Addition of side chain interactions to modified Lifson-Roig helix-coil theory: Application to energetics of Phenylalanine-Methionine interactions

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1 Protein Science (1995), 4: Cambridge University Press. Printed in the Copyright The Protein Society USA Addition of side chain interactions to modified Lifson-Roig helix-coil theory: Application to energetics of Phenylalanine-Methionine interactions BENJAMIN J. STAPLEY, CAROL A. ROHL, AND ANDREW J. DOIG Department of Biochemistry and Applied Molecular Biology, University of Manchester Institute of Science and Technology, Manchester M60 IQD, United Kingdom Depar:ment of Biochemistry, Stanford University Medical Center, Stanford, California (RECEIVED July 18, 1995; ACCEPTED September 11, 1995) Abstract We introduce here i, i + 3 and i, i + 4 side chain interactions into the modified Lifson-Roig helix-coil theory of Doig et al. (1994, Biochemistry 33: ). The helixkoil equilibrium is a function of initiation, propagation, capping, and side chain interaction parameters. If each of these parameters is known, the helix content of any isolated peptide can be predicted. The model considers every possible conformation of a peptide, is not limited to peptides with only a single helical segment, and has physically meaningful parameters. We apply the theory to measure the i, i + 4 interaction energies between Phe and Met side chains. Peptides with these residues spaced i, i + 4 are significantly more helical than controls where they are spaced i, i + 5. Application of the model yields AG for the Phe-Met orientation to be kcal.mol, whereas that for the Met-Phe orientation is kcal.mol-. These orientational preferences can be explained, in part, by rotamer preferences for the interacting side chains. We place Phe-Met i, i + 4 at the N-terminus, the C-terminus, and in the center of the host peptide. The model quantitatively predicts the observed helix contents using a single parameter for the side chain-side chain interaction energy. This result indicates that the model works well even when the interaction is at different locations in the helix. Keywords: a-helix; helix-coil; hydrophobic; protein folding; protein stability; side chain Many de novo designed peptides exhibit partial cy-helix formation in water (reviewed by Scholtz & Baldwin, 1992). The be- 1991; Merutka et al., 1991; Stellwagen et al., 1992; Huyghues- Despointes et al., 1993; Scholtz et al., 1993), hydrogen bonds havior of such molecules indicates that helix stability is a (Scholtz et al., 1993; Huyghues-Despointes et al., 1995), and function of the intrinsic helix-forming tendencies of constituent amino acids (Chakrabartty et al., 1994; reviewed by Scholtz et al., 1991; Chakrabartty & Baldwin, 1994), capping preferences at the carboxyl and amino termini (Presta & Rose, 1988; Richardson & Richardson, 1988; Serrano & Fersht, 1989; Lyu et al., 1992; Forood et al., 1993; Doig et al., 1994; Doig & Baldwin, hydrophobic interactions (Shoemaker et al., 1990; Armstrong et al., 1993; Padmanabhan & Baldwin, 1994a, 1994b; Viguera & Serrano, 1995). Charged residues may also interact with the helix-dipole (Hol et al., 1978; Armstrong et al., 1993; Huyghues- Despointes et al., 1993; Scholtz et al., 1993), but these energies are partly subsumed into capping preferences (Doig et al., 1994). 1995), and specific side chain-side chain interactions. These side Specific stabilizing interactions between nonpolar side chains chain interactions include salt bridges (Marqusee & Baldwin, spaced i, i + 4 have been reported for Tyr-Leu, Tyr-Val, Leu- 1987;Lyuetal., 1989;Andersonetal., 1990;Fairmanetal., 1990; Leu, Val-Leu, Ile-Leu, Phe-Leu, and between Tyr and non- Horovitz et al., 1990; Cans et al., 1991; Merutka & Stellwagen, natural, straight-chain amino acids (Padmanabhan & Baldwin, 1994a, 1994b). Side chain interactions between two nonpolar amino acids spaced i, i + 4 are favored when one of the pair has Reprint requests to: Andrew J. Doig, Department of Biochemistry and Applied Molecular Biology, University of Manchester Institute of limited side chain flexibility (Padmanabhan & Baldwin, 1994b; Science and Technology.. P.O. Box 88. Manchester M60 IOD..I UK: Creamer Rose, 1995). These interactions are thought to re doig@umist.ac.uk. sult from the hydrophobic effect, which makes a major contri- 2383

2 2384 B. J. Stapley et al. bution to protein stability. Exclusion of water from hydrophobic surfaces is thought to occur early in the folding pathway (Dill et al., 1993), and there is evidence that hydrophobic clusters arise in denatured states (Neri et al., 1992; Shortle, 1993) and in folding intermediates (Martensson et al., 1993). A recent statistical study of protein crystal structures has revealed strong correlations for specific pairs of amino acids spaced i, i + 4 in a-helices; the strongest preferences are for salt bridges, Leu-Leu and Phe-Met (Klingler & Brutlag, 1995). In folded proteins, more than 50% of methionines are in contact with aromatic groups, with the sulfur generally approaching the edge of the aromatic ring, suggesting a possible electrostatic interaction in addition to the hydrophobic effect (Reid et al., 1985). The free energy of interaction between disulfide derivatives and aromatic groups has been calculated to be as strong as -3.3 kcal.mol" (Rodner et al., 1980; Nemethy & Scheraga, 1981). In this study we examine how the helicity of alanine-based peptides changes with the position, spacing, and orientation of phenylalanine and methionine. Peptides are based on the prototype sequence (Marqusee & Baldwin, 1987; Chakrabartty et al., 1993): Ac-YGAAKAAAAKAAAAKAA-NH, Such sequences have been shown to be monomeric in aqueous solution (Padmanabhan et al., 1990). Peptide sequences are designed to avoid i, i + 4 interactions between lysine and methionine or between lysine and phenylalanine. Tyrosine is placed at the N-terminus to permit accurate determination of peptide concentration, and glycine is placed between tyrosine and the remainder of the peptide to prevent interaction of the aromatic chromophore with the helix, which may contribute to the circular dichroism signal (Chakrabartty et al., 1993). Peptide sequences are designed to be approximately 50% helical, where ellipticity is most sensitive to small changes in side chain interaction energy. We analyze the data using a further modified form of the Lifson-Roig helix-coil theory (Doig et al., 1994) that includes terms for i, i + 3 and i, i + 4 side chain interactions. An estimation of the free energy of interaction of Phe and Met in an a-helix is made by fitting this modified theory to the experimental data. Theory The thermodynamics of helix formation in water are complex, and several models of the helix-coil transition have been formulated based on statistical mechanics (Zimm& Bragg, 1959; Lifson & Roig, 1961; Doig et al., 1994; Muiioz & Serrano, 1994). Lifson-Roig-based models describe the helix-coil transition as two-state on the level of individual residues (reviewed by Qian & Schellman, 1992). The conformation of a peptide can be described by assigning each component residue to either an h (helical) or c (coil) conformation as defined by its backbone dihedral angles (4, $). The N- and C-terminal units are not residues, as they are not flanked by peptide bonds on both sides. Consequently, they cannot be constrained to the helical conformation and are in the c state by definition. The statistical weight of each peptide conformation is calculated as the product of statistical weights of the component residues, where statistical weights are assigned to residues based on their own conformation and the conformation of the two nearest neighbors. The coil conformation flanked by two coil neighbors is defined as the reference state and assigned a statistical weight of unity. In the original Lifson-Roig model, only two non-unity statistical weights are assigned. Nucleating residues, assigned weight u, are residues in the h conformation with one or more of the nearest neighbors in the c conformation. Propagating residues, assigned weight w, are those in the h conformation whose nearest N- and C-terminal neighbors are also in the h conformation. These weights and assignments lead to a physical meaning for the statistical weights. u is the equilibrium constant, relative to a nonhelical conformation, for constraining a residue to the helical region of the Ramachandran map. w is the equilibrium constant for constraining a residue to the helical region of the Ramachandran map when it is part of an a-helix. Consequently, each helix-propagating residue assigned weight w is associated with an i, i + 4 main chain-main chain hydrogen bond. The original Lifson-Roig model has been previously modified to include N- and C-capping by defining new statistical weights for the coil residues adjacent to helical neighbors (Doig et al., 1994). Residues in the c state with an h state C-terminal neighbor are assigned weight n, and those with an N-terminal helical neighbor are assigned weight c. Residues in the c state that have both an N-terminal and C-terminal h state neighbor are assigned the geometric mean of the two capping weights, Jnc. To include side chain-side chain interactions in this model, we have defined an i, i + 4 interaction parameter, p, and an i, i + 3 interaction parameter, q. The free energy of interaction is related to these parameters via the relations AG(1,,+4, = -RT Inp and AG(,,,+3, = -RT In q; p and q are thus equilibrium constants for formation of an interaction in an a-helix. In the model, two side chains are considered to be interacting when these residues, and all intervening residues, are in the h state. For an i, i + 4 interaction to occur, therefore, five consecutive residues must be in the h state. When this condition is met, the interaction parameter contributes to the conformational statistical weight, and one residue contributes w*p to the partition function rather than w. For mathematical simplicity, we have assigned weight w * p to the central residue in the hhhhh quintet, where the first and last residues are the interacting residues. For i, i + 3 interactions, four residues must be in the h state, and we have assigned weight w * q to the central residue in the quintet hhhhc, where the first and fourth residues are the interacting ones. When both an i, i + 3 and an i, i + 4 interaction are present, the central residue of the hhhhh quintet is assigned weight w * p * q. The statistical weight of each residue in the helix can thus be assigned by correlating the conformations of the residue of interest and the two nearest N-terminal and two nearest C-terminal neighbors. A 16 x 16 matrix is required to describe all possible conformations of five consecutive residues (not shown). Because many of the quintets have degenerate statistical weights, however, this correlation matrix, mi, can be simplified:

3 ctions chain Phe-Met side peptides 2385 hhhh m. h m = ' (h U c ) m hhh hhhc hhc(h U c) ( h ( h U c) ch(h(h U c) ( h ( h U c) - w*p*q w*q hhhc 0 0 U Jiic 0 C U 0 ch(h U c)(h U c) W W U m 0 C (h U c ) m n Weights in the matrix are for residue i. Row labels are residues i - 2, i - 1, i and i + 1. Column labels are residues i - 1, i, i + 1, and i + 2. h U c indicates the union of the helix and coil conformations. The partition function, 2, for the system is the sum of statistical weights of the molecule in every possible conformation and is given by Figure 2 illustrates the effect of varying the interaction free energy on the mean helix content of a 19-residue homopolymer with a single i, i + 4 side chain interaction either near the N-terminus or in the peptide center. This dependence is sigmoidal, with favorable interactions (p > 1) increasing mean helix content and unfavorable interactions ( p < 1) destabilizing the helix. Even whenp is very large, helix formation is not complete because fraying still occurs outside the region of the favorably interacting residues. The peptide with an interaction near the helix terminus reaches a higher helix content than that with an interaction near the peptide center because fraying occurs more When all the interaction parameters are set equal to unity, this partition function is equivalent to that determined from the model of Doig et al. (1994). Many properties of the system can be calculated from the partition function, including the average number of residues in helical conformation, the average number of helical hydrogen bonds, and the probability of an individual residue being in a given conformation. The probability that a given i, i + 4 side chain interaction is present can be determined by calculating the probability that residues i, i + l, i + 2, i + 3, and i + 4 are all simultaneously in the h conformation: x ;. e YI The predicted effect of a side chain interaction on helix stability can be understood by comparing the helix content on a residue-by-residue basis in the presence and absence of an interaction. Calculated helix profiles for two of the peptides in this study are shown in Figure 1. A favorable side chain interaction stabilizes the helical conformation of the interacting residues and the intervening residues. Because the helix-coil transition is partially cooperative, however, this stabilizing effect is propagated away from the site of interaction. Consequently, a favorable side chain-side chain interaction enhances helix formation along the entire length of the peptide, although the stabilization decreases with increasing distance from the site of interaction. For peptide F8M12, the largest increase in helix content occurs at the peptide center, with smaller effects at the helix termini (Fig. 1A). F3M7, with an interaction at its N-terminus, has significantly reduced helix fraying relative to the predicted helix profile in the absence of an interaction (Fig. 1B). Relatively little stabilization is observed at the C-terminus. A c Y G F A K A M A A K A A A A K A A A m l d e Residue Fig. 1. Predicted helicity as a function of position. A: F8M12 with (pfm = 4, shaded bars) and without (p1" = 1, hatched bars) side chain interaction. B: F3M7 with ( pfm = 4, shaded bars) and without (PFM = 1, hatched bars) side chain interaction.

4 ~~~ 2386 B. J. Stapley et al. Table 2. Theoretical helicities loo 7 Theoretical helicity (070) Experimental - "- ~~~ Peptide helicity (070) p = 1.0 ~ F = M 4.0 p~~ = '11'111' I O AG(L,,+1) (kcalmol") Fig. 2. Mean helix content as a function of the side chain interaction energy. Calculations are made for a 19-residue blocked homopolymer with ( w) = 1.4, ( u2) = , (n) = (c) = 1.O. Mean helix content is determined as the percentage of residues in helical conformation. Curves are shown for a peptide with an interaction between residues near the N-terminus (residues 2 and 6; narrow line) or the peptide center (residues 8 and 12; bold line). at the helical ends. A favorable interaction near the end of a peptide is positioned to stabilize an intrinsically less stable part of the helix. For unfavorable interactions, the largest effect on mean helix content is observed when an interaction is near the center of the peptide, because the interaction is positioned to destabilize the most stable part of the helix. Similar, but smallermagnitude effects are observed for peptides with i, i + 3 interactions (data not shown). Results The sequences of the synthesized peptides with their respective mean residual ellipticities and helix contents are shown in Table l. F8M12, which contains phenylalanine and methionine spaced i, i + 4, is considerably more helical than the F7M12 control peptide, which contains Phe and Met spaced i, i + 5 (A0222 = 5,100 deg cm*.dmol"). Likewise, M8F12 is more helical than M7F12 (A0222 = 4,800 deg cm2.dmol"). These differences are outside the range of experimental error (-+ 1,OOO deg cm2.dmol") and clearly demonstrate qualitatively that Phe-Met and Met-Phe interactions stabilize isolated a-helices in water. To quantitatively determine the free energy of these interactions we have fitted the model described above to experi- the mental data. A comparison of the experimental helicities of the Table 1. Name F3M7 F8M12 F7M12 F13M17 M8F12 M7F12 "- Sequences and helix contents of peptides Sequence [0lzzza 070 Helicityb AC-YGFAKAMAAKAAAAKAA-NH ,600 Ac-YGAAKAAEAKAMAAKAA-NH ,700 Ac-YGAAKAFAAKAMAAKAA-NH ,600 AC-YGAAKAAAAKAAFAKAM-NH2-16, Ac-YGAAKAAMAKAEAAKAA-NH2-1 1, Ac-YGAAKAMAAKAFAAKAA-NH ,300 "Ellipticities in units of deg cm2.dmol" at 222 nm. bcalculated as [e]222cobrer"ed,/[-40,000(l - 2.5/n)], where n is the number of amino acids in the peptide. F3M NA 41.O 56.7 F8M NA F13M NA F7M NA M8F NA 31.9 M7F NA I reference peptides with those predicted by our algorithm (Table 2) reveals that the two peptides containing an i, i + 5 spacing between Phe and Met both have helicities in good agreement with theory, indicating that the values for w, u, c, and n accurately reproduce the experimental data. Peptides containing Phe-Met spaced i, i + 4 are considerably more helical than predicted by the model in the absence of a side chain interaction ( pfm = pmf = 1). Both orientations increase helicity, although the Phe-Met orientation is more helix stabilizing than the Met- Phe orientation. Values of p for the Phe-Met and Met-Phe side chain-side chain interactions were determined by least-squares fitting of the model to the experimental data (see Materials and methods). p for the Phe-Met i, i + 4 interaction was determined to be 4.0 while that for Met-Phe i, i + 4 interaction is 2.7, corresponding to free energies of interaction of and kcal. mol", respectively. By allowing the experimentally measured helix contents to increase or decrease by 3% and refittingp (Materials and methods), we have made an estimate of the error in AG. AG for the Phe-Met interaction is between and kcal.mol-l while that for the Met-Phe interaction is in the range to kcal.mol-l. Discussion A stabilizing interaction between phenylalanine and methionine spaced i, i + 4 in an a-helix was proposed based on statistical correlations observed in protein structures (Klingler & Brutlag, 1995). Here we have quantitatively measured the energetics of this interaction in both orientations using an isolated peptide helix in which the effect of the interaction can be dissected away from other contributions to stability. In the peptides studied here, the interaction between Phe and Met is the only side chainside chain interaction present in the helix, allowing the free energy of this interaction to be accurately determined. The measured free energies (-0.75 kcal.mol" and kcal.mol-l for the Phe-Met and Met-Phe orientations, respectively) are slightly larger than the interaction energies determined by Viguera and Serrano (1995) for similar interactions in peptides containing several different side chain-side chain interactions analyzed with AGADIR (Muiioz& Serrano, 1994). Muiioz and Serrano's results ( kcal.mol-' for Phe-Met and t 0.2 kcal.mol-l for Met-Phe) may differ principally as a result of using AGADIR (see discussion below). The interaction energies found by AGADIR from globally fitting data on peptides are given as -0.3 kcal.mol-' for Phe-Met and -0.0 kcal.mol-'

5 Phe-Met side chain interactions in a-helical peptides 2387 for Met-Phe in Muiioz and Serrano (1994) and -0.4 kcal.mol-l for Phe-Met and -0.2 kcal.mol-' for Met-Phe in Muiioz and Serrano (1995). Both these sets of results differ from the results found here and by Viguera and Serrano (1995). occupancy,a A possible Fraction reason for this is that AGADIR has many more parameters (768 in Muiioz & Serrano [ and 868 in Muiioz & Serrano [ 19951) than peptide data (323 in Muiioz & Serrano [I9941 and 423 in Muiioz & Serrano [1995]). The authors were, therefore, forced to assume that many side chain interactions have identical free energies. It is not stated exactly which parameters were assumed to be identical. Table 3. Populations of rotamer states in the absence of side chain interactions Rotamer a Calculated from Lee et al. (1994) f,, Comparison to statistical preferences observed in protein helices The significant free energies measured for Phe-Met interactions in peptide helices indicate that the statistical preferences observed in proteins reflect a real, stabilizing interaction rather than a fortuitous correlation. Phe-Met spaced i, i + 4 in a-helices occurs 2.3 times more often than might be expected from the normal distribution of these amino acids in a-helices, while Met- Phe spaced i, i + 4 occurs 1.6 times more frequently (Klingler & Brutlag, 1995). The statistical occurrence of side chain pairs in protein a-helices can be related to the thermodynamics of the putative interaction between the side chains by the Boltzmann equation: Phe-Met and Met-Phe interactions, we have applied the method suggested by Sharp and Englander (1994) to calculate AGmic, the free energy of interaction corrected for the rotamer preferences of the interacting side chains. We define a completely helical system consisting of the four rotamer states of the x1 angles of residues i and i + 4 in an.. a-helix: irr~nsi+~lruns, ~rmns~+4guuche+ 3 iguuche+ i+4gouche+, and iguuchc+ i+4rru,,,. We assume that side chain interactions occur only in the irrunsi+4guuche+ state. When the side chains do not interact, fractional occupancy, f,,, of the nth state can be related its the energy level, E,,, and the partition function for the noninteracting system, Z(no : where Pobserved is the observed frequency of side chain pairs, Pexpecred is the frequency expected due to chance, and AG,,, is the free energy of interaction. This correlation may not be strictly followed because other factors can result in side chain pairs occurring with nonrandom frequency. By implementation of such a "Boltzmann device" (Sippl, 1990), the Phe-Met interaction has a free energy approximately 0.2 kcal.mo1-' lower than the Met-Phe. The absolute values for free energies cannot be calculated by this method because the reference state is not clearly defined; the free energy of interaction between two residues that are i, i + 4 as often as expected by chance is unknown. The experimentally determined difference in free energy between Phe-Met and Met-Phe interactions of about 0.2 kcal.mo1-l is in excellent agreement with statistical distributions in folded proteins. The rotameric populations in the absence of interactions, f,,, are calculated from the data of Lee et al. (1994)(Table 3). AG, is defined as the difference in free energy between the system with an interaction and the system lacking an interaction, and is related to the ratiof the partition functions for the two systems: AG,,, is the free energy for forming an interaction in the i,,,,,i+4g,uch,+ rotameric state. The partition function for the interacting system can be written in terms of the noninteracting system by adjusting the energy of the interacting rotameric state: Orientation preference of interactions between Phe and Met The energetics of many frequently observed side chain-side chain interactions can be rationalized on the basis of preferred rotamers of side chains in a-helices (McGregor et al., 1987; Lee et al., 1994). For aromatic side chains, the trans rotamer is preferred, whereas the gauche+ rotamer is favored for aliphatic, non-0-branched side chains. The gauche- rotamer is very rarely observed. Since side chains interact primarily when residue i is trans and i + 4 is gauche+, the helical rotamer preferences of Phe and Met suggest that the Phe-Met orientation will be more helix stabilizing than the Met-Phe orientation. To rationalize quantitatively the difference in free energy between the Substituting Equation 3 into Equation 1 and making use of Equation 2, we obtain: From Equation 5, AG,, of the Phe-Met interaction is -1.O kcal. mol" and that of the Met-Phe interaction is -1.7 kcal.mol-i. At a microscopic level, a stronger interaction occurs in the Met-Phe orientation than the Phe-Met orientation (AG$-Phe >

6 2388 B. J. Stapley et al. A G Phe-Met m,c ). AG, is smaller for the Met-Phe orientation, however, because the rotamer state in which the interaction occurs is less populated. It is not clear why ACmjc for the Met-Phe orientation is larger than for the Phe-Met orientation. Although the side chains may indeed have better van der Waals contact or electrostatic interactions in the Met-Phe orientation, part of this difference may be accounted for by the presence of favorable side chain-side chain interactions in rotamer states other than the i,ru,,si+4guuche+. Such interactions are not accounted for in our treatment. Additionally, the rotamer populations, f,, are calculated on the basis of a rigid bond approximation that may not be applicable to interacting side chains in an a-helix. When two side chains come into contact in an a-helix, some strain in bond angles is probably induced that is offset by more favorable packing of the side chains. Although thermodynamics of interacting side chains is probably very complex, this analysis demonstrates that the difference between free energies of Phe-Met and Met-Phe interactions can be partly explained by the rotamer preferences of the side chains. Incorporation of capping interactions in helix/coil models Statistical mechanical models of the helix-coil transition have been demonstrated to accurately reproduce the experimentally observed characteristics of the helix-coil transition in both helical polymers and isolated peptide helices (see reviews by Scholtz & Baldwin, 1992; Chakrabartty & Baldwin, 1994). In their original formulation, however, only intrinsic propensities of the different amino acids are included in the Lifson-Roig and Zimm-Bragg models. Several groups have proposed models based on these statistical mechanical treatments, which incorporate sequencedependent effects, including capping interactions, charge-dipole interactions, and side chain-side chain interactions. The model described here incorporates the capping model of Doig et al. (1994) in which capping is described by a single statistical weight assigned to the N-cap or C-cap residue. It is important to consider the validity of including all capping phenomena in a single parameter. The n- and c-values give the effect on helix stability of varying an amino acid with nonhelical dihedral angles that is immediately adjacent to a helical segment. There are several structures found at helix termini that merit a more sophisticated treatment. The N-terminal capping box (Harper & Rose, 1993) includes a side chain-backbone hydrogen bond from N3 to the N-cap (i, i - 3). A side chain interaction with this geometry cannot be assigned a q-value despite having the appropriate spacing, as a q-value describes side chain interactions between residues that are all in a helical geometry. A side chain interaction between residues in the conformation chhh requires a unique statistical weight, and we have further extended the Lifson-Roig model to include this (B.J. Stapley & A.J. Doig, unpubl.). At the C-terminus, two distinct structures, named Schellman and al, have been observed when Gly is at a C-cap position in a helix (Schellman, 1980; Baker & Hubbard, 1984; Milner-White, 1988; PreiRner & Bork, 1991; Dasgupta & Bell, 1993; Nagarajaram et al., 1993; Aurora et al., 1994). The Schellman structure is characterized by 6+1, 5-2 hydrogen bonds between NH and C=O groups in the backbone. The al conformation has a 5+1 backbone hydrogen bond. These structures again merit special treatment with new statistical weights. Finally, a short section of 3,0-helix is commonly observed as an N- or C-terminal extension of an a-helix (Baker & Hubbard, 1984; Barlow & Thornton, 1988). We have developed a statistical mechanical model that allows the formation of pure 310- helix, pure a-helix, and mixed 310-/a-helices (C.A. Rohl & A.J. Doig, unpubl.). Although complex capping structures have been experimentally observed and can be incorporated into the Lifson-Roig for- malism, we have not included such interactions in the model described here. The peptides considered here have been designed to minimize the possibility of the formation of these more unusual helix terminal structures. In particular, they do not have any of the sequence requirements for the formation of the capping box, Schellman motif, or al conformation. Comparison of helixkoil models incorporating side chain interactions Several groups have recently proposed the addition of side chain-side chain interactions into helix-coil transition theory. These models make different assumptions and use significantly different parameter definitions, warranting an in-depth discussion and comparison. Scholtz et al. (1993) used a model based on the one-helical-sequence approximation of the Lifson-Roig model to quantitatively analyze salt bridge interactions in alanine-based peptides. Only a single interaction between residues of any spacing was considered. In the one-helical-sequence approximation, peptide conformations containing more than one helical segment are assumed not to be populated and are ex- cluded from the partition function (i.e., assigned statistical weights of zero). Because helix nucleation is difficult, conformations with multiple helical segments are expected to be rare in short peptides. This approximation has been demonstrated to be valid for peptides up to 50 residues (Qian & Schellman, 1992). As peptide length increases, the approximation is no longer valid because multiple helical segments can be long enough to overcome the initiation penalty. Mufioz and Serrano (1994) have proposed a one-helicalsequence helix-coil model (AGADIR) that includes side chain interactions. By invoking the one-helical-sequence approxima- tion, conformations that contain multiple helical segments are excluded from the partition function. In addition, the authors stated that the minimum helical length is four residues in helical angles plus two caps (Mufioz & Serrano, 1994), although three residues in helical angles (Pauling et al., 1951), plus two caps, are sufficient to form a single turn of helix. The effect of this assumption is to exclude all helices that contain a single hydrogen bond; only helices with two or more hydrogen bonds are allowed. These assumptions should have minimal effect on the predicted helix contents, as helices with only one hydrogen bond and short peptides with multiple helical segments are rare. The most significant difference between AGADIR and the model described here is in the definition of the reference state. In Lifson-Roig-based models, the reference state is the pure coil conformation and is independent of position and residue identity. In AGADIR, the reference state for a given residue includes all conformations in which the residue of interest is not part of a helix. This reference state will clearly depend on the position and identity of the residue being considered. For example, the reference state for the N-terminal residue of a peptide includes all helical conformations that do not extend to the N-terminus, as well as the complete coil conformation. The majority of the peptide conformations will fall into this group. For a residue at

7 Phe-Met side chain interactions in a-helical peptides 2389 the peptide center, however, the reference state includes the complete coil conformation and conformations with helical segments that do not extend over this central residue. At this position, consequently, most helical conformations will not be part of the reference state. Although the reference state of AGADIR is different for each position and amino acid type considered, the authors assume that these differences in the reference state are small and can be ignored. The validity of this assumption, however, has not been demonstrated. The parameters derived from AGADIR will depend on the location of the amino acid or side chain interaction in the peptides used to derive the parameters, whereas they should be independent of such considerations. Recently, Shalongo and Stellwagen (1995) have also proposed incorporating side chain interaction energies into the Lifson- Roig model. As with the model presented here, they included every possible conformation of the peptide in their partition function. Whereas in our model we have considered only i, i + 3 and i, i + 4 side chain interactions, Shalongo and Stellwagen (1995) more generally considered side chain interactions of any spacing. In their implementation of the Lifson-Roig formalism, however, they changed the definition of the propagating and initiating (capping) weights such that the physical meaning of these parameters is lost and the number of propagating residues (w) no longer correlates with the number of hydrogen bonds formed. Their capping parameters are associated with residues in helical conformations instead of coil. In proteins, however, N- and C-cap residues have nonhelical dihedral angles (Richardson & Richardson, 1988). This loss of physical meaning means that their energies for substituting residues at capping and interior positions in peptide helices are not directly transferable to helices in proteins. In contrast, the parameters in the original Lifson-Roig formalism, the model presented here, and our previous capping model (Doig et al., 1994) are physically meaningful. Results obtained using these models can be used for sub- stitutions in proteins, as illustrated by the good correlations between parameters measured in peptides and free energies determined in proteins (Chakrabartty et al., 1994). The model described here is a complete Lifson-Roig-based treatment of the helix-coil transition in the sense that it does not exclude any peptide conformations from the partition function. Conformations with multiple helical segments are allowed, and many side chain interactions, including overlapping interactions, are included simultaneously. Our model fits the experimental data well, even when the interaction is in different positions in the peptide. This model has also been used successfully to interpret the energetics of an i, i + 4 Asp-Gln hydrogen bonding interaction in alanine-based peptide helices (Huyghues-Despointes et al., 1995). In that study, a single interaction parameter was sufficient to predict the helix content of peptides with single interactions in different locations and of peptides with multiple noninteracting Asp-Gin interactions. The ability to reproduce the observed helix contents of peptides with interactions with a single interaction parameter, even when interactions are in dif- ferent locations or multiple interactions are present, is a sensitive test of the validity of the model. Because the great majority of the naturally occurring amino acids are intrinsically helix breakers (Chakrabartty et al., 1994), additional sequence-dependent interactions, including side chain-side chain interactions, are clearly important in determining helix stability. Favorable side chain interactions increase the stability of conformations containing five consecutive helical res- idues, and consequently may be also important in initiating helix formation by stabilizing a single turn of the helix from which propagation can occur. Here we have examined the helixstabilizing effect of interactions between Phe and Met side chains in different locations and orientations. Thenergetics of these interactions are well fitted by a modified version of the Lifson- Roig model that incorporates intrinsic propensities, capping interactions, and side chain interactions. This model is generally applicable to any i, i + 3 or i, i + 4 interaction observed in peptide helices, has physically meaningful parameters, and accurately predicts the effect of multiple, independent interactions (Huyghues-Despointes et al., 1995). With a knowledge of the helix parameters for all residues and the interaction parameters for all possible interactions, it is expected that the helix content of any isolated peptide or unfolded protein can be predicted, in the absence of tertiary interactions, complex capping interactions, and strong aromatic artifacts. Materials and methods Peptide synthesis Peptides were synthesized by the solid phase method using Fmoc chemistry. Pentafluorophenyl esters of 9-fluoroenylmethoxycarbonyl amino acids (Millipore) were coupled to rink amide resin (Millipore) using TBTU (benzotriazole-l-yl-tetrameckyluronium; Peninsula Labs, Inc.) in the presence of N,N-diisopropyl-ethylamine. N-termini were acetylated using pyridine and acetic anhydride. Cleavage from the resin was achieved by 95% trifluoroacetic acid/5% anisole. After precipitation of the peptide/trifluoroacetic acid mixtures into dry-ice-cold diethyl ether, the peptides were purified by CI8 reverse-phase fast protein liquid chromatography (Pharmacia) using a water/acetonitrile gradient of 10-30% containing 0.1 % trifluoroacetic acid. Molecular weights were confirmed by electrospray mass spectrometry at the Michael Barber Center for Mass Spectrometry, UMIST, Manchester, UK. Circular dichroism measurements CD measurements were made using a Jobin Yvon CD6 spectropolarimeter at Zeneca Pharmaceuticals (Alderley Park, UK). Measurements were made in a 1.O-cm quartz cell at 0 C in 10 mm NaCl, 5 mm sodium phosphate buffer at ph 7.0. Concentrations of stock solutions of the peptides were determined by diluting aliquots in 6.9 M guanidine hydrochloride and measuring tyrosine UV absorbance at 275 nm using c~,~,,,,, = 1,450 M. cm (Brandts & Kaplan, 1973). Absorbencies were the mean of three measurements differing by less than 3%. CD measurements are given as mean residue ellipticity at 222 nm ([O],,,) in units of deg cm2.dmol. Helix content was calculated as [O1222(observed)/[-40,000(l- 2.5/n)], where n is the number of amino acids in a peptide with blocked termini (Chakrabartty et al., 1991). Phe has a weak UV absorption and has been shown to contribute a small but significant positive ellipticity when placed at an interior position in an a-helix (Chakrabartty et al., 1993). Such aromatic effects involving Phe are presumably complex functions of helicity, position within the peptide sequence, and orientation of aromatic chromophore. Because :his contribution is expected to be small, probably within the experimental error,

8 2390 B. J. Stapler et al. Table 4. w-, u-, n-, and c-values for prediction of helicities c-vaheb n-vaheb w-valuea Residue U-value supported by grants from the BBSRC (SC103358) and the Royal Society ( G501). Ala LYS+ Phe Met GlY Tyr Acetyl Amide o a Chakrabartty et al. (1994). Doig and Baldwin (1995). Rohl et al. (1992). dthis residue never occurs at a position requiring this statistical weight in the peptides considered here. we have not attempted to correct for aromatic effects. If the phenylalanine contributes a positive ellipticity, however, the p-values determined here will be underestimates. Application of Lifson-Roig-based theory A computer program implementing the model described in the theory section was written in FORTRAN. The program evaluates the partition function of any sequence given w, u, n, c, p, and q-values for the constituent amino acids and predicts the fractional helicity, capping, and side chain interaction probabilities. Values for the statistical weights of residues in various states are given in Table 4. To determine a free energy for an interaction in an a-helix, the p-value of an i, i + 4 interaction is varied by intervals of 0.1 until the sum of residuals is at a minimum. We estimate the greatest experimental error to be in the measurement of helicity and to be about +3%. The error in p was evaluated by repeating the fitting procedure using experimental helicities uniformly increased or decreased by 3%. A computer program, SCINT, implementing the model described in the theory section was written in FORTRAN. SCINT is available via anonymous ftp from cmgm.stanford.edu in the directory /pub/helix/scint. The program evaluates the probability that each residue has w-, u-, n-, c-, p-, or q-weightings from inputs of the peptide sequence and tables of w-, u-, n-, c-, p-, or q-values. Note added in proof Sulfur-aromatic interactions have also been studied by Auld et al. (1993). 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Huyghues-Despointes BMP, Scholtz JM, Baldwin RL Helical pep- We thank Derek Barratt at Zeneca Pharmaceuticals, Alderley Edge, for tides with three pairs of Asp-Arg and Glu-Arg residues in different orithe use of the CD spectropolarimeter and Ian Fleet and Simon Gaskell entations and spacings. Protein Sci 2: at the Michael Barber Centre for Mass Spectrometry, UMIST, for ver- Klingler TM, Brutlag DL Discovering structural correlations in proification of peptide identity. B.J.S. is the grateful recipient of a BBSRC teins. Protein Sci 3: (UK) studentship. We thank Tod Klingler, Robert Baldwin, Luis Ser- Lee KH, Dong X, Freire E, Amzel LM Estimation of changes in side rano, and Beatrice Huyghes-Despointes for helpful discussions and Luis chain configurational entropy in binding and folding: General methods Serrano for a preprint of Viguera and Serrano (1995). This work was and application to helix formation. Proteins Srmct Funct Genet 20:68-84.

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