water interface - American Chemical Society

3600 Boulevard Casavant Ouest, St. Hyacinthe, Quybec, Canada J2S 8E3. Received November 19, 1991. In Final Form: March 27, 1992. In order to mimic the...
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Langmuir 1992,8, 1988-1993

1988

Packing of Hydrophobic a-Helices: A Study at the Air/Water Interface? Pierre Lavigne,j Pierre Tancr&de,*'*Fransois Lamarche,$ and Jean-Joseph Max$ Dkpartement de chimie- biologie, Universitk du Qukbec h Trois-Rivikres, P.O.Box 500, Trois-Rivikres, Qukbec, Canada G9A 5H7,and CRDA, Agriculture Canada, 3600 Boulevard Casavant Ouest, St. Hyacinthe, Qukbec, Canada J2S 8E3 Received November 19,1991. In Final Form: March 27, 1992 In order to mimic the packing of a-helices in a-helical proteins, two hydrophobic homopolypeptides, poly-L-alanine (PLA) and poly-L-leucine(PLL), in their a-helical conformation were spread at the air/ water interface and studied by the monolayer technique. The surface pressure and surface potential isotherms of both polypeptides were recorded at 22 f 2 "C. In addition to the arrest point already identified in the literature as the starting point for the monolayedbilayer transition, the surface pressure isotherms also showed an inflection point at 12.8 &/residue for PLA and 17.0A2/residuefor PLL. Starting from these residual areas, asurface potential pseudoplateau was encountered for both polypeptides. From calculations of the internal and external radii of the PLA and PLL a-helices, we have rationalized the organizationof the hydrophobic helices at the inflection point. It is demonstrated that, at these residual areas, the hydrophobic a-helices are organized such that their side chains are interdigitated. From the residual areas at the inflection point, we calculated the interhelical distance to be 8.5 and 11.0 A for PLA and PLL, respectively. These values agree with the interhelical distances found in a variety of a-helical proteins as well as those obtained from theoretical studies. On the basis of these results, and from what is known in the literature on the packing of the a-helicesin such proteins, we suggest that the hydrophobic a-helices of PLA and PLL are organized as coiled-coil structures at the aidwater interface.

Introduction Earlier studies on the properties of hydrophobic homopolypeptides at the air/water interface have led to interesting results about the organization of these molecules. For example, many of these hydrophobic homopolypeptidesare found to exist and to be stable in their Also, a-helical conformation at the aidwater interfa~e.l-~ the a-helices are adsorbed with their long axis parallel to the aidwater interface, the long axis predominantly being perpendicular to the direction of compre~sion.~*~ In addition, it appears that their mutual orientations could be either parallel or antiparallel.2 Finally, such monolayers give rise to a monolayer/bilayer transition and, in certain cases, to the formation of further layer^.^?^ Although these results are important inasmuch as the physical chemistry of these molecules at the interface is concerned, they also provide a good model system for structural studies on proteins becausethere is no ambiguity concerning the secondary structure of the proteinic material under study. For this reason, it is our opinion that the monolayer model has not been sufficiently exploited so far in the literature for studies related to the structural aspects of proteins. In recent years much attention has been paid to the interactions between a-helices from a theoretical point of view.@ Analysis of the packing of a-helices can provide

* To whom correspondence should be addressed. f

CRDA Contribution No. 256.

* Universite du Quebec B Trois-RiviBres.

8 CRDA, Agriculture Canada. (1) Malcolm, B. R. h o c . R. SOC.London, A 1968,305,363.

(2) Malcolm, B. R. h o g . Surf. Membr. Sci. 1973, 7, 183. (3) Cornell, D. G.J. Colloid Interface Sci. 1979, 70, 167. (4) Gabrielli,G.; Baglioni,P.;Ferroni,E. J. Colloidlnterface Sci. 1981, 81, 139. (5) Takeda, T.; Matsumoto, M.; Takenaka, T.; Fujiyoshi, Y.; Uyeda, N. J . Colloid Interface Sci. 1983, 91, 267. (6) Chou, K.-C.;Nbmethy, G.;Scheraga, H. A. J. Phys. Chem. 1983, 87., 2869. ---(7) Chou, K.4.; Maggiora, G. M.; NBmethy, G.;Scheraga, H. A. h o c . Natl. Acad. Sci. U.S.A. 1988,85, 4295. (8)Furois-Corbin, S.; Pullman, A. Chem. Phys. Lett. 1986, 123,305. ~

important information on the structure of proteins, especiallythose for which the secondarystructure is strictly a-helical such as the four a-helix bundle proteins (e.g., haemerythrinlO),or the two-stranded coiled-coilproteins (e.g., tropomyosinell). The main features related to the interactions between the a-helices within these two types of proteins are the hydrophobic nature of the residues involved in the packing and the antiparallel or parallel orientations of the a-helices.12-14 The interhelicaldistance is also of utmost importance if one wants to gain insight into the structure of these a-helical proteins. Obviously, the interhelical distance is likely to depend on the size of the residues involved in the packing. In this context, the monolayer technique was used to obtain the surfacepressure and surface potential isotherms of two hydrophobic homopolypeptides, poly-L-alanine (PLA) and poly-L-leucine(PLL), known to exist in their a-helical conformation at the aidwater i n t e r f a ~ e . ~ ~ J ~ Surface pressure (II)-residual area (A) isotherms of such polypeptides can provide information about the packing of the a-helices at the air/water interface since the technique allows control of the molecular density at the aidwater interface. However, to extract interhelical distances from the 11-A isotherms, one needs to transform the residual areas obtained into distances. To do so, we used a simple geometrical model that includes the translation distanceper residue alongthe helix axis,the effective internal radius of the helix backbone, and the effective external radius of each polypeptide studied. According to the model, the 11-A isotherms obtained suggest the existence of side-by-sidearrangements for both PLA and PLL at the surface pressure liftoff. For further decrease (9) Bruccoleri, R. E.; Novotny, J.; Keck, P.; Cohen, C. Biophys. J. 1986, 49, 79. (IO)Weber, P. C.; Salemme, F. R. Nature (London)1980,287, 82. (11) Smilie, L. B. Trends Biochem. Sci. 1979, 4, 151. (12) Ho, S. P.; DeGrado, W. F. J. Am. Chem. SOC.1987, 109, 6751. (13) Hodges, S. H.; Saund, A. K.; Chong, P. C. S.; St.-Pierre, S. A.; Reid, R. E. J . Biol. Chem. 1981,256, 1214. (14) Talbot, J. A.; Hodges, R. S. Acc. Chem. Res. 1982, 16, 224. (15) Malcolm, B. R. Adu. Chem. Ser. 1975,145, 338.

0743-7463/92/2408-1988$03.00/00 1992 American Chemical Society

Langmuir, Vol. 8, No. 8,1992 1989

Packing of Hydrophobic a-Helices

of the residual areas interdigitation of the side chains of adjacent helices is found to occur. The completion of this interdigitation process is also observed to coincide with the beginning of the pseudoplateau recorded in the surface potential isotherms. The interhelical distances in the interdigitated states are consistent with those observed in native proteins and in other model systems. We also discw the possibility that a coiling process of the a-helices accompanies the intergiditation process at the aidwater interface. Materials and Methods Materials. PLA (MW 18 800, DP 265) and PLL (MW 10 200, DP 91) were supplied by Sigma Chemical Co. (St. Louis, MO). The PLA solutions were obtained by dissolving about 2 mg of the polymer in 0.6 mL of dichloroacetic acid (99+% purity, Anachemia Canada Inc., Montrhl, Qubbec),used without further purification. The volumes of the solutions were increased to 10 mL withdistilled CHCls (Accusolv,AnachemiaCanadaInc., Montrbal, QuBbec). The PLL solutions were made by dissolvingabout 1 mg of the polymer in CHCl3 containing 20% trifluoroacetic acid (99+ 5% purity, Anachemia Canada Inc., Montrbal, Qubbec). From these solutions, PLA and PLL are known to exist in their a-helical conformation when spread at the aidwater i n t e r f a ~ e . ~ J ~ All the weighings were made using a microelectrobalance (model M25D, Sartorius, Gottingen, Germany). Monolayer Methodology. The polypeptide monolayerswere spread, using a microsyringe (series A gas syringe, Dynatech Precision Sampling Corp., Baton Rouge, LA), by successively applying small drops of the solution at the surface of a doublequartz-distilled water (Hereaus Quarzschmelze Model Bi18 T, Hanau, Germany) subphase until residual areas of 25 A2/residue for PLA and 30 AZ/residue for PLL were attained. The specific resistivity of water was greater than 17 X 106Q.cm and the surface tension greater than 70 "em-1. All the measurements were made at 22 f 2 "C. The Langmuir trough used in this study was 15 cm wide and 30 cm long. The trough was built from glass and covered with autoadhesive Teflon. Surface pressure measurements were obtained witha Langmuir film balance.le The Langmuir balance float was made from Mylar and attached to the borders of the trough by two pieces of flexible Teflon tape. The force applied on the effectivelength of the float was measured by a displacement magnetic transducer (model ZDCDT-050,Hewlett-Packard, Andover Division, Andover, MA) with a linear output voltage up to 1.2 V. The sensitivity of the torsion balance is found to be 41.0 mN-m-1.V-1, and was used to convert the displacement measurements as obtained from the transducer into surface pressures. The surface pressure isotherms were recorded by reducing the residual area in a stepwise fashion (0.5 (A2/residue)/step),the film being left to relax in between each step until the rate of the surface pressure decrease was 1/20thto 1/5Othof the initial rate. This corresponds to a rate of surface pressure decrease of about 0.1 "am-1.min-1. This was done in order to minimize the important surface pressure gradients that could otherwise exist in the film. The surface potential was recorded simultaneously to the surface pressure by using the ionizing electrode method.17 An 2 4 1 A m (Nuclear Radiation Developments, Grand Island, NY) electrode was positioned at 3 mm above the water surface. The platinum reference electrode (modelp101, Radiometer electrodes, Copenhagen, Denmark) was immersed into the subphase behind the float and grounded. The 241Amelectrode was connected to a high impedance input (1014Q) operational amplifier (model ADBllK, Analog Devices, Norwood, MA) mounted in the noninverting voltage amplifier mode with a fixed gain. The experimental error on the surface potential is estimated to be *20 mV.

Results and Discussion The surface pressure isotherms obtained for PLA and PLL are plotted in Figure 1. The results presented for (16) Gaines, G. L.Insoluble monolayers at liquid-gas interface;John Wiley & Sons: New York, 1966; p 54. (17)Gaines, G.L. Insoluble monolayers at liquid-gas interface; John Wiley & Sons: New York, 1966; p 75.

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Figure 1. Surface pressure vs residual area isotherms of PLA and PLL (2' = 22 f 2 "C; subphase, double-distilled water).

each polypeptide are the average of three different isotherms, the maximum deviation between the surface pressure being of the order of fl mN.m-l and the error on the residual area being k0.3 A2/residue. As it can be seen, both isotherms show the typical arrest point encountered for the monolayer/bilayer transition already reported for the two hom~polypeptides.~~~J~ We find these two points at approximately 10.4A2/residuefor PLA and 14.5&/residue for PLL. These residual areas more likely correspond to close-packed organizations of the a-helices beyond which the so-calledmonolayer to bilayer transition is initiated. The II-A isotherm obtained for PLA is in agreement with previously published r e ~ u l t s . ~Gabrielli *~ et aL4 obtained comparable results with shorter PLA a-helices, although their isotherm was recorded at 30 "C. Malcolm2 also obtained a very similar isotherm on a 10 mM KC1 subphase at 20 "C.On the other hand, the surfacepressure isotherm obtained here for PLL is quite different from the one obtained by Malcolm.l5 Both the surface pressure and the residual areas reported by Malcolml5 are much lower than the results obtained here. However, Malcolm reports problems in the spreading of his PLL solution. Apparently, drops of the solution were creeping to his syringe, and to his saying, the residual areas obtained were probably low. This problem was not encountered, and our results, inasmuch as the residual area is concerned, are therefore more likely to be correct. It seemed important to us to investigate further the possible organizations of the a-helices as the residual area was reduced. The rationale of such an investigation was to obtain an insight into the packing of a-helices at the aidwater interface and hence to serve as a model approach to study the structure of a-helical proteins. The relevance of this approach relies on the ability to transform the experimental data, residual areas, into interhelical distances (A), since the interhelical distance is an important parameter to describe the packing of a-helices in a-helical proteins. To achieve this task, one needs to know the effective internal radius of an a-helix (pi), the effective external radius (re)of PLA and PLL, and the translation distance per residue along the a-helix. The latter quantity being known (1.5 &residue'*), we need to calculate pi and re. Pi is taken to be the smallest distance an atom of an a-helix can approach the axis of another a-helix. This distance is directly given by the polar cylindricalcoordinates of the backbone atoms of an a-helix plus the van der Waals radius for the corresponding atom.6 The cylindrical coordinates (18) Pauling, L.;Corey, R. B. Nature (London) 1953, 171, 59.

1990 Langmuir, Vol. 8, No.8,1992

for an alanyl residue being given in refs 19-21, one finds that Q = 2.9 A. recorrespondsto the distance of the farthest atom of the side chain from the helix axis, including the van der Waals radius of the atom. To calculate re,we used the geometry of L-amino acid residues as described in ref 22 and took into account the projection of the side chain, this side chain pointing from Ca toward the direction of the amino terminus of the helix. For PLA and PLL, we calculated re for the fully extended conformation of the side chains (i.e., x1 = 180' for PLA and x1 = x2 = x3 = 180' for PLL). The values obtained for PLA and PLL are 5.5 and 7.9 A, respectively. The values obtained for the external radius of PLA and PLL are in very good agreement with comparable values in the literature. For instance, Furois-Corbin and Pullman8cite a value of 5.3 A for the external radius of PLA while Chou et aL7give a value of 5.55 A. It has not been possible to find directly the correspondingradius for PLL. However, Richmond and Richards23found, from crystallographic data, that in myoglobin the Cbof leucine is about 7.3 A (includingthe van der Waals radius) away from the a-helix axis. For the same carbon (with x1 = x2 = 180') our calculations give 7.6 A, thus suggesting that our re value for PLL, which concerns the outmost H atom, is reasonable. Once the pertinent molecular dimensions were known, the characteristic points of the surface pressure isotherms were rationalized in terms of the organization of the ahelices at the air/water interface. It is well known that long hydrophobic a-helices lie flat when spread on a water surface and that they tend to orient themselves predominantly parallel to the movable barrier when reducing the area a~ailable.~,~ These facts led to consideration of a side-by-sidearrangement of the a-helices at the air/water interface. Therefore, when the surface available is gradually reduced, the a-helices are gradually ordered until the surface is covered by helices with their side chainsjust coming into contact (Figure 2A,B). The parameters obtained above allow calculation of the residual area of such an organization. The residual area should correspond to the diameter of the helix (i.e., 2re) times the translation distance (1.5 &residue). Using the values of re obtained above for PLA and PLL, calculationsshowthat the residual area for a side-by-sideorganization of the helices is 16.5 A2/residuefor PLA and 23.7 A2/residuefor PLL. These values are very close to the residual areas observed at the surface pressure liftoffs for PLA and PLL, i.e., around 16.0 and 24.0 A2/residue,respectively. Table I summarizes the experimental residual areas and distances for PLA and PLL (columns 2 and 3, lines 2 and 4, respectively)and the corresponding calculated values for the two polypeptides (lines 1 and 3). Considering the excellent agreement between the experimental and calculated values of the residual areas at the surface pressure liftoff, it is therefore likely that the hydrophobic a-helices at this point are indeed organized side by side. The next step was to characterize what happens when the a-helices are forced to pack upon further reduction of the available area. One can have insights about this from what is known to occur in proteins as well as what has been learnedfrom theoretical considerationson the energy (19) Parry, D. A. D.; Suauki, E. Biopolymers 1969, 7,199. (20) Arnott., S.; Dover, S. D. J . Mol. Biol. 1967, 30, 209. (21) Elliot, A. In Poly-a-amino acids, protein models for conformationalstudies; Fasman, G. D., Ed.; Marcel Dekker, Inc.: New York, 1967, p 23. (22) Momany, F. A.; McGuire, R. F.; Burgess, A. W.; Sheraga, H. A. J. Phys. Chem. 1975, 79, 2361. (23) Richmond, T. J.; Richards, F. M. J . Mol. Biol. 1978,119, 537.

Lavigne et al. B

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Figure 2. Organization of the a-helices at various steps of the compression isotherms: (A) and (B) side-by-sideorganization of the helices occurringat the liftoff point (16.0and 24.0 A2/residue for PLA and PLL); (C) and (D) fully interdigitated state found at the inflection point (12.8 and 17.0 A2/residue for PLA and PLL); (E) and (F) close-packed structure found at the arrest point (10.4 and 14.5 A2/residuefor PLA and PLL) (d = interhelical distance).

of interaction of pairs of a-helices. Richmond and Ric h a r d established ~~~ that, for myoglobin, interpenetration occurs between side chains of adjacent helices. On the other hand, Chou et aL7and Furois-Corbinand Pullman,8 from theoretical considerationson interactingpairs of PLA a-helices, independently found that the lowest energy packing state for such a pair has an interhelical distance much lower than twice the external radius of the PLA helix. As both pointed out, this implies that important interpenetration occurs. From the model presented above and the parameters calculated, it was possible to determine the actual residual area at which complete interdigitation occurs. Figure 2C presents a cross sectional view of the closest approach of the helix axes in the plane of the interface. Thus, the interdigitation is such that the side chain of an a-helix is allowed to penetrate to a distance corresponding to the minimal effective internal radius Q of an adjacent a-helix. Figure 2C clearly shows that the interhelical distance (i.e., the distance between the axis of each helix) for this organizationis given by ri + re. As shown above,the values of ri for PLA and PLL are identical, 2.9 A, while re = 5.5 and 7.9 A for PLA and PLL, respectively. This yields an interhelical distance of 8.4 A for PLA and 10.8 A for PLL (Table I, column 5). Converted into residual areas, one finds 12.6 and 16.2 A2/residuefor PLA and PLL (Table I, column 4). A close examination of the surface pressure isotherms of Figure 1 shows that these residual areas coincide with an inflection point in the isotherms. These inflection points are more clearly highlighted if one plots -A dn/dA, the surface compressional modulus, as a function of the residual area. Figure 3 shows that the compressional modulus reaches a maximum value at 12.8 f 0.3 and 17.0 f 0.3 A2/residuefor PLA and PLL (Table I, column 4). Thermodynamically,this implies that from the inflection points a second-order phase transition is initiated when the area is further decreased, which is an indication that reinforces the fact that a process is completed at these points, in occurence interdigitation. In order to obtain further information, the surface potential isotherms were recorded. Figure 4 presents the surface potential, AV, as a function of the residual area. The AV-residual area isotherms are similar to those obtained by Malcolm2J5for PLA and PLL. It can be seen that, for both polypeptides, AVincreaseswhen the residual

Langmuir, Vol. 8, No.8, 1992 1991

Packing of Hydrophobic a-Helices

Table I. Residual Areas (A) and Interhelical Distances (a) for Various Organizations of the PLA and PLL a-Helices (Experimental and Calculated Values) ~~

PLA

side-by-side organization A (&/residue) d (A) 16.5 11.0 16.0 10.7 23.7 15.8 24.0 16.0

calculated experimental cdculated experimental

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interdigitated organization A (Azlresidue) d (A) 12.6 12.8 16.2 17.0

8.4 8.5 10.8 11.0

closely packed interdigitated organization A (Atlresidue) d (A) 10.7 7.1 10.4 6.9 13.8 9.2 14.5 9.7

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area is reduced until a pseudoplateau occurs. The pseudolateau is initiated at about 17 A2/residuefor PLL and 13 2/residue for PLA. These residual areas correspond exactly to the residual areas at which the inflection points are found to occur on the surface pressure isotherms. However, Malcolm argues that the surface potential reaches a plateau only when the so-called monolayer/bilayer transition is initiated, i.e., at the arrest p ~ i n t . We ~J~ clearly show here that the plateaus are rather reached at residual areas larger than the residual areas corresponding to the onset of the monolayer/bilayer transition. In fact, if one closely examinesthe published surface potential results, one finds that the plateaus observed in the surface potential isotherms recorded for PLA (Figure 7 of ref 2) and PLL (Figure 3 of ref 15) are indeed initiated where we find an inflection point in the surface pressure isotherms, and not at the onset of the monolayer/bilayer transition as claimed. To rationalize the surface potential data and their implications, one needs to recall the origin of the surface potential when hydrophobichomopolypeptidesare spread at the aidwater interface. As discussed by Malcolm? the sole contribution that has to be considered is the reorientation of the water molecule dipoles as a consequence of their interaction with the hydrophobic helices. In this context, the fact that the surface potential increases up to the inflection point when reducing the residual area (Figure 4) simply results from an increase of the water

x

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Figure 5. Perpendicular dipolar moment (solid line) and

corrected perpendicular dipolar moment (dottedline)vs residual area. Arrows show the onsets of the correction,corresponding to the onset of the pseudoplateausin Figure 5.

dipole density at the aidwater interface. This point holds, however, only if one assumes that there is no contribution arising from a distortion of the a-helix or its side chains under compression. As shown in the followingparagraph, this is likely to be so. The inflection point in the isotherms therefore represents a limit case for which the packing of the helices in the plane of the interface is maximal, corresponding to the fully interdigitated state referred to above (Figure 2C). Additional information is obtained if one plots the perpendicular dipolar moment per residue, pl, as a function of the residual area, p, being obtained from the experimental results through (mD) = A AV/12* (1) where A is given in angstroms squared per residue and AV in milliv01t.s.~~ The results are presented in Figure 5 (solid line) which shows, as inferred by Malcolm? that p~ is constant for PLA and PLL from the initial residual areas at which the polypeptideswere spread until the AVplateau is reached. The fact that p L is constant suggests that the amount of reoriented water molecules around the hydrophobic helices does not change upon compression until the complete interdigitation of the side chains occurs. It also suggests that indeed no important contribution originates from a distortion of the a-helix under compression, a point also discussed by Malcolm.2 Figure 5 (solid line) also shows that when the residual area is decreased beyond the inflection point, p, decreases. Again, by using eq 1, one sees that AV being almost constant for residual areas smaller than the residual area at the inflection point (Figure 4), the decrease of p l is necessarily associated with a decrease of the residual area, A. One has to recall that the residual area at the inflection point represents the minimal residual area in the plane of the interface resulting from the interdigitation of the side chains of adjacent a-helices. The residual areas used in the calculation of p l through eq 1 are in fact apparent residual areas (i.e., total number of residues divided by p,

(24) Gaines, G. L. Insoluble monolayers at liquid-gas interface; John Wiley & Sons: New York, 1966;p 190.

Lavigne et al.

1992 Langmuir, Vol. 8, No. 8, 1992 the available area), which do not take into account the way the helices are packed at the aidwater interface. Instead, beyond the inflection point the residual areas for the residues in direct contact with water should remain constant, Le., 12.8 and 17.0 &/residue for PLA and PLL, these areas representing minimal areas. Using these minimal areas, one can calculate, through eq 1, the perpendicular dipolar moment. Figure 5 shows the corrected dipolar moment, plC (dotted line), plotted as a function of the apparent residual area. The results show that the dipolar moment is mostly constant for the entire residual areas covered, thereby indicatingthat the a-helix/ water interface does not changemuch in nature throughout the entire isotherms. To rationalize even more the type of organization that can prevail at the aidwater interface when hydrophobic a-helices are forced to pack, we have turned to what is known in the literature on the packing of a-helicalproteins. It has been proposed several years ago that the structure of elongated a-helical proteins can be described as a multistranded coiled-coil structure.181zs Since then, a-helical coiled-coil structureshave been proposed as the preferred mode of organization of rodlike and moderately extended multistranded a-helical proteins such as the four a-helix bundle proteins and the intrinsic membranar proteins.z6 If one considers the simplest case when only two adjacent helices are involved, one obtains a two-stranded a-helical coiled-coil structure. This structure is organized as a superhelix built from two slightly twisted a-helices packed accordingto the knobs into holes model, the angle between the individual axes of the a-helices being about 20° as a consequence of the a-helix nonintegral number of residues per turn.25J7 In this context, the eventuality that the packing of ahelices at the aidwater interface could lead to an array of two-stranded a-helical coiled-coilstructureswas explored. This had to be taken into account considering the length of the a-helices used in the present study as well as the fact that homopolypeptides were used for which one expects the knobs into holes mode of packing to hold.8 In this mode of packing, each knob (i.e., the side chain) on an a-helix packs into the hole created by residues n, n + 3, n + 4, and n + 7 on the adjacent a-helkZ7 As a consequence, the two interacting a-helices get coiled with only minor deformations of each a-helix.zs~z7Indeed, as Crickz7pointed out "It is impossible to pack such models of the a-helix closely side by side since a good fit in one place produces a bad fit somewhere else, due to the nonintegral nature of the a-helix." Figure 2D gives a schematic view of the organization of such a coiled-coil structure. It shows clearly that the interhelical distance in the plane of the inbrface for this kind of structure is compatibleto that calculated for the interdigitated state (Figure 2C). It is important to note that the actual packing of the helices at the aidwater interface may not lead to the perfect structure shown in Figure 2D since the helices involved do not have necessarily the same length. However, considering the way a-helices are known to pack when they come into close contact, and considering also the excellent agreement between the experimental and calculated results at the inflection point, the model presented is plausible. The organization model of the helices proposed in Figure 2D also allows one to predict the molecular organization of the helices at the arrest point (Figure 2F) and hence to ~~

~

(25) Crick, F. H. C. Acta Crystallogr. 1953,6, 689. (26) Cohen, C.; Parry,D. A. D.Trends Biochem. Sci. 1986, 1 1 , 245. (27) Crick, F. H. C. Nature (London)1952, 170, 882.

gain possible insight into the molecular process associated with the second-orderphase transition taking place a t the inflection point. It is indeed possible for an arrangement of coiled-coil helices to pack even more closely, as shown in Figures 2E,F. In this kind of organization,the contacts between a pair of adjacent coiled-coil helices are made alternately in and abovethe plane of the air/water interface (Figure 2E). At the points of contact (indicated as a solid line in Figure 2F), the two-stranded coiled-coil helices are rotated by an angle of 45O with respect to the plane of the interface (Figure 2E). The mean interhelical distance in the plane of the interface in this case is simply given by ((ri + r,)(l + COS 45'))/2. This distance is equal to 7.1 and 9.2 A for PLA and PLL (Table I, column 7) corresponding to an apparent residual area of 10.7 and 13.8 &/residue (Table I, column 61, for PLAand PLL, respectively. These values are very close to those experimentally determined for PLA and PLL at the arrest point, Le., 10.4 &/residue for PLA and 14.5 &/residue for PLL (Table I, column 6), supporting the close-packed molecular arrangement proposed at the arrest point. Since interpenetration is known to occur between ahelices in natural proteins23 and in model systems,B-8how do the values obtained for the interhelical distance in such systems compare to the values obtained experimentally in the present work for the interdigitation of the two hydrophobic a-helices studied? For a number of antiparallel four a-helical bundle proteins found in vivo, the interhelical distances of closest approach are reported to be in the range of 9.3 f 1.3 A7 or, similarly, 9.6 f 1.4 A.10 These distances are obtained for a-helices that contain a variety of hydrophobic residues. Interestingly the interhelical distances experimentally obtained here at the inflection point (where interdigitation is complete) are 8.5 A for PLA and 11.0 A for PLL (Table I, column 5), in good agreement with the values reported for proteins. It is also interesting to note that the a-helices in bacteriorhodopsin, an integral membranar protein, are packed with an interhelical distance ranging from 10 to 12 A.28 In addition, within the three-stranded coiled-coil portion of the haemagglutinin glycoprotein of the influenza virus, the a-helices are separated by a distance of approximately 10 A, the side chains participating in the packing being mainly isoleucine, leucine, and valine.29 Thus, our experimental values are again consistent with the interhelical distances measured in actual proteins. The PLA values are also consistent with the interhelical distance obtained from the theoretical work done by Furois-Corbin and Pullmanawho found an interhelical distance in the range of 7.4-7.9 A for PLA a-helices packed according to the knobs into holes model. Similarly Chou et al.7 also from theoretical considerations, find an interhelical distance in a range from 7.5 to 8.5 A for a four a-helix bundle of PLA. In conclusion, the present work has shown that the surface potential pseudoplateau recorded in the surface pressure isotherms of PLA and PLL is not initiated at the arrest point, as suggested in the literature, but rather at the inflection point observed in the surface pressure isotherms. From our calculations of the internal and external radii of PLA and PLL a-helices, it has been shown that, at the residual areas corresponding to this inflection point, the a-helices of PLA or PLL are interdigitated. On the basis of what is known in the literature about the packing of elongated a-helical proteins, it is proposed that, at the inflection point, the helices are arranged in a ~~

~~

~~

~~

(28) Henderson, R.; Unwin, P. N. T. Nature (London)1976,257,28. (29) Wilson, I. A.; Skehel, J. J.; Wiley, D. C. Nature (London)1981, 289,366.

Packing of Hydrophobic a-Helices

structure closely related to a two-stranded coiled-coil pattern. Admittedly, the packing model of the helices at the aidwater interface presented here may seem speculative. But, the excellent agreement between the interhelical distances experimentally obtained here and those reported in the literature for native proteins or from theoretical considerations strongly supports the mode of packing proposed. Work is now in progress (e.g., electron microscopic observations of Langmuir-Blodgett films of

Langmuir, Vol. 8, No. 8,1992 1993

PLA and PLL) in order to further characterize the organization of a-helicalhomopolypeptidesat the airlwater interface. Acknowledgment. This work was supported by agrant from the Natural Sciences and Engineering Research Council of Canada (NSERC). P.L. is grateful to the NSERC and the Fonds pour la Formation de Chercheurs et l’Aide la Recherche (QuBbec) for postgraduate fellowships.