Estimation of Molecular Environment for Amino Acids in Proteins by

Langmuir 2001, 17, 2493-2496

2493

Estimation of Molecular Environment for Amino Acids in Proteins by Monolayer Behaviors of the Amino Acid Derivative Polymers on a Water Surface Makoto Ide and Tokuji Miyashita* Institute for Chemical Reaction Science (ICRS), Tohoku University, Katahira 2-1-1, Aoba-ku, Sendai 980-8577, Japan Received July 24, 2000. In Final Form: November 14, 2000 The behaviors of polymer monolayers on a water surface were investigated by the measurement of surface pressure-area isotherms. A good correlation of the collapse pressure of the monolayer with the hydropathy scales of the natural amino acid rather than the hydrophobicity scales was observed. It is concluded that the amino acid residue in the polymer monolayer on the water surface exists in a similar molecular environment to that of the polypeptides. The hydropathy scale for the amino acid residue can be obtained experimentally from the collapse pressure of the corresponding monolayer, and that for unnatural amino acid residues also was examined. Moreover, the dependence of the hydrophobic character of the amino acid on temperature is also investigated by the change in the collapse pressure of the amino acid derivative polymer monolayers.

Introduction Proteins have to keep a special conformation to fulfill their functions. It is well-known that noncovalent bondings working in polypeptides, such as hydrogen-bonding and hydrophobic interaction, are important factors for formation of a special conformation of protein.1-7 Especially, hydrophobic interaction between apolar amino acid sidechains that induces the folding of the secondary structure of the polypeptide plays an important role.4-7 Many researchers have often studied the hydrophobic character of amino acid side-chains. Since real hydrophobicity of amino acid side-chains placed in actual proteins or a membrane intrinsic protein is usually difficult to determine directly, most of the hydrophobicity of amino acids has been determined thermodynamically from the water-organic solvent or water-vapor partition coefficients, Kd, of free amino acid molecules or anologues.8-13 However, the scales for hydrophobicity often differ strongly from each other, especially when the residues having lone pair and π electrons are considered. Moreover, the hydrophobicity determined by the method does not consider the molecular environment of the amino acid in actual proteins. In this situation, Kyte and Doolittle proposed hydropathy scales14 to modify the hydrophobicity (1) (a) Fasman, G. D. In Prediction of Protein Structure and the Principles of Protein Conformation; Plenum Press: New York, 1990. (b) Chou, P. Y.; Fasman, G. D. J. Mol. Biol. 1973, 74, 263. (2) Garnier, J.; Osguthorpe, D. J.; Robson, B. J. Mol. Biol. 1978, 120, 97. (3) Janin, J.; Chothia, C. J. Mol. Biol. 1980, 143, 95. (4) Tanford, C. In The Hydrophobic Effect: Formation of Micelles and Biological Membranes, 2nd ed.; Wiley: Chichester, 1980. (5) Pain, R. H. In Mechanisms of Protein Folding; Oxford University Press: Oxford, 1994. (6) Kauzmann, W. Adv. Protein Chem. 1985, 14, 1. (7) Kauzarides, T.; Ziff, E. Nature 1988, 336, 646. (8) Roseman, M. A. J. Mol. Biol. 1988, 200, 513. (9) Fauchere, J. L.; Pliska, V. Eur. J. Med. Chem. Chim. Ther. 1983, 18, 369. (10) Nozaki, Y.; Tanford, C. J. Biol. Chem. 1971, 246, 2211. (11) Wolfenden, R. V.; Andersson, L.; Cullis, P. M.; Southgate, C. C. B. Biochemistry 1981, 20, 849. (12) Cohn, E. J.; Edsall, J. T. In Protein, Amino Acids, and Peptides as Ions and Dipolar Ions; Reinhold: New York, 1943. (13) Hine, J.; Mookerjee, P. K. J. Org. Chem. 1975, 40, 292. (14) Kyte, J.; Doolittle, R. F. J. Mol. Biol. 1982, 157, 105.

scales10-13 by considering the position of the amino acid in a known protein structure.15 The protein structures in lipid membranes have been successfully discussed by the hydropathy scale of a given amino acid sequence. However, it is necessary for the estimation of the hydropathy scale to get the information of the exact protein structure. Therefore, the hydropathy scale of unnatural amino acid side-chains cannot be estimated by the method which was proposed by Kyte and Doolittle. In this paper, we described a novel method for experimental estimation of hydropathy scales of amino acid sidechains without the structural data of proteins. LangmuirBlodgett monolayers and multilayers are known to offer molecular environments similar to that in a biomembrane.16 The behavior of a Langmuir monolayer on the water surface strongly relates with a hydrophobichydrophilic balance of the compound. Through the previous studies on a series of polyacrylamide monolayers and LB films, we showed that the polyacrylamide structure is suitable for the formation of a high-quality monolayer where hydrogen-bonding of the amide groups plays an important role for self-assembly.17-20 In this work, based on this excellent property of the polyacrylamide, we prepared the monolayer of polyacrylamide modified by various amino acid derivatives (Figure 1) and measured the monolayer behavior with a variety of amino acids. Depending on the property of the amino acid in the polymer monolayer, whether the amino acid tends to participate in the water phase or the hydrophobic phase, the properties of the polymer monolayers were changed. We found that the change in hydropathy scales of the amino acids can be estimated experimentally from the change in the (15) The term hydropathy was first introduced by Kyte and Doolittle as a neutral term to describe the relative affinity that a solute has for water as compared to another phase. (16) Birdi, K. S. In Lipid and Biopolymer Monolayers at Liquid Interfaces; Plenum Press: New York, 1989. (17) Miyashita, T. Prog. Polym. Sci. 1993, 18, 263. (18) Matsui, J.; Mitsuishi, M.; Miyashita, T. Macromolecules 1999, 32, 381. (19) Taniguchi, T.; Fukusawa, Y.; Miyashita, T. J. Phys. Chem. B 1999, 103, 1920. (20) Feng, F.; Miyashita, T.; Okubo, H.; Yamaguchi, M. J. Am. Chem. Soc. 1998, 120, 10166.

10.1021/la001044o CCC: $20.00 © 2001 American Chemical Society Published on Web 03/23/2001

2494

Langmuir, Vol. 17, No. 8, 2001

Ide and Miyashita

Table 1. Properties of P-(Amino Acid)-Ac Monolayers ASAa (nm2)

polymer

Mw/103 (Mw/Mn)

ALA (nm2)

P-Gly-Ac P-Ala-Ac P-Val-Ac P-Leu-Ac P-Ile-Ac P-Met-Ac P-Phe-Ac P-Trp-Ac

6.91 (1.65) 5.60 (1.46) 4.50 (1.67) 4.99 (1.40) 4.55 (1.34) 5.48 (1.60) 4.99 (1.73) 5.40 (1.21)

0.291 0.434 0.507 0.488 0.508 0.477 0.511 0.531

Natural Amino Acids 0.526 56.1 0.937 45.1 1.57 30.7 1.74 33.2 1.82 25.3 1.98 43.0 2.29 41.3 2.72 48.5

P-Chx-Ac P-Nva-Ac P-Bcy-Ac

3.98 (1.48) 4.32 (1.56) 4.64 (1.89)

0.589 0.522 0.580

Unnatural Amino Acids 24.6 29.0 32.4

a

πcol (mN/m)

hydrophobicityb (kcal/mol)

hydropathyc

0.00 0.67 2.18 3.02 3.02 1.30 3.24 2.86

-0.4 1.8 4.2 3.8 4.5 1.9 2.8 -0.9

Reference 21. b Reference 8. c Reference 14.

Figure 1. Chemical structures of P-(amino acid)-Ac and amino acid side-chain.

monolayer behaviors of the amino acid derivative acrylamide polymers. Experimental Section Materials. We employed poly(long-alkylacrylamide) as a base polymer, and natural and unnatural amino acid residues (glycine (Gly), L-alanine (Ala), L-valine (Val), L-leucine (Leu), L-isoleucine (Ile), L-methionine (Met), L-phenylalanine (Phe), L-tryptophan (Trp), L-cyclohexylglycine (Chx), L-norvaline (Nva), and S-benzylL-cysteine (Bcy)) were introduced chemically into the polymers (P-(amino acid)-Ac, Figure 1). The amino acid derivative polymer, for example, poly[N-{1(tetradecylcarbamoyl)ethyl}acrylamide] (P-Ala-Ac), was prepared by usual radical polymerization of the corresponding monomer, NR-acryloyl-N-tetradecylalanine amide. The molecular weights and dispersions of the polymers determined by gel permeation chromatography are given in Table 1. NR-Acryloyl-N-tetradecyl-(amino acid) amide monomers were synthesized by the following procedure. As an example, the case of NR-acryloyl-N-tetradecyalanine amide monomer is described; NR-tert-butoxycarbonylalanine (5.0 mmol) (SIGMA), n-tetradecylamine (5.0 mmol) (Nacalai), and 1-hydroxybenzotriazole (5.5 mmol) (Nacalai) were dissolved in tetrahydrofuran (THF) (150 mL), and then N,N-dicyclohexylcarbodiimide (5.5 mmol) (Nacalai) was added to the solution at 0 °C. The mixture was allowed to react at 0 °C for 1 h and at room temperature for 8 h. The solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate (EtAc) (300 mL) and washed with 1 M citric acid aqueous solution, saturated NaCO3 aqueous solution, and water. After being dried over Na2SO4, the mixture was concentrated to about 80 mL. After cooling to 0 °C, NR-tert-butoxycarbonyl-Ntetradecylalanine amide was obtained as a precipitate. The precipitate was dissolved in trifluoroacetic acid (TFA) (3 mL). The mixture was stirred at room temperature for 1.5 h, followed by evaporation of TFA under reduced pressure. The residue was dissolved in EtAc and washed with saturated NaCO3 aqueous solution and water. After being dried over Na2SO4, the solvent was removed under reduced pressure. The residue was chromatographed on SiO2 (4:1:1 CHCl3/EtAc/MeOH) to give Ntetradecylalanine amide. The N-tetradecylalanine amide (3.0 mmol) was reacted with acryloyl chloride (3.0 mmol) (TCI) in the presence of triethylamine (3.3 mmol) in CHCl3 (90 mL) at 0 °C. After stirring at 0 °C for 0.5 h and at room temeperature for 5

Figure 2. Surface pressure-area isotherms for natural (A) and unnatural (B) amino acid derivative polymers: P-Gly-Ac (a); P-Ala-Ac (b); P-Val-Ac (c); P-Leu-Ac (d); P-Ile-Ac (e); P-MetAc (f); P-Phe-Ac (g); P-Trp-Ac (h); P-Chx-Ac (i); P-Nva-Ac (j); P-Bcy-Ac (k). h, the mixture was washed with saturated NaCO3 aqueous solution, saturated NaCl aqueous solution, and water. After being dried over Na2SO4, the solvent was removed under reduced pressure. The residue was chromatographed on SiO2 (8:2:1 CHCl3/ EtAc/MeOH) to give NR-acryloyl-N-tetradecylalanine amide. Measurement of Surface Pressure-Surface Area Isotherm. Measurement of surface pressure (π)-surface area (A) isotherms was carried out with a automatic Langmuir trough (Kyowa Kaimen Kagaku, HBM-AP) equipped with a Wilhelmy balance at a compression speed at 14 cm2/min. Milli-Q grade water (resistivity > 17 MΩ‚cm) was used as subphase. The subphase was thermostated by a water jacket that was maintained by circulation of water from a constant-temperature bath. The polymers were spread from chloroform solution (10-3 M) on the water surface.

Results and Discussion Monolayer Behaviors of Natural Amino Acid Derivative Polymers on the Water Wurface. The π-A isotherms of natural amino acid derivative polymers (P(amino acid)-Ac) were measured at 25 °C (Figure 2A). The steep rise in surface pressure and the high collapse pressure in the isotherms indicate the formation of condensed monolayers. Despite only the amino acid sidechain (R) being different in the chemical structure of the polymers, the isotherms are drastically varied with the amino acid derivatives. Extrapolation of the steeply rising part of the π-A curve to zero surface pressure gives the

Estimation of Molecular Environment for Amino Acids

Figure 3. Correlation of the collapse pressures of the monolayers with the hydrophobicity scale (A) and with the hydropathy scale (B) of the amino acid side-chain: P-Gly-Ac (a); P-Ala-Ac (b); P-Val-Ac (c); P-Leu-Ac (d); P-Ile-Ac (e); P-Met-Ac (f); P-Phe-Ac (g); P-Trp-Ac (h).

average molecular occupied surface area per repeating unit (ALA) in the monolayer (Table 1). The ALA values varied with the amino acid side-chain, depending on the molecular size of the side-chain. The amino acid side-chain in the monolayer is placed at the water surface with its own surface area. The surface areas (ALA’s) are compared with the accessible surface area (ASA) of the amino acid sidechain,21 that is calculated from the van der Waals radii in the tripeptide (Gly-X-Gly, where X is the amino acid in question) (Table 1). The molecular orientation of the side-chain in the monolayer where the long axis of the amino acid side-chain is parallel to the long alkyl chain (tetradecyl moiety) is suggested. Correlation of Collapse Pressure for Amino Acid Derivative Polymers with Hydrophobicity and Hydropathy Scales. A. Natural Amino Acid. The collapse surface pressures (πcol) of the monolayers were dependent on the amino acid derivatives. The glycine derivative shows the highest pressure, and the lowest pressure is for the isoleucine derivative. The πcol values decreased with the amino acids in the order Gly > Trp > Ala > Met > Phe > Leu > Val > Ile (Table 1). The order in the collapse pressure cannot be apparently explained by steric hindrance of the amino acid side-chains, because P-Trp-Ac, having an imdolyl substituent, and P-Ala-Ac, having a methyl one, show similar πcol values. It is well-known that a monolayer property strongly depends on a hydrophobichydrophilic balance of the compound.16,22 The πcol values are expected to correlate with the hydrophobicities of the amino acid side-chains,8 which are determined by a partition coefficient, Kd, of amino acid derivatives between water and various organic solvents. The πcol value is plotted as a function of the hydrophobicity in Figure 3A. Apparently, no correlation between them is obtained. Next, the πcol value was plotted against the hydropathy scale of amino acid side-chains in Figure 3B. The hydropathy scales are determined by computer calculations based on known protein structures in which the amino acid residue is found to be in a water phase or in a protein-membrane phase and on the partition coefficients of amino acid (21) Chothia, C. J. Mol. Biol. 1976, 105, 1. (22) Gaines, G. L. In Insoluble Monolayers at Liquid-Gas Interfaces; John Wiley & Sons: New York, 1966.

Langmuir, Vol. 17, No. 8, 2001 2495

molecules or analogues between water and organic solvents.14 A good correlation between the πcol value and the hydropathy is observed (Figure 3B). In the following we attempt to describe a possible correlation of hydrophobicity with the collapse pressure of the amino acid derivative polymer monolayers. Most of the hydrophobicity values of the amino acids have been determined thermodynamically from the Kd values of amino acid derivative compounds. The hydrophobicity determined by the Kd measurement varies by the amino acid derivatives employed for the measurement; that is, it is dependent on the chemical form, ionic species or neutral form. For example, free amino acid exists as the zwitterionic (carboxylate and ammonium groups) form in pure water, and three basic formations (H2NCH(R)COO-, H3N+C(R)COO-, and H3N+C(R)COOH) can be considered. Therefore, it is difficult to estimate the hydrophobicity of the side-chain (R) itself in the amino acid. Therefore, the hydropathy scale is proposed to modify the hydrophobicity obtained from the Kd values by computer calculation considering the position of the amino acid in the globular or intrinsic membrane protein.14 For the calculation it is necessary to know the protein sequence and structure exactly. The monolayer properties of the present amino acid derivative polymers show a good correlation with the hydropathy scale, which means that the amino acid residue in the present monolayer on the water surface exists in a similar molecular environment to that of the polypeptides. The good correlation in Figure 3B suggests that the hydropathy scale for an amino acid residue can be obtained experimentally by measuring the π-A isotherm, especially the collapse pressure of the corresponding monolayer of the amino acid derivative acrylamide polymer. In this method, it is not necessary to get information on the exact protein structure. Moreover, it is valuable that this method is applicable to the determination of a hydropathy scale of an unnatural amino acid. B. Estimation of Hydropathy Scale for Unnatural Amino Acid Side-Chains. As described above, the collapse pressures of the amino acid derivative polymer monolayers are correlated with the hydropathy of the side-chain. Therefore, it is expected that the hydropathy scale of an unnatural amino acid side-chain can be estimated by the behavior of the corresponding polymer monolayers. The monolayer behaviors of unnatural amino acid (L-cyclohexylglycine (Chx), L-norvaline (Nva), and S-benzyl-Lcysteine (Bcy)) derivative polymers on the water surface were investigated (Figure 2B). The π-A isotherms showed a steep rise in surface pressure, which means that their polymers also form a densely packed monolayer on the water surface, similar to that of a natural amino acid. The collapse pressures (πcol) for P-Chx-Ac, P-Nva-Ac, and P-Bcy-Ac were determined to be 24.5, 29.1, and 32.4 mN/m at 25 °C, respectively (Table 1). The hydropathy scale for the unnatural amino acid side-chain can be estimated from the πcol; and their hydropathy scale is placed in the order Trp (-0.9) < Gly (-0.4) < Ala (1.8) < Met (1.9) < Phe (2.8) < Bcy < Leu (3.8) < Val (4.2) < Nva < Ile (4.5) < Chx. C. Effect of the Structure of the Side-Chain on the Hydropathy Scale. We discuss here the relation between the πcol values (hydrophathy) and the chemical structure of the amino acid side-chain. In general, hydrohobicity is related to alkyl chain length or the number of carbons. The πcol values for the amino acid derivative monolayers having a linear alkyl side-chain (Ala, Nva, and Gly) decrease with the number of carbons (Cn). On the other hand, the πcol values for amino acids having a branched, cyclic, aromatic, or sulfone side-chain, and especially

2496

Langmuir, Vol. 17, No. 8, 2001

amino acids having lone pair and/or π electrons (Met, Phe, Trp, and Bcy), are much lager than the expected values from the Cn. This is explained by the fact that the lone pair and π electrons are able to act as proton acceptors; therefore, they enhance the affinity of the side-chain for water. It was previously reported by Hine and Mookerjee13 that aromatic and other unsaturated systems are consistently more hydrophilic than their saturated counterparts, and moreover, Radzicka and Wolfenden23 suggest that the lone pair electrons of an indole ring could serve as a proton acceptor. Our experimental result is consistent with the results of Hine and Radzicka. In the case of a branched or cyclic alkyl side-chain such as Val, Leu, Ile, and Chx, their πcol values were slightly larger than the expected values. Suzuki et al. investigated the hydration shell around the amino acid side-chain (Nva, norLeucine, Val, Leu, and Ile) by the microwave dielectric method.24 They reported that the number of water molecules in the hydrophobic hydration shell around the branched sidechain (Val, Leu, and Ile) is smaller than that around the linear alkyl counterpart (Nva and norLeucin), regardless of the fact that the accessible surface areas of the sidechains have almost the same value. Their result shows that the hydrophobic field forming the branched sidechain has greater overlap with the hydrophilic field forming the amide groups in the amino acid molecule than does that forming the linear side-chain. It is therefore considered that the branched structure of the side-chain makes the hydrophobic character of itself in the amino acid molecule weak. The consideration by Suzuki et al. is not in conflict with our result that the πcol values for amino acids having branched or cyclic side-chains are larger than those for amino acids having linear chains. After all, the lone pair and π electrons, and the branched structure of the amino acid side-chain, tend to decrease the hydropathy scale. The collapse pressures of the monolayers are nicely related with the molecular structure. We can say that the molecular environment of each amino acid in the protein is easily discussed by measuring the collapse pressure of the amino acid monolayer on the water surface. The effect of temperature on πcol values for amino acid derivative polymers is given in Figure 4. It is seen that (23) Radzicka, A.; Walfenden, R. Biochemistry 1988, 27, 1664. (24) Suzuki, M.; Shigematsu, J.; Fukunishi, Y.; Kodama, T. J. Phys. Chem. B 1997, 101, 3839.

Ide and Miyashita

Figure 4. Dependence of the collapse pressure of the polymer monolayers on the temperatures: P-Gly-Ac (b); P-Ala-Ac (O); P-Val-Ac (2); P-Leu-Ac (4); P-Ile-Ac (0); P-Met-Ac (9); P-PheAc (3); P-Trp-Ac (1).

at 40 °C the magnitude of πcol is the same for different polymers. This may suggest a relationship to the thermal denaturation of proteins. Conclusion Hydropathy scales for amino acids are determined experimentally by the measurement of surface pressurearea isotherms of the amino acid derivative polymer monolayers. The hydropathy, so far, has been determined by modifying the hydrophobicity value obtained from the partition coefficients of amino acid derivatives by the means of a computer calculation considering the position of the amino acid in the globular or intrinsic membrane protein. Therefore, for determination of the hydropathy scale of the amino acid side-chain, it is necessary to get the information of the exact protein structure. On the other hand, in the case of the method that is described in this paper, the hydropathy scale of the amino acid sidechain can be determined without the information of the exact protein structure. It becomes feasible to estimate the hydropathy scales of unnatural amino acids. Moreover, the change in the hydrophobic character of the amino acid with temperature can be monitored by the change in the collapse pressure of the amino acid derivative polymer monolayers. It is also suggested that there is a certain temperature where the difference in the hydrophobic properties among the amino acid residues disappears, which would be related to the thermal denaturation of proteins. LA001044O