptide and contained a trace of TSP to provide a reference signal, which was set at 0.0 ppm. A trace of TMS provided the reference signal in the 89% and 100% methanol samples. Instrumention and Procedures. NMR spectra were collected at 500 MHz using a Varian INOVA instrument and a Nalorac triple resonance probe equipped with xyz pulsed field gradient coils. Pulse sequences used for the solvent-peptide intermolecular NOE experiments involved suppression of both water (OH) and alcohol (CH2 or CH3) signals and were based on those published by Dalvit,35 with 25 or 50 Hz bandwidth solvent selective inversion pulses used during the DPFG-SE part of the experiment. An excitation sculpting scheme43,44 with 15 Hz bandwidth square pulses was used for selective inversion of the solvent signal at the start of the NOE mixing time, rather than the approach described by Dalvit. Mixing times for intermolecular NOE experiments ranged from 50 to 800 ms. TOCSY, ROESY, and NOESY spectra (4K × 4K, 6000 Hz sweep width in both dimensions) were obtained using pulse sequences based on those of Fulton and Ni,45 with double solvent signal suppression during detection that used Dalvit’s method. A mixing time of 70 ms was used for TOCSY experiments while typical mixing times for ROESY and NOESY were 50, 100, and 200 ms. Multiply selective RF pulses were generated with the Pbox function of the Varian software. Carbon-13 decoupling was present during the application of any selective RF pulse and during fid acquisition. Self-diffusion coefficients of sample components were determined by bipolar double stimulated echo pulsed field gradient experiments,46 following the procedures previously described.28 Magic angle gradients were used during the detection phase in all experiments.47 Radiation damping was controlled by imposing a weak field gradient during delays in the pulse sequence used. Proton T1 relaxation times were determined by the nonselective inversion-recovery method.48 Sample temperatures were determined using a standard sample of methanol (Wilmad) and are believed to have been constant to better than (0.1 °C during the course of an experiment and accurate to better than (0.5 °C.

J. Phys. Chem. B, Vol. 114, No. 19, 2010 6723 Data analysis and Calculations. Molecular modeling and visualizations employed SYBYL (Tripos), PYmol (DeLano Scientific LLC) and MOLMOL.49 Intramolecular NOE spectra were analyzed with the aid of the program SPARKY.50 Assigned cross peaks led to conformation-sensitive H-H distances for the peptide. The programs DYANA or CYANA were used to find conformations consistent with these experimental distance constraints.51,52 Structures so defined were relaxed in the AMBER 4.1 force field using SYBYL 8.0 (Tripos). Solvent-peptide intermolecular cross relaxation rate constants 53 (σNOE HH ) were estimated as described previously. NOE intensity data, obtained as a function of mixing time, were analyzed to obtain initial slopes. If spin diffusion effects can be neglected, NOE . All cross relaxation these initial slopes can be equated to σHH data were corrected for the extent of solvent line inversion. NOE depend on the separation of Probable errors for a specific σHH a signal of interest from others in the spectrum and the S/N ratio of the data. It is estimated that the reliability of the reported NOE values is about (25%. σHH The program used to predict observed intermolecular Overhauser effects took into consideration both intramolecular and intermolecular spin dipolar interactions since intramolecular spin diffusion effects can modify observed solvent-derived cross relaxation parameters. A “full-relaxation matrix” treatment of intramolecular 1H-1H interactions was used,54–56 with 1H-1H dipolar interactions within the network of solute spins being characterized by an overall rotational correlation time (τR) and a correlation time (τM) for methyl group rotation. The correlation times τR and τM were estimated by simulating inversion-recovery experiments with the peptide spin system as a function of these parameters, with values of τR and τM being adjusted by trialand-error until reasonable agreement between initial slopes of experimental and calculated magnetization recovery curves was obtained. The approximate correlation times estimated in this way are significantly smaller than those obtained by considering the molecular dimensions of the peptide and use of the Stokes-Einstein equation. However, it is unlikely that the peptide rotates as a rigid body, and the local τRcharacterizing a given intramolecular proton-proton interaction must reflect the effects of local motions.57,58 Thus, τR and τM obtained by this procedure are effective correlation times. In this light, the values of these parameters reported are probably not universally appropriate for the entire peptide. Fortunately, the effective correlation times obtained are small enough that spin diffusion effects are predicted to have minimal influence on intermolecular NOEexperimentsandconclusionsdrawnregardingsolvent-peptide NOEs are not sensitive to the exact values of τR and τM. Theoretical estimates of solute-proton/solvent-proton inNOE termolecular cross relaxation parameters (σHH ) were based on the formulation of Ayant et al.59 In their model, the peptide proton of interest is considered to be located in a sphere of radius rH while the solvent spin is situated in a sphere of radius rS. The intermolecular cross relaxation rate is given by

NOE σHH

3γH4h2NS ) (6J2(2ωH) - J2(0)) 10πDr

(1)

where ωH is the proton Larmor frequency, NS is the number of inverted solvent spins per mL, D is the sum of the diffusion coefficients for the molecules containing the peptide and solvent spins (D ) DH + DS), r is their distance of closest approach (r ) rH + rS) and J2(ω) is a spectral density function given in eq 2

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J2(ω) ) 5 ωτ + (ωτ)1/2 + 4 √2 (ωτ)3 + 4√2(ωτ)5/2 + 16(ωτ)2 + 27√2(ωτ)3/2 + 81ωτ + 81√2(ωτ)1/2 + 81

Neuman and Gerig

)

(2)

where the correlation time τ ) r2/D. For the present study, we define a reduced or scaled cross relaxation parameter σR,

σR )

NOE σHH 3γH4h2 ) (6J (2ωH) - J2(0)) NS 10πDr 2

(3)

pulse sequence on the magnetizations of all spins of the peptide at the start of the NOE mixing time. These initial solute spin magnetizations were then used in the intermolecular NOE simulation program and the initial slopes of computed NOE vs mixing time curves compared to the corresponding values of the intermolecular cross relaxation parameter. Significant disagreement between these would signal that an experimental initial slope might be an unreliable indicator of the corresponding cross relaxation term. For all observations reported here, such calculations indicated that the errors in equating initial slopes of NOE vs mixing time plots to the corresponding intermolecular cross relaxation parameters were at most a few percent. Results

While independent of the concentration of inverted alcohol spins, it is seen that the magnitude of σRdepends in a complex way on the diffusion of the peptide and solvent and their interaction distance. The sign of σR depends on the value of τ relative to the Larmor frequency. It can be shown that at a proton frequency of 500 MHz, the value of τ at which the sign of σNOE HH or σR changes is 8.13 × 10-10 s. The equation for σR shown in eq 3 is based on the assumption that solvent molecules can approach the sphere representing the solute proton equivalently from all directions. In actual molecules, the three-dimensional structure of the solute will make solvent approaches from different directions nonequivalent. To take into account the shape of the solute in its interactions with solvent molecules, the empirical approach described previously was used.53,60 We imagine a large number of equi-spaced rays extending in all directions from the peptide hydrogen atom of interest. Each ray intersects the surface of the peptide at a particular distance from the hydrogen nucleus, and at that distance, the characteristic contribution to σR given by eq 3 is calculated. Averaging the contributions associated with all rays was assumed to give the aggregate σR. We used the Connolly algorithm61 to obtain a representation of the surface of [val5]angiotensin in a particular conformation. This procedure generates a collection of points that correspond to locations where the sphere representing a solvent molecule is able to make contact with the van der Waals surface of the solute. At a density of 200 dots Å-2 there was a “Connolly surface dot” 0.1 Å or closer to each ray extended from a hydrogen atom. Because they rotate rapidly, solvent molecules were represented by spheres.32 It was assumed that all hydrogen atoms of an alcohol molecule are located at the center of its representative sphere.62 The radii of spheres representing methanol and ethanol were estimated to be 2.08 and 2.40 Å, respectively, by a method based on the van der Waals surfaces of the alcohols and described previously.21 The radii of ethylene glycol in the trans and gauche conformations were predicted by that method to be 2.54 and 2.49 Å, respectively. The gauche conformation is favored due to formation of an intramolecular hydrogen bond,63 and the smaller radius was used for the calculations described below. Peptide protons with chemical shifts close to that of the alcohol resonance could potentially be perturbed by pulses used to invert the solvent line. Such unwanted effects on these spins would lead to an initial slope in a NOE vs mixing time plot, and a value of σR that is influenced by T1 relaxation and intramolecular NOEs. Using observed chemical shifts and estimated T1 and T2 relaxation times, the Bloch equations were employed to calculate the effects of the solvent line inversion

General Approach. Proton NMR data for [val5]angiotensin in various alcohol-water mixtures were collected at a sample temperature of 0 °C since previous work indicated that unanticipated alcohol interactions with the peptide would most likely be observed at temperatures below room temperature.28,36 As was the case in earlier studies, signals were observed for a minor conformation of the peptide which presumably arise by rotational isomerism at the his6-pro7 peptide bond. The minor conformation was ignored in the present work. Spectra of the major conformation were assigned using TOCSY, NOESY, and ROESY data. Assigned proton chemical shifts for all systems discussed here are given in the Supporting Information. Forty-five to 119 conformation-defining intramolecular 1 H{1H} NOEs and ROEs were detected for the peptide, depending on the identity of the alcohol cosolvent. The distance constraints developed from the available NOE and ROE data for each system are summarized graphically in the Supporting Information. For each system, peptide conformations consistent with the interproton distances indicated by the observed NOEs were developed. The conformational energies of ten or more of these structures were minimized in the AMBER99 force field.64 As was the case in our earlier studies of [val5]angiotensin, the number of available distance constraints in each system was insufficient to indicate a single dominant conformation of the peptide. Rather, many somewhat folded conformations of the peptide were found to be consistent with distance restraint data in each solvent system (Figure 1). Observed vicinal coupling constants (3JNHCRH) for the backbone of [val5]angiotensin are given in the Supporting Information. For the peptide dissolved in solvent mixtures with a high water concentration (25% methanol-water, 35% ethanol-water, 36% ethylene glycol-water), these coupling constants averaged 8 Hz, consistent with rapid interconversion between multiple conformations of the peptide.65 In methanol-rich solutions (89% methanol-water, 100% methanol), the average 3JNHCRH was 9.5 Hz, possibly suggesting an increase in the amount of extended conformations present in these systems compared to the waterrich solvents. Translational diffusion coefficients were measured for the components of samples studied. The results are given in Table 1. Translational diffusion of a peptide is sensitive to the hydrodynamic size and shape of the peptide.33,66,67 The translational diffusion constant (Dtrans) can be related to molecular dimensions by means of the Stokes-Einstein equation. This equation is most easily applied to estimating molecular sizes by comparing the diffusion constant of a species of interest to that of a reference material of known dimensions present in the same solution. The ratio of diffusion constants thus provides a

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Figure 2. Intermolecular NOEs on low field signals of [val5]angiotensin resulting from inversion of the solvent methyl resonance in a mixture of 88.6% CH3OH/11.4% CD3OH (v/v) (“100% methanol”) at 0 °C (top). A control spectrum is shown at the bottom. The mixing time for the NOE experiment was 400 ms.

Figure 1. Conformations of [val5]angiotensin found to be consistent with interproton distance constraints derived from observed NOEs and ROEs in (A) 100% methanol, (B) 25% methanol/water, (C) 35% ethanol/water, and (D) 36% ethylene glycol/water. Typically, the rmsd of backbone atom positions in a family of structures was ∼2 Å while the rmsd of all heavy atoms was ∼3 Å.

TABLE 1: Diffusion Constants (Dtrans) at 0° C (× 106 cm2 s-1) component [val5]angiotensin solvent acetate TSP or TMS a

36% 89% 25% 35% ethylene 100% methanol- methanol- ethanolglycolmethanol water water water water 2.08 9.07 a 8.84

1.96 8.62 a n.d.

0.67 4.97 3.19 1.98

0.41 2.18 2.02 1.43

0.33 1.68 1.59 0.93

Acetate (impurity) signal not detected in these samples.

value for the hydrodynamic radius of the peptide relative to that of the reference material (eq 4). Reference Dtrans Peptide Dtrans

)

rPeptide rReference

(4)

Hydrodynamic radii of [val5]angiotensin were estimated from the data in Table 1 using the radii of acetate, TMS, and TSP

(2.26,68 2.84,69 3.62 Å,21 respectively) as references. There is evidence that TSP can form stable complexes with biomaterials.70 Complex formation would render TSP unreliable as a diffusion reference. However, the hydrodynamic radius for the peptide found in each system was approximately the same within experimental uncertainty, 11 ( 1 Å, regardless of which reference compound was used. While the hydrodynamic radii obtained by these experiments are not highly accurate, it is clear that the radius of [val5]angiotensin varies little is going from a water-rich to a water poor solvent and that it is unlikely that TSP forms stable complexes with [val5]angiotensin. The radii of the various conformations of [val5]angiotensin found to be consistent with the observed 1H-1H intramolecular NOE data shown in Figure 1 were estimated using the method previously described. These calculated radii averaged ∼7.7 Å for high water systems and ∼8.4 Å for the methanol-rich systems. 100% Methanol. Figure 2 shows intermolecular NOEs on the low field proton NMR signals of [val5]angiotensin dissolved in methanol at 0 °C produced by inversion of the solvent methyl resonance. The NOEs are easily detected in this system because the concentration of solvent (methyl) spins is high (65.6 M). The portion of the spectrum shown includes the side chain protons of the tyr4, his6, and phe8 residues; resonances for the other side chain protons of the peptide (not shown) also show positive NOEs. Initial slopes of intermolecular NOE vs mixing time plots for the peptide signals were determined. The reduced cross relaxation rates derived from these for the peptide backbone and some side chain protons, along with an indication of errors, are presented schematically in Figure 3. Calculation of σR values were done for ten conformations of the peptide found to be consistent with the intramolecular 1H-1H NOEs and ROEs observed in 100% methanol (Figure 1A). It

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Figure 3. Initial slopes of intermolecular NOE vs mixing time plots for various protons of [val5]angiotensin, scaled by the concentration of methanol CH3 protons in the “100% methanol” solvent mixture at 0 °C (red squares). The red bars indicate a (25% error range for these initial slopes. The blue squares represent the average of σR calculated for ten conformations of the peptide, each conformation having 1H-1H distances consistent with distance constraints indicated by the observed 1H-1H intramolecular NOEs and ROEs. The blue bars represent the range of σR calculated for these structures.

was estimated that intramolecular 1H-1H dipolar interactions are described by effective correlation times (τR, τM) of 0.15 and 0.01 ns, respectively. Figure 3 compares calculated cross relaxation parameters (σR, blue symbols) to the experimental observations. The lengths of the blue bars in the figure provide an indication of the sensitivity of a calculated σR to the conformation of the peptide. It is apparent that when experimental errors and the conformational dependence of calculated cross relaxation rates are considered, the experimental and predicted methanol-peptide intermolecular NOEs in “100% methanol” are in good agreement. 89% Methanol-Water (v/v). Experiments and calculations analogous to those described above were carried out for [val5]angiotensin in a solvent containing 89% methanol/11% water at 0 °C. Results for this system are given in the Supporting Information. In summary, similar to observations made with the 100% methanol system described here, both the experimental scaled initial slopes and calculated σR overlap when experimental errors and the conformational sensitivity of the σR parameters are considered. The effective rotational correlation times (τR, τM) were found to be the same as those characteristic of the 100% methanol system. 25% Methanol-Water (v/v). Intermolecular NOEs resulting from interactions of the protons of [val5]angiotensin with methanol protons in a solvent composed of 25% methanol/water (v/v) are shown in Figure 4. Experimental values for the scaled initial slopes are compared to σR predicted for various conformations of the peptide in Figure 5. Although weak, the solvent NOEs on the peptide backbone protons are close to those expected. With the exception of the protons of the histidine side chain, the solvent NOEs for the side chain protons of the peptide are about 25-50% larger than expected. The effective correlation times (τR, τM) were estimated to be 0.45 and 0.01 ns, respectively. The increased value for τR and the smaller translational diffusion coefficients are consistent with the increased viscosity of 25% methanol-water compared to that of neat methanol.71 35% Ethanol-Water (v/v). We have previously reported intermolecular NOE studies of [val5]angiotensin in 35% ethanol1,1-d2/water.36 The experiments with this system at 0 °C were repeated for the present study. Experimental determinations of diffusion coefficients were made, rather than relying on the extrapolated values used in the previous study. The effective correlation times (τR, τM) used for calculations of the cross relaxation terms were 0.55 and 0.07 ns, respectively. Figure 6 compares observed initial slopes and calculated σR values.

The present study confirms the results previously reported for this system. The experimental cross relaxation terms for the peptide backbone protons are substantially more negative than those predicted. Cross relaxation terms for the side chain protons are close to the expected values, with the exception of the phe8 phenyl ring protons, where the observed positive cross relaxation term is about twice as large as the predicted (positive) value. 36% Ethylene Glycol-Water (v/v). Intermolecular NOEs at the protons of [val5]angiotensin arising from interactions with the methylene protons of ethylene glycol at 0 °C in a solvent composed of 36% ethylene glycol/water (v/v) are shown in Figure 7. The results are reminiscent of those found for the peptide in ethanol/water at this temperature in that the NOEs to the peptide backbone protons are negative, NOEs to the side chain protons of his6 and phe8 are positive and the NOEs at the tyr4 Hδ and Hε protons have opposite signs. The effective correlation times (τR, τM), used for calculations of σR, were estimated to be 0.80 and 0.10 ns, respectively. In contrast to the results in 35% ethanol-water, Figure 8 shows that the reduced cross relaxation terms for the backbone σR protons observed in this solvent mixture are in agreement with expectations based on the experimental diffusion coefficients. Except for the valine methyl protons, cross relaxation terms observed for the side chain protons of the peptide are significantly larger than those predicted. Water-Organic Solvent Cross Relaxation. The intermolecular NOEs described herein were produced by inversion of magnetization associated with spins of the alcohol cosolvent. In most systems, there was a large concentration of water protons in contact with the cosolvent, as well as the peptide. Peptides and proteins are presumably solvated by water in a highly aqueous solution,31 and it could be argued that the Overhauser effects produced by inversion of the organic solvent resonance are indirect effects. That is, inversion of the cosolvent magnetization results in a change in the polarization of the water magnetization, which then perturbs the magnetizations of the peptide spins. This possibility was explored by determining water proton/cosolvent proton NOEs and by computation. For example, experimental values for the intersolvent cross relaxNOE ) in 36% ethylene glycol-water were 0.060 ation terms (σHH -1 and 0.097 s when the glycol methylene or water resonance was inverted, respectively. Given these cross relaxation rates, the water magnetization would be changed by only a few percent over the course of the mixing times used in the intermolecular NOE experiments. Calculations of σR that employed experimental solvent-solvent cross relaxation rates showed that the

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Figure 4. Intermolecular NOEs on signals of [val5]angiotensin resulting from inversion of the solvent methyl resonance in a mixture of 25% CH3OH-water (v/v) at 0 °C (top). A control spectrum is shown at the bottom. The mixing time for the NOE experiment was 300 ms.

Figure 5. Initial slopes of intermolecular NOE vs mixing time plots for various protons of [val5]angiotensin, scaled by the concentration of methanol CH3 protons in 25% methanol-water at 0 °C (red squares). The red bars represent a (25% error range for these quantities. The blue squares indicate the average of calculated σRobtained for ten conformations of the peptide indicated by distance constraints derived from 1H-1H intramolecular NOE and ROE data. The blue bars represent the range of calculated σR found with these structures. Experimental data for the CA proton of His6 could not be obtained since the (suppressed) water resonance is very close to the signals of this proton.

effects of the indirect mechanism in producing alcohol-peptide proton NOEs would be so small as to be undetectable by our experiments. Even with solvent-solvent cross relaxation terms 10 times larger than the experimental values, calculations indicated that the effects produced by solvent-solvent cross relaxation would be less than 0.1% of experimentally observed σR values. Thus, there is no support for the notion that perturbing the magnetization of the alcohol cosolvent produces the intermolecular NOEs observed in our work by altering the magnetization of the more abundant water spins.

Discussion Drawing conclusions from the data presented here turns on the reliability of the methods employed to predict solvent-peptide 1 H-1H cross relaxation terms. The methods make several assumptions. These include (1) the solvent mixture is compositionally and dynamically homogeneous, (2) solvent molecules can be represented by spheres that rotate rapidly enough that solvent protons can be regarded as being at the center of the sphere, (3) the sizes of the representative solvent spheres used are correct, (4) the translational diffusion coefficient of a species

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Figure 6. Initial slopes of intermolecular NOE vs mixing time plots for protons of [val5]angiotensin, scaled by the concentration of ethanol CH3 protons in 35% ethanol-d2-water (v/v) at 0 °C (red squares). The red bars represent a (25% error range for these quantities. The blue squares represent the average of σR values calculated for ten conformations of the peptide that are consistent with distance constraints derived from observed 1 H-1H intramolecular NOES and ROEs. The blue bars represent the range of calculated scaled initial slopes found with these structures. A reliable experimental value for σR for the valine methyl resonances could not be obtained due to baseline distortions resulting from suppression of the nearby ethanol signal at 1.16 ppm.

Figure 7. Intermolecular NOEs on signals of [val5]angiotensin resulting from inversion of the solvent CH2 resonance in a mixture of 36% ethylene glycol-water (v/v) at 0 °C (top). A control spectrum is shown at the bottom. The mixing time for the NOE experiment was 200 ms.

is independent of the proximity of that species to other molecules in the sample and can be equated to the experimentally measured coefficient, (5) there is no preferred solvent orientation for interactions with the peptide, and (6) the known aggregation behavior of hydrocarbon alcohols in water can be neglected. While these considerations are numerous and substantial, there is evidence that the approaches used nonetheless produce reliable predictions for the systems studied. For example, σR calculated by our methods for the 100% methanol and 89% methanol-water systems agree with the experimental values to well within the errors of the experiments. Similarly, observed and calculated σR for acetate are in agreement in all systems where the acetate

peak is present. The prediction of the reduced cross relaxation term for the TMS reference in 100% methanol (Figure 3) is not good, although the agreement between experiment and calculation for TMS is better in the 89% methanol-water system (Supporting Information). Overall, the reasonable agreement between calculated and experimental σR values observed suggests that significant divergences of observed and calculated cross relaxation terms probably indicate a breakdown of one or more of the assumptions listed and thereby provide some clues regarding interactions between solvent and peptide. The radii of methanol, ethanol, and ethylene glycol used in the calculations were 2.08, 2.40, and 2.49 Å, respectively. These

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Figure 8. Initial slopes of intermolecular NOE vs mixing time plots for protons of [val5]angiotensin, scaled by the concentration of methylene protons in 36% ethylene glycol-water (v/v) at 0 °C (red squares). The red bars represent a (25% error range for these quantities. The blue squares represent the average of σR values calculated for ten conformations of the peptide that are consistent with distance constraints based on the observed 1 H-1H intramolecular NOES and ROEs. The blue bars indicate the range of calculated σR found with these structures. The signals for the CA protons of Asp1 and Arg2 overlap, as do signals for the CA protons of Tyr4 and Phe8. Data shown for these protons are averages.

were estimated by a method based on modeling of the van der Waals surface of the alcohols. Hard sphere radii of methanol and ethanol indicated by gas solubility data are 1.8 and 2.2 Å,72 respectively, while NMR studies of transport processes in liquid methanol indicate a hard sphere radius of 1.8.73 Considerations of peptide solubilities in alcohol/water mixtures lead to estimates of the methanol and ethanol radii of 2.0 and 2.4 Å.74 Neutron scattering studies have suggested a radius of 1.5-2 Å for neat ethylene glycol.75 However, hydrogen bonding when ethylene glycol is dissolved in water probably makes this estimate unreliable for aqueous systems.63,76 Changing the radius of an alcohol over the ranges indicated by these studies in our calculations had relatively small effects on the σR values predicted and in no case changed the sign of a predicted σR. The NOE and coupling constant data do not point to a single structure for [val5]angiotensin in the alcohol-water solutions used. Rather, the peptide appears to be present in a variety of partially extended conformations. The intent in calculating σR values for a family of conformations was not to sample completely the conformational space available to the peptide but rather to obtain an idea the sensitivity of σR to conformation. Presuming that conformational interconversions of the peptide are rapid on a time scale defined by spin-lattice (T1) relaxation, the predicted value of σR that should be compared to an experimental result is a correctly weighted average of the values. Unfortunately, we have no information from our data regarding the dominant conformations of [val5]angiotensin present or their relative populations, so that calculation of a correct, averaged σR is not feasible. Chemical shifts for protons of [val5]angiotensin (shown in the Supporting Information) are sensitive to the amount and the nature of alcohol present in the solvent. The maximum shift changes with solvent for most peptide N-H protons ranged from 0.1 to 0.25 ppm. The shift of the phe8 N-H is an exception, varying by up to 0.8 ppm as the solvent is changed from 100% methanol to 35% ethanol-water. The heightened sensitivity of this shift may signal atypical interactions of solvent components with the peptide near this residue. In light of the chemical shift sensitivity observed for the phe8 N-H, it is interesting that experimental intermolecular NOEs arising from interactions of alcohol protons with the ring protons of phe8 at 0 °C are considerably larger than those predicted for the 35% ethanol-water, 36% ethylene glycol-water, and 25% methanol-water systems. The imidazole ring of his6 is reasonably close to the aromatic ring of phe8 in all conformations of [val5]angiotensin and the experimental σR for the histidine Hε1

ring proton is also larger than predicted in each of these systems. A possible mechanism for increasing the σR for the phe8 and his6 side chains is preferential accumulation of the alcohol component of the solvent mixture near them.77 In the case of ethanol-water, an enhancement by up to a factor of 3 is possible by this process, so the observed values of σR for the his6 and phe8 side chains in 35% ethanol-water could be accounted for in this way. However, in 36% ethylene glycol-water, σR for the phe8 aromatic ring protons is expected to be small and negative while a large, positive σR is observed. Even if the sign is neglected, an increase in the local concentration of ethylene glycol around this residue from 6.5 to ∼52 M would be required to account for the observed cross relaxation term. Since the molar concentration of pure ethylene glycol is only 18 M, the large σR for the phe8 ring protons in this solvent mixture cannot be due exclusively to a preferential solvation mechanism. Observed and calculated alcohol-peptide cross relaxation terms for the peptide backbone protons are in agreement for all methanol-containing systems and for the 36% ethylene glycol system at 0 °C. Although positive σR values for the methanol systems are both observed and predicted, the σR become negative for the ethylene glycol system, primarily due to the reduction of diffusion coefficients by the higher viscosity of the ethylene glycol-water mixture. Confirming previously reported results,36 experimental peptide backbone intermolecular NOE cross relaxation terms for 35% ethanol-water are strikingly more negative than predicted values (Figure 6). It was suggested previously that these negative σR values could be consistent with association of ethanol molecules with the peptide for times in the nanosecond range.36 The hydrodynamic radius of angiotensin as a random coil is expected to be about 9 Å at 25 °C.78 The average radii computed for the peptide conformations used to define the blue bars in Figures 3, 5, 6, and 8 were all ∼8 Å, in agreement with determinations of the peptide radius in 35% ethanol-water36 and 42% trifluoroethanol-water28 at 25 °C. The difference between the radius expected for a random coil and these values is consistent with some folding of the conformations present in solution. In contrast, the hydrodynamic radius for [val5]angiotensin at 0 °C found in the alcohol-water mixtures described here is larger (∼11 Å). In each water-rich solvent system studied, there are indications from the intermolecular NOEs of preferential association of the alcohol with some part of the [val5]angiotensin structure. Such association could contribute to increasing the hydrodynamic radius of the peptide. However, there are no indications of alcohol-peptide associa-

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tion in the 100% methanol mixture and yet the peptide radius is still near 11 Å. This lower dielectric solvent could favor some aggregation of the peptide, thereby enhancing its radius. Further experimental effort (and more precise experimental data) will be required to understand the hydrodynamic radii in all of these systems. It is clear that alcohol-peptide interactions with [val5]angiotensin depend in some way both on the structure of the alcohol and on the amount of water in an alcohol-water solvent mixture. At 25 °C these interactions appear to be welldescribed by a model in which the solvent mixture is regarded as homogeneous in composition, with the dynamics of encounters between alcohol and peptide molecules characterized sufficiently by the observed bulk translational diffusion coefficients. However, at temperatures below 25 °C, the dynamics of alcohol-peptide interactions appear to become more complicated, with alcohol molecules in some cases spending more time in contact or near-contact with peptide molecules than would be predicted solely on the basis of mutual diffusion. In addition, selective accumulation of the organic component of the solvent near regions of the peptide may take place. Finally, it is possible that alcohol molecules interact with the peptide in such a way that they cannot be represented by rapidly rotating spheres. It is beyond the capabilities of the intermolecular NOE experiments reported here to elucidate these possibilities and it is hoped that all-atom MD simulations now getting underway in our lab will illuminate them. Summary Investigation of intermolecular NOEs arising from spin dipolar interactions of alcohol protons with [val5]angiotensin protons at 0 °C in alcohol-water mixtures indicate that alcohol-peptide interactions are different in each solvent system. Solvent NOEs at the peptide backbone protons in methanol-water and ethylene glycol-water mixtures indicate that alcohol interactions near these groups appear to be adequately described by a standard hard sphere model. In contrast, in ethanol-water, NOEs indicate substantial slowing of the dynamics of peptide backbone-ethanol interactions in a waythatissimilartowhathasbeenobservedintrifluoroethanol-water mixtures.28 The side chain protons of the phe8 and his6 residues of the peptide exhibit intermolecular NOEs that are larger than those expected on the basis of the hard sphere model in all alcohol-water systems. Such enhanced NOEs could indicate preferential accumulation of alcohol near these side chains, but this cannot be the only reason for these larger-than-anticipated effects. Additional reasons for enhanced NOEs could include alteration of the dynamics of side chain-alcohol interactions or peptide-alcohol collisions that involve specific orientations of the alcohol molecule. Acknowledgment. We thank the National Science Foundation for support of this work (Grant CHE-0408415). Supporting Information Available: Listings of proton chemical shifts for all systems studied, observed coupling constants (3JNH-CRH), peptide hydrodynamic radii, and further details of the intramolecular NOEs observed in each solvent system, along with plots for studies of the peptide in 89% methanol-water analogous to Figures 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Gomez-Puyou, A. Biomolecules in Organic SolVents; CRC Press: Boca Raton, FL, 1992.

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