Langmuir 1994,10, 2731-2735
2731
Monolayer Properties of Hydrophobic a-Helical Peptides Having Various End Groups at the Airmater Interface? Katsuhiko Fujita, Shunsaku Kimura, and Yukio Imanishi* Department of Material Chemistry, Faculty of Engineering, Kyoto University, Yoshidahonmachi, Sakyo-ku, Kyoto 606-01, Japan
Elmar Rump and Helmut Ringsdorf Institute of Organic Chemistry, Johannes Gutenberg University Mainz, J.-J. Becherweg 18-20, 55099 Mainz, Germany Received January 18, 1994. I n Final Form: April 18, 1994@ A hydrophobic peptide,Boc-(Ala-Aib)s-OMe(BAlGM),and its end-modified derivativeswere synthesized, and the pressure-area (n-A) isotherms ofthe peptides spread at the airlwater interface were studied from the viewpoint of interhelix interactions. All n-A isotherms of the synthetic peptides showed an inflection and weak irregular bumping at a surface areas of about 240 and 230 A21molecule,respectively, indicating that the helix axis of the peptide is oriented parallel to the interface. A small mound was observed at around 300 A2/moleculein the n-a isotherm of BAlGM, which was ascribed to the phase transition from a liquid to a solid state. The monolayer of an equimolar mixture of the peptides having an opposite kind of charge in the end group underwent the phase transition in the n-A isotherm, which was not observed with one of the two peptides. The electrostatic interaction between the end groups should stabilize the molecular packing at the interface.
Introduction Hydrophobic helical peptides are often found in watersoluble globular proteins (e.g., hemog1obin)l and the transmembrane region of membrane proteins (e.g., bacteriorhodopsin).2 Several helices associate together in the protein, forming a so-called helix bundle, which is one of the major structural motifs of naturally-occurringproteins. It is, therefore, important to study interactions between helices in terms of the formation of a tertiary structure or the stability of the p r ~ t e i n . ~ - ~ The monolayer technique is a unique means to investigate intermolecular interactions, because it detects intermolecular forces acting in a low-dimensional array of molecules.6 When amphiphilic molecules are spread at the airlwater interface, they show various states such as gas-analogous, fluid-expanded, fluid-condensed, and solid-condensed phases, depending on the surface pressure.' These phases can be analyzed on the basis of the pressure-area (n-A)isotherm which reflects electrostatic interactions or molecular packing between amphiphilic molecules in each phase.6zs The relationship between the structure of amphiphiles, especially the polar head group,
and the surface properties of monolayer^^^^ has been investigated. Synthetic poly(a-amino acid)s have been shown to form monomolecular films at the aidwater interface.1°-16 However, their surface pressure-area isotherms did not show phase transitions or sharp collapses. A possible reason is the distribution of molecular weight, which causes irregularities of molecular packing. Another possible reason is instability of the secondary structure against the surface pressure. In the present study, hydrophobic helical peptides having a defined chain length and various terminal groups were synthesized, and their behavior at the airlwater interface was investigated. The monolayer formation of a mixture of different types of peptides was also analyzed to know the nature of the interhelix interactions. Since the primary sequence of one helix in the helix bundle is not necessarily the same as the other, there should be several factors which contribute to the stability of the helix-bundle structure depending on the composing amino acids. This knowledge will be important for construction of a helix monolayer with a novel function, where a few helices with different functional groups associate together. Peptides containing a-aminoisobutyricacid (Aib)to tend t Abbreviations: Aib, 2-aminoisobutyric acid; BAlGB, Boc-(AlaAib)s-OCHzC&; BAlGM, Boc-(Ala-Aib)s-OCH3; Boc, tert-butylto form a helical structure because of the steric hindrance oxycarbonyl; BS3A16M, Boc-Sar3-(Ala-Aib)s-OCH3;CD, circular around the a-carbon atom of Aib residues in other dichroism; COOHAXM, HOOC(CH2)2CO-(Ala-Aib)s-OCHs; DCC, conformations.17 For example, a hexadecapeptide, Bocdicyclohexylcarbodiimide; HAlGB, T F A . H - ( A ~ ~ - A ~ ~ ) ~ - O C H Z C ~ H ~ ; (Ala-Aib)s-OMe (BAlGM), has been revealed to take an HAlGM, TFA-H-(Ala-Aib)s-OCH3;HOBt, l-hydroxybenzotriazole; HS3A16M, TFA.H-Sar3-(Ala-Aib)s-OCH3; TFA, trifluoroacetic acid; n-A isotherm, surface pressure-area isotherm. Abstract published in Advance A C S Abstracts, July 1, 1994. @
(1)Fermi, G.; Perutz, M. F.Atlas ofMolecularStructures in Biology. 2. Haemoglobin and Myoglobin; Clarendon Press: Oxford, 1981. (2)Michel, H. J . Mol. Biol. 1982,158, 567. (3)Alberts, B.; Bray, D.; Lewis, J.; Raff,M.; Roberts, K.; Watson, J. D.Molecular Biology of the Cell, 2nd ed.; Garland Publishing: New York and London, 1989;Chapter 3. (4)Rao, S. T.; Rossmann, M. G. J . Mol. B i d . 1973,76, 241. (5) Ho, S. P.; DeGrado, W. F. J . Am. Chem. Soc. 1987,109, 6751. (6)Gains, G. L., J r . Insoluble Monolayers at liquid interface; Interscience: New York, 1966;Chapter 5. (7) Birdi, K. S.Lipid and biopolymer monolayers at liquid interfaces; Plenum Press: New York, 1989;Chapter 3. (8)Heath, J. G.; Amett, E. M. J . Am. Chem. Soc. 1992,114, 4500.
(9) Ikeura, Y.; Kurihara, K.; Kunitake, T. J . Am. Chem. Soc. 1991, 113, 7342. (10) Malcolm, B. R. h o c . R. Soc. London, Ser. A 1968,305,363. (11)Loeb, G. I.; Baier, R. E. J . Colloid Interface Sci. 1986,27, 38. (12)Takeda, T.; Matsumoto, M.; Takenaka, Y.; Fujiyoshi, Y. Uyeda, N. J . Colloid Interface Sci. 1983,91, 267. (13)Malcolm, B.R. Biochem. J . 1968,110, 733. (14)Birdi, K. S.Lipid and biopolymer monolayers at liquid interfaces; Plenum Press: New York, 1989;Chapter 5. (15)Lavigne, P.; Tancrede, P.; Lamarche, F.; Max, J.-J. Langmuir 1992,8, 1988. (16) Weisenhorn, A. L.; Romer, D. U.;Lorenzi, G. P. Langmuir 1992, 8,3145. (17) (a) Burgess, A. W.; Leach, S. J. Biopolymers 1973,12,2599.(b) Karle, I. L.;Balaram, P. Biochemistry 1990,29, 6747.
0743-746319412410-2731$04.50/0 0 1994 American Chemical Society
2732 Langmuir, Vol. 10, No.8, 1994 Table 1.
Fqjita et al. the Synthetic
Strpctpre and Abbredation of
HelieriPeptldea abbreviation BAl6M BAl6B HAl6M HAl6B BAlGOH COOHAl6M BS3Al6M HS3A16M
Table 2. Molar JCEpticity (0) of the Helical Peptides at 888 n m in Ethaidmatex (Wbvhr) compound (0) (degcmzYdmol) compound (e) (degunzKdmol) BAl6M -328x105 BA16OH -2.36 x 105 HAl6M
-2.47 x 105 -2.63 x 10s''
COOHA16M
-2.68 x 105 b -3.18 x 105 -3.22 x 105'
Addition of equimolar NaOH. Addition of equimolar HC1. 30 A
a-helical structure in the crystalline state by X-ray di%action.18 The dimensions of the unit cell were 9.3 x 9.3 x 25.7 which are in good agreement with the calculated values for a complete a-helical str~cture.~~.~~ This peptide and the derivatives having various end groups were synthesized, and their monolayer properties at the &/water interface were investigated.
r
E
1 E
0 3
20-
m
ea 0
+! 1 0 a v)
Materials and Methods Peptide preperation. The tdzuchw of synthetic peptides with their abbreviations are shown in Table 1. BAl6M and BAl6B were synhaized by the previous pmcedUrea0 with some modifications. TheBocgroupo€BAl6MandBAl6Bwasremoved with trifluoroacetic acid ("FA) to obtain HA16M and HAlGB, respectively. HAl6M was reacted with Bfold M succinic anhydride in pyridine at 40 "C for 144 followedby gel 6lhtion with a Sephadex LH-20W '91 Up& Sweden) column
byuaingmethanolastheelutionaolvent,tooMainCoOHAl6M. Boc-Sar&)Hwassyntimizedbystepwiseelongationasreported HAl6M was connected to Boc-Sar&)H using a couplingreagent, DCc/ROBt,~indimethyKodde,followed by purification with gel tiltration as described above to obtain BS3Al6M. HS3Al6MwasprepamdhmBS3Al6Minthesame way as the synthesis of HAl6M h m BAl6M. Method. The peptide samples were dissolved in CHCWOH (9llvh) (Mer& analyticalgrade)at concentrations of (1.53) x lo-' M. The n-A iaotherm was m r d e d at a constant rate of reducing area of 0.37 cm% with a homemade Ignrrmuir hugh.= The peptide solution was spread on theaqueousphase by using-a and equilibrated for 15 min before compreamon. The Mdlqore water, 50 mM citrate buffer (pH 3.5-5.0), and 50 mMphosphase bder(pH5.5-11.0) were d fortheaqueoussubphase. Eachexpezhentwasrepeaklseveral times to check the reproducibility. Circular dichroism (CD) of the peptides was measured in ethanolkater (95.6 vhr) at room temperature on a Jasa, 5-600 CD spectropolarimeter using an optical cell of l-cm path length.
-
200
400
600
(A2) F'igure 1. n-A isotherm of BA16M on a water subphase at 10 (- - -), 20 (-), and 30 (- - -) "C. Molecular a r e a
Table 3. Infiection Point and Phase Transition Point in the Presaue-Area Isotherms of the Completely Protected Peptide W 6 M temp subphase water water water 0.5MNaCl
inflection point area pressure (Az) (mN/m)
top of mound area pressure
254.2 255.6 256.3 257.8
312.8 299.5 282.0 292.8
(m~~/m) ~~
10
20 30 20
11.6 11.5 11.8 15.6
4.7 6.1 8.9 9.0
solvent. The Cotton effeds of HAlGM, BAlGOH, and COOHA16M at 222 n m were scarcely affected by the addition of hydrochloric acid or aqueous sodium hydroxide under different ionization states of the end groups (Table 2). It is, therefore, concluded that each peptide takes an a-helical conformation in ethanol containing 5% water and that the conformation is stable irrespective of the charge state of the helix terminal. This observation is in sharp contrast to the previous reports"3 that the helix codormation is affktedby the interaction of the terminal charge with the macrodipole of the helix. Results Fully protected Helical Peptide, BAlGNI, at the Interface. n-A isotherms of BA16M on the water Conformation. CD spectra of all the synthesized subphase at 10,20, and 30 "Care shown in Figure 1.The peptides showed a doubleminimum profile, which is inflection point appearing in the n-A isotherm, which charaderistc of an a-helical sgucture.u The helix content corresponds to the maximum value of the area elastic of BAl6M was estimated to be ca.60% h m the molar modulus, -A(WdA),was independent ofthe temperature ellipticity at 222 while it forms a perfect a - h e l i d (Table 3). The irregular bumping was observed in smaller structurein the solid state." It is known that the h e l i d areas than the inflection point, indicating collapse of the structureof peptides is destabilized in a highly dielectric monolayer.30 The area at the inflection point coincides The low helix content in solution should with the sectional area of this helical peptide along the represent a conformational destabilization in the polar helix axis (239k), which has been determined by X-ray analy~is.'~The n-A isotherm, therefore, indicates that (18)otoda,K; Kitagawa, Y.; Kimura, S.; rmnninhi,Y. B w p d y m m the peptides take an a-helical structure at the &/water l-. 33,1337. (19)Brauden, C.; Tooee, J. Zntroductro . n to Prvtein Structure. interface and orient the helix axis parallel to the interface. Garland: New York, 1991. . (zo)Otota,IL;Kimura,S.;TmaninhiY.BiocAim.Biqplrys.Actal~,In the region between the inflection point and the starting point of the irregular bumping, inte-tation should 1145,33. (21) Sugihara, T.;rmaninhi,Y.; Higashimura,T. Biopolymers 1976, occur such that the side chain of the a-helix is allowed to 14,723. penetrate to the neighbor.15 Further compression col(22)KO*, w.; Geiger, R C h .Ber. 1970,103,788. lapsed the monolayer. (23)AIbrecht, 0. Thin sdid Films 1983.99.227.
w,
(24)Holewarth, G.; Doty, P. J . Am. C h .Soc. 1965,87,218. (25)Greenfield, N.; Fasman, G. D. Biochemisby 1969,8,4108. (26)Chen, Y.-H.; Yaag, J. T.;Chau, E H. BiocAemistry 1974,13,
3350.
(2'7)Balaram, H.; Sukumar, M.;Balaram, P. Bwpdymers l%36,25,
2209.
(28)Wada, A. Adu. Biophys. 1976,9,1. (29)Shoemaker, K R; Yo& E. J.; Stewart, J. M;Baldwin, B. L. Nahue (London) lW7,326,563. (30)Gains, G. L,Jr. Insoluble Mo~whyersat liquid in&+; Interscience: New York, 1966, Chapter 4, Section 3.
Langmuir, Vol. 10,No.8,1994 2733
Helical Peptide Monolayer at the Air / Water Interface In the n-A isotherm, a mound was observed at around 300 Az. The local maximum appeared a t higher pressure as the temperature was raised. The same sort of temperature dependence has been observed with the phase transition from the fluid-expanded state to the fluidThe condensed state of lipid or fatty acid mono1aye1-s.~~ mound was observed either in the compression experiment after aging equilibration (160 min) or in both compression stages during a compression to 13mN/m, depression and compression process. It was found to become smaller with decreasing compression speed, indicating that the mound is due to kinetic effects as reported on the monolayer of azacrown derivative^.^^ The mound nearly vanished and a plateau level appeared in the n-A isotherm at extremely slow compression. Therefore, the mound should not result from destruction of monolayers but from rearrangement of the molecules in the monolayer. The depression from the top of the mound to the bottom represents the phase transition from a liquid to a solid state. I t is well-known that the phase transition of an insoluble monolayer can be observed with a fluorescence microscope by using a fluorescence probe added to the m ~ n o l a y e r . ~The ~,~~ domain formation of the monolayer containing a fluorescence-labeled helix at a concentration of 1or 2 mol % was observed under a fluorescence microscope when the compression was held at the mound.35 In addition, fluorescenceanisotropy was observed. These observations support the phase transition from a liquid to a solid state around the mound region in the n-A curve. In the solid state, the helix rods should be arranged regularly, while they might be dispersed irregularly or form small crystalline domains in the liquid state.36 The surface pressure at the inflection point and at the top of the mound was higher on the 0.5 M NaCl aqueous subphase than on the water subphase at the same temperature, 20 "C (Table 3). It is hard to explain the higher collapse pressure or phase transition pressure in terms of the interaction of ions with BAlGM, which is a nonionizable peptide molecule. It is considered that the collapse pressure was increased by the "salting out7) e f f e ~ tunder ~ ~ , high ~ ~ ionic strength in the subphase. Sodium diphosphate has been shown to affect the protein structure strongly.39 The surface pressure in the n-A isotherm began to increase at a large surface area in the presence of 100 mM sodium diphosphate in the aqueous subphase (Figure 21, suggesting that the peptide underwent the transition from an a-helix to a random-coil conformation. Amphiphilic Helical Peptides with a Terminal Amino Group, HA16M and HA16B. In the n-A isotherms of HAl6M and HA16B on a water subphase or on a buffered subphase, the phase transition from a liquid to a solid state was not observed. The surface area at the inflection point was similar to that of BA16M. Therefore, HA16M and HA16B also take an a-helical conformation and orient the helix axis parallel to the interface. The collapse pressure at higher pH than 7.7 was higher than that at low pH at the same NaCl concentration (Table (31)Forexample: Chi, L.F.; Johnston, R. R.; Ringsdorf, H.Langmuir 1991,7,2323. (32)Mertesdorf, C.; Ringsdorf, H. Liq. Cryst. 1989,5, 1757. (33)Meller, P.; Peters, R.; Ringsdorf, H . Colloid Polym. Sci. 1989, 267.97. (34) (a) Losche, M.; Rabe, J.; Fischer, A.; Rucha, U.; Knoll, W.; Mohwald, H. Thin Solid Films 1984,117,269.(b) McConnell, H. M.; Tamm, L. K.; Weis, R. M. Proc. Natl. Acad. Sci. U S A . 1984,81,3249. (35)Fujita, K.;Kimura, S.; Imanishi, Y.; Rump, E.; Ringsdorf, H . Submitted for publication t o J . A m . Chem. Soc. (36) Kajiyama,T.; Oishi,Y.; Uchida,M.; Morotomi,N.; Ishikawa,J.; Tanimoto, Y. Bull. Chem. SOC.Jpn. 1992,65,864. (37)Hofmeister, E.Arch. Exp. Pathol. Pharmakol. 1888,24, 247. (38)Ciferri, A.;Orofino, T. A. J . Phys. Chem. 1966,70,3277. (39)Goto, Y.;Aimoto, S. J . Mol. Biol. 1991,218,378.
\. \
,
I
'2%2. ((
P-
,
I
,
,
I
,
I
I
1000 M o l e c u l a r a r e a (81')
0
1500
500
Figure 2. n-A isotherm of BA16M on 50 mM phosphate buffer (pH 11) (- - -) and 25 mM (- - -) and 100 mM (-) sodium diphosphate solution (pH 11) at 20 "C. Table 4. Inflection Point in the Pressure-Area Isotherms of the Amphiphilic Peptides with an Amino Group at 20 "C inflection point compound
subphase
HA16M HA16M HA16M HA16B HA16B HA16B HA16B HA16B HA16B HA16B HA16B
water pH 9.3a pH 4.0b water pH 9.3" pH 5.5a pH 4.0b pH 9.3c pH 7.7c pH 5.5c pH 4.W
area
(Az)
pressure (mN/m)
253.7 255.2 258.5 253.5 255.6 250.0 256.4 252.0 255.6 255.6 254.2
5.5 8.8 6.0 8.5 10.9 9.5 10.0 14.5 11.0 9.0 9.3
a 50 mM sodiumphosphate buffer. 50 mM sodiumcitrate buffer. 50 mM sodium phosphatd0.5 M NaCl buffer. 50 mM sodium citratd0.5 M NaCl buffer.
Table 5. Inflection Point and Phase Transition Point in the Pressure-Area Isotherms of BAl6OH at 20 "C inflection point subphase water pH 9.3a pH 5.5a pH 4.0b pH 9.3c pH 5.5c pH 5.od pH4.od pH 3.5d
top of mound
area
pressure (mN/m)
area
pressure (mN/m)
262.5 253.9 262.5 264.5 243.8 256.3 262.5 264.8 268.7
12.5 12.2 11.1 12.2 17.2 15.2 16.1 14.6 15.6
320.5 273.7 276.5 324.5 253.9 271.1 290.9 306.8 310.7
4.3 10.8 8.8 3.4 15.5 11.3 8.9 6.2 6.3
(k)
(Az)
a 50 mMsodium phosphate buffer. 50 mMsodium citrate buffer. 50 mM sodium phosphate/0.5 M NaCl buffer. 50 mM sodium citratd0.5 M NaCl buffer.
4, bottom four lines). This is probably due to disappear-
ance of electrostatic repulsion between ammonium groups at high pH. The terminal amino group of both peptides might be deprotonated at pH 9.3, which is much higher than the pKa value of the a-amino group of amino acids. Under any conditions HA16B collapsed at higher surface pressure than HA16M. Amphiphilic Helical Peptides with a Terminal Carboxyl Group, BA16OH and COOHA16M. The inflection point in the n-A isotherms of the above peptides (Tables 5 and 6) was observed at the area comparable to that of BA16M (Table 3). These peptides should take an a-helical conformation at the aidwater interface and orient the helix axis parallel to the interface. The phase transition from a liquid to a solid state was observed always in the isotherm ofBA16OH and sometimes in that of COOHA16M.
Fujita et al.
2734 kngmuir, Vol. 10, No. 8, 1994 Table 6. Inflection Point and Phase Transition Point in the Pressure-Area Isotherms of COOHA16M at 20 "C
subphase
water pH 9.3" pH 5.5"
pH4.e
pH 9.3' pH 5.5c pH5.W pH 4.5d pH 3.5d
intlection point area pressure (Az) (mN/m)
top of mound area pressure (AZ) (mN/m)
236.2 265.7 238.7 249.1 256.5 230.4 239.3 243.4 236.9
319.5
2.3
253.2 325.1
8.7 3.6
260.6 263.0 282.3 306.3
10.2 10.0 6.8 3.5
8.6 8.4 11.7 11.7 13.3 14.8 14.0 12.8 12.5
50 mMsodium phosphatebuffer. 50 mMsodium citratebuffer. 50 mM sodium phosphatdO.5M NaCl buffer. 50 mM sodium citratd0.5 M NaCl buffer.
In the case of BAlGOH, the local maximum pressure of the mound increased with increasing pH of the aqueous subphase in the presence of 0.5 M NaCl (Table 5). The increasing surface pressure at the phase transition should be explained by repulsion between likely charged end groups. The terminal carboxyl group of the peptide may be ionized around pH 5.5, through the pK. value of the carboxyl group a t the C terminus of peptides is 3.4. Since the PK, value of polyelectrolytes deviates from the standard value due to dense distribution of the dissociative group along the molecule,* the pKavalue of the terminal group of peptides should shift to a higher value in the monolayer. The inflection point, however, was not influenced significantly by changing the pH of the subphase. On the other hand, the n-A isotherm of COOHA16M changed dramatically with the change of pH (Table 6). The phase transition occurred at low pH, but did not occur at pH 9.3. This observation suggests that the monolayer of COOHA16M undergoes phase transition only when it is unionized. Amphiphilic Helical Peptide with Sar Residues, BS3A16M. The n-A isotherm of BS3A16M did not show any evidence for phase transition, and it was similar to that of HA16M or COOHA16M a t high pH. In the case of the deprotected compound, HS3A16M, the surface pressure scarcely increased with compression. Mjxture of Peptida Although the monolayer HAl6M did not undergo phase transition from a liquid to a solid state in the n-A isotherms, the monolayer of an equimolar mixture of HA16M and BA16OH underwent phase transition on a pure water subphase (Figure 3). However, the phase transition was detected neither at pH 4.0 nor in the presence of 100 mM NaCl. These results suggest that the electrostatic interaction between the ammonium group of HA16M and the carboxylate group of BA16OH stabilizes the molecular packing in the monolayer. At pH 4.0 the slope of the n-A isotherm began to increase at the which is halfthe molecular area molecular area of 130k, at the inflection point in the x-A isotherm of pure BA16OH. Therefore, the monolayer ofthe mixture is fully phase-separated under this condition. In the case of the mixture of HAl6M and COOHA16M (l/l), the phase transition was not observed under any conditions (Figure 41, in contrast to the mixtureofHA16M and BAlGOH, though the same kind of interaction might occur in both mixture systems. Since the monolayer of HA16M or ionized COOHA16M did not undergo phase transition, the electrostatic interaction alone is not enough for the phase transition of the monolayer. At pH 4.0, the area at the inflection point is apmximately half that in (40) For example: Nagasawa, M.;Holtzer, A J . Am. Chem. Soc. 1964,86,538.
,
I
200 400 Molecular a r e a
0
(A2)
600
n-A isotherm of the equimolar mixture of HAl6M and BA16OH on water (-), 100 mM NaCl (- - -1, 50 mM phosphate buffer of pH 9.3 (- - -), and 50 mM citrate buffer of pH 4.0 (- - -) at 20 "C.
Figure3.
-. E
2
-E 2 0 3 u)
?
n 0
p
10-
3 v)
200
0
400
600 . .
(A2) Figure 4. n-A isotherm of the equimolar mixture of HAl6M and COOHAl6M on water (-),50 mM phosphate buffer (pH 9.3)(- - -1, and 50 mM citrate buffer (pH 4.0) (- - -) at 20 "C. Molecular area
30r
. E
-E z
E
20-
3
a u1
2
0. 0
2
10-
3 v)
I
0
I
1
400 Molecular area
200
(A2)
600
Fignre5. n-A isotherm of the equimolar mixture of BA16OH and COOHA16M on water (-), 50 mM phosphate buffer (pH 9.3)(- - -), and 50 mM citrate buffer(pH 4.0) (- - -1 at 20 "C.
the pure COOHA16M monolayer, suggesting that the phase separation of the monolayer as observed in the mixture of HA16M and BA16OH took place. The monolayer of an equimolar mixture of BA16OH and COOHA16M underwent phase transition on a pure water subphase and an aqueous solution buffered at pH 4.0 (Figure 5). The monolayer of each component peptide underwent the phase transition under these conditions. Onthe other hand, a t pH 9.3,the monolayer ofthe mixture did not show phase transition. This is probably because the molecular packing of COOHA16M in the monolayer is not so stable as to undergo phase transition at pH 9.3. The monolayer of an equimolar mixture of BA16M with BA16OH on a water subphase gave a n-A isotherm having the phase transition (Figure 6) similar to that of the BA16M or BA16OH monolayer. However, the mixture of
Helical Peptide Monolayer at the Air / Water Interface 30
r
1
0
200
400
Molecular area ( A 2 )
600
Figure 6. n-A isotherm of the equimolar mixture of BA16M and COOHA16M on water (- - -1, BA16M and BA16OH (- - -1, and BA16M and HA16M (-1 on water at 20 "C. BA16M and COOHA16M shows a different behavior. Apparently, the interaction of BA16OH with a fully protected peptide, BAlGM, is different from that of COOHA16M with BAlGM, although both BA16OH and COOHA16M have a terminal carboxyl group. In the case of the mixture of BA16M and HAlGM, the x - A isotherm has an inflection point a t a molecular area of around 130 A2,which is half the molecular area at the inflection point of the pure BA16M monolayer, indicating a complete phase separation in the monolayer. To compare Figure 6 with Figure 3, the electrostatic interaction between the end groups of HA16M and BA16OH should promote the molecular packing in the monolayer.
Discussion The peptides synthesized in the present investigation were shown to take a perfect a-helical conformation at the aidwater interface, although the helix content was around 60% in ethanollwater (9515dv). Since the helical structure is stabilized in the environment of low dielectric constant, the major part of the peptide should be exposed to air when the peptide molecules are spread at the air/ water interface. It is worth noting that the peptides oriented the helix axis parallel to the aidwater interface. The helical structure is stabilized by intramolecular hydrogen bonds. However, three amide protons and carbonyl groups, respectively, at the N- and C-terminal ends of the helix are free from intramolecular hydrogen bonds.41 These amide groups should be hydrated at the aidwater interface. This should be the reason for the helices to take an orientation parallel to the interface. The extent of hydration at both ends of a peptide chain should be different according to their hydrophilicity, and should also affect the molecular packing of the monolayer. The strong hydration at the chain ends should be the reason for the absence of phase transition in the monolayers of HAlGM, HAlGB, BS3A16M, HS3A16M, and COOHA16M (at high pH), although these peptides take an a-helical conformation (Table 2). In general, the negative charge at the C-terminus and the positive charge at the N-terminus scarcely destabilize the helical structure as a result of the interaction with the macrodipole of the he lice^.^^!^^ The monolayer of COOHA16M formed only expanded aggregates at higher pH (Table 6),but condensed aggregates at a lower pH, even though the negative charge
Langmuir, Vol. 10, No. 8, 1994 2735 on the N-terminus might stabilize the helical structure at high pH. Therefore, the deformation of the helical structure due to the terminal charge cannot explain the expansion of the monolayer. On the other hand, the monolayer of HA16B carrying a terminal benzyl group, which is bulky and hydrophobic to inhibit hydration of the terminal amide bonds, showed much higher surface pressure at the inflection point than HA16M. This observation gives support for the explanation considering destabilization of the monolayer by the hydration of the peptide terminus. In the case of HS3A16M, a strong hydration of the N-terminal region prohibited monolayer formation of the peptide molecule. The n-A isotherm of the BA16M monolayer showed a mound at the molecular area of around 300 A2. The isotherm was reversible at larger areas than that at the mound. Therefore, the mound is not caused by the formation of a multilayer as previously reported for homopolymers of a-amino acids.12,13 The mound should have resulted from the molecular rearrangement from a liquid to a solid state. The peptides might be dispersed irregularly in the liquid state. However, it is considered that the peptide forms smaller aggregates in the liquid state, which grow to a larger crystal in the solid state. This explanation is based on the high tendency of the peptide to associate together, which was shown by the previous report that the hexadecapeptide and the longer peptides associate together in an ordered form when incorporated in a lipid bilayer membrane.42 The behavior of the binary peptide system is complex. When the monolayers ofindividual peptides show a mound in the x - A isotherm, the two components mix homogeneously at the aidwater interface. Examples are the combinations of BA16M and BAlGOH, and BA16OH and COOHA16M a t low pH. However, phase separation occurs, when one component shows a mound in the n-A isotherm and the other does not. Examples are the combinations of BA16M and HAlGM, BA16OH and HA16M a t low pH or high ionic strength, BA16OH and COOHA16M a t high pH, and COOHA16M and HA16M at low pH. However, the combination of BA16OH and HA16M is an exception of this category. Although the monolayer of BA16OH shows a mound in the n-A isotherm and the monolayer of HA16M does not, these peptides are mixed homogeneously due to electrostatic attraction acting between the two components. This is the first interesting example showing that the nature of the terminal group of a helix peptide has a strong influence on the interhelix interaction, and hence the packing of helices. Proteins forming an ion channel such as a Na+ channel have been shown to be composed of several strands of helices.43 Since these helices are embedded in the phospholipid bilayer membrane, each helix should suffer on internal pressure of ca. 30 mN/m.44 However, each helix does not necessarily have to be resistant to this pressure by itself, if interhelix interactions take place favorably as exemplified by the monolayer of a mixture of BA16OH and HA16M on the pure water subphase.
Acknowledgment. This research was financially supported by the New Energy and Development Organization (NEDO), Tokyo. (42)Otoda,K.;Kimura,S.;Imanishi,Y.Biochim.Biophys.Acta 1993, 1150, 1.
(41)Branden, C.; Tooze, J. Introduction to Protein Structure; Garland: New York, 1991; Chapter 2.
(43) Catterall, W.A. Science 1988,242, 50. (44)Pink,D.A. Can. J . Biochem. Cell Biol. 1984, 63, 767.
