Positional preference of proline in a-helices - Europe PMC

Protein Science ~1999!, 8:1492–1499. Cambridge University Press. Printed in the USA. Copyright © 1999 The Protein Society

Positional preference of proline in a-helices

MEE KYOUNG KIM and YOUNG KEE KANG Department of Chemistry, Chungbuk National University, Cheongju, Chungbuk 361-763, Korea ~Received January 8, 1999; Accepted March 31, 1999!

Abstract Conformational free energy calculations have been carried out for proline-containing alanine-based pentadecapeptides with the sequence Ac-~Ala!n-Pro-~Ala!m-NHMe, where n 1 m 5 14, to figure out the positional preference of proline in a-helices. The relative free energy of each peptide was calculated by subtracting the free energy of the extended conformation from that of the a-helical one, which is used here as a measure of preference. The highest propensity is found for the peptide with proline at the N-terminus ~i.e., Ncap 1 1 position!, and the next propensities are found at Ncap, N9 ~Ncap 2 1!, and C9 ~Ccap 1 1! positions. These computed results are reasonably consistent with the positional propensities estimated from X-ray structures of proteins. The breaking in hydrogen bonds around proline is found to play a role in destabilizing a-helical conformations, which, however, provides the favored hydration of the corresponding N–H and C5O groups. The highest preference of proline at the beginning of a-helix appears to be due to the favored electrostatic and nonbonded energies between two residues preceding proline and the intrinsic stability of a-helical conformation of the proline residue itself as well as no disturbance in hydrogen bonds of a-helix by proline. The average free energy change for the substitution of Ala by Pro in a a-helix is computed to be 4.6 kcal0mol, which is in good agreement with the experimental value of ;4 kcal0mol estimated for an oligopeptide dimer and proteins of barnase and T4 lysozyme. Keywords: a-helices; conformational energy calculations; positional preference; proline

The proline ~Pro! residue is unique in that its side chain is covalently bonded to the nitrogen atom of peptide backbone. Consequently, the backbone of Pro cannot form a hydrogen bond and its N–C a rotation is rigid. The pyrrolidine of Pro residue is a fivemembered ring, which may adopt two distinct down- and uppuckered conformations ~Momany et al., 1975! that are almost equally favorable ~DeTar & Luthra, 1977; Madison, 1977; Némethy et al., 1992; Kang, 1996!. The highly constrained structure on the backbone of proline relative to other amino acids leads to that Pro occurs in b-turns, nonrepetitive structures, and at the ends of strands and helices ~Richardson & Richardson, 1989!. In particular, Pro is frequently found at the N-terminus but not in the middle of a-helices, because it breaks at least two adjacent hydrogen bonds and its pyrrolidine ring pushes the preceding turn of backbone away by about 1 Å ~Richardson & Richardson, 1988!. The higher preference of Pro at the N-terminus was suggested that Pro is perhaps better described as a helix initiator rather than a helix breaker ~Richardson & Richardson, 1988!. Several studies have been done on the conformational role of prolines in a-helices by analyzing X-ray structures of proteins ~Piela et al., 1987; Sankararamakrishnan & Vishveshwara, 1992! Reprint requests to: Young Kee Kang, Department of Chemistry, Chungbuk National University, Cheongju, Chungbuk 361-763, Korea; e-mail: [email protected].

and by theoretical computations on proline-containing peptides ~Piela et al., 1987; Sankararamakrishnan & Vishveshwara, 1990; Yun et al., 1991; Hurley et al., 1992; Némethy et al., 1992; Polinsky et al., 1992; Sankararamakrishnan & Vishveshwara, 1993!. In particular, Piela et al. ~1987! have studied the a-helical conformations of proline-containing alanine-based peptides in the absence of hydration in order to see the effect of Pro on several preceding residues and the extent of distortion of the a-helix caused by the presence of Pro. Only a limited number of works have been focused on the positional preference of proline in a-helices by analyzing X-ray structures of proteins ~Richardson & Richardson, 1988; Aurora & Rose, 1998; Kumar & Bansal, 1998!. These X-ray structures of proteins give us three questions: ~1! Why proline has the highest preference at the beginning of a-helix? ~2! What determines the conformational role of proline in the middle of a-helix? ~3! What controls the positional propensity of proline in a-helices? To answer these questions, we have carried out the conformational study on the extended and a-helical proline-containing pentadecapeptides by varying the position of proline in the unhydrated and hydrated states.

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Positional preference of proline in a-helices Materials and methods Model peptides In this work, the peptide PRi stands for the pentadecapeptide Ac~Ala!n-Pro-~Ala!m-NHMe, where n 1 m 5 14 with a proline residue at the i th full residue. The position was defined as N9, Ncap, N1, N2, N3, N4, N5, M, C5, C4, C3, C2, C1, Ccap, and C9 along the a-helix, where N1 through C1 belong to the helix and the primed residues belong to turns that bracket the helix at either end ~Richardson & Richardson, 1988; Aurora & Rose, 1998!. Ncap and Ccap are bridge residues that belong both to the helix and an adjacent turn. The all alanine-containing peptide, Ac-~Ala!15NHMe, was represented by PA that was chosen as a reference peptide, because Ala has almost uniformly favorable distributions along the helix ~Richardson & Richardson, 1988; Aurora & Rose, 1998!. In each a-helical conformation, two residues beyond Nand C-termini were assumed to be extended, i.e., the residues at N9, Ncap, Ccap, and C9 positions. For example, the a-helical sequence of the peptide PA is Ac-~Ala!2 e -~Ala!11 h -~Ala!2 e -NHMe, where e and h designate the extended and a-helical conformations, respectively. Force fields Conformational energy calculations were carried out using the Empirical Conformational Energy Program for Peptides, version 3 ~ECEPP03! force field ~Némethy et al., 1992!, in which the total conformational energy ~Etot ! is the sum of the electrostatic energy ~Ees !, the nonbonded energy ~Enb !, and the torsional energy ~Etor !. The hydrogen bond energy is included in the nonbonded energy component. In addition, the internal conformational energy ~Epro ! of pyrrolidine ring, depending on the puckering and the cis0trans imide bond, was added to the total energy ~Némethy et al., 1992!. For both puckerings of the proline residue in the ECEPP03 force field, a common set of averaged bond lengths and bond angles was used, except around the C g atom. Small adjustments in some geometrical parameters about the C g atom occurred to provide an exactly closed pyrrolidine ring, which may result in the difference in internal energies of the down and up puckerings ~Némethy et al., 1992!. However, the internal conformational energy of the proline residue in the ECEPP03 algorithm was computed as only the sum of nonbonded and electrostatic interactions between all atoms separated by three or more bonds in the pyrrolidine ring. The hydration shell model improved by Kang et al. ~1987a, 1987b, 1987c, 1988! was used to calculate the hydration free energy ~Ghyd ! for each conformation of pentadecapeptides in the hydrated state, where the hydration free energy was obtained as the sum of two contributions from water accessible volume and polarization. The total free energy ~Gtot ! for each conformation of pentadecapeptides in the hydrated state is given by the sum of its total conformational energy ~Etot ! and hydration free energy ~Ghyd !. A quasi-Newton algorithm Secant-type Unconstrained Minimization problem SoLver ~SUMSL! ~Gray, 1983! was used for energy and free energy ~i.e., a sum of the energy and hydration free energy! minimizations in the unhydrated and hydrated states, respectively. All torsion angles of the peptide backbone, side chains, and end groups of each pentadecapeptide were allowed to move during minimization. In particular, the torsion angle f of Pro was fixed at 268.88 and 253.08 for the down and up puckerings,

1493 respectively, due to the ring constraint ~Némethy et al., 1992!. The conformation for each residue of pentadecapeptides was denoted in terms of the conformational letter code of Zimmerman et al. ~1977!, which was assigned to each residue specifying its location on a f–c map. Starting conformations To generate starting conformations for minimization, the conformations with letter codes E and A were chosen for the extended and a-helical conformations of the Ala residue, respectively, where f 5 21558 and c 5 1578 for the former conformation, and f 5 2748 and c 5 2358 for the latter one ~Vásquez et al., 1983!. On the other hand, the letter codes for extended and a-helical conformations of the Pro residue were chosen as F and A, where f 5 268.88 and c 5 1598 for the former conformation, and f 5 2538 and c 5 2378 for the latter one ~Némethy et al., 1992!. In addition, the down and up puckerings were chosen for the Pro residues of extended and a-helical conformations, respectively, which is based on X-ray structures of proline-containing sequences of proteins ~Milner-White et al., 1992!. In particular, for the starting f and c values of the Ala residue preceding Pro only four letter codes of C ~f 5 2808, c 5 768!, D ~f 5 21518, c 5 468!, E ~f 5 21558, c 5 1578!, and F ~f 5 2758, c 5 1398! were selected. This is because the residues preceding Pro have nonhelical conformations ~Piela et al., 1987! and the entire region of 21808 , c , 608 on the Ramachandran plot is completely inaccessible to the Ala residue preceding proline ~MacArthur & Thornton, 1991!. Positional propensity Here, the positional propensity of the a-helical peptide PRi with a proline at the i th position was calculated using the Boltzmann i equation, i.e., exp~2DDGtot 0RT !. The relative total free energy of i the peptide in the hydrated state DDGtot was obtained by subtract0 i ing DGtot ~extrhlx! from DGtot ~extrhlx!, which are free energy changes for a-helical conformations relative to extended conformations for the peptides PA and PRi, respectively. Because these propensities are too small, they were normalized so as to make the propensity of the peptide PN1 be 2.00 that is almost the mean value of propensities estimated by analyzing X-ray structures of a-helices in proteins ~Richardson & Richardson, 1988; Aurora & Rose, 1998; Kumar & Bansal, 1998!. The kink angle The kink angle of proline-containing helical peptide can be calculated from two helical axes located before and after proline residue, each of which is defined as a least-squares line computed from the coordinates of all C a atoms of the helix ~Chou et al., 1984!. Only for the peptide PM that has at least five or more residues in each half of the helix, the kink angle was computed. Although an analysis of a-helices in proteins gave the mean helix length as approximately 10 residues, the helix length at the maximum distribution was found to be less than and equal to five residues ~Barlow & Thornton, 1988!. Results Conformational properties in the unhydrated state The Ala residue preceding Pro ~i.e., Ala ~ p21! ! of the minimized a-helical conformation of each proline-containing pentadecapep-

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tide PRi is found to have one of the conformations D, E, F, and A in the unhydrated state, which is shown in parenthesis following the designation of each peptide in Table 1. For most pentadecapeptides, the conformation D of the Ala ~ p21! residue dominantly occurred, which is in good agreement with previous computations ~Piela et al., 1987! and X-ray structures ~Karplus, 1996! for prolinecontaining a-helices. In particular, for the peptide PC2 the Ala residue at the Ccap position in the nonhelical part has peculiarly the conformation C after minimization in spite of the starting conformation assigned to be E. The order of peptides for helix-forming propensity in the unhydrated state is calculated to be PN1 . PNcap ' PN9 . PC9 by the values of DDEtot in Table 1. The favored electrostatic and nonbonded energies contribute to stabilizing more a-helical conformations of the peptides PN1, PNcap, PN9, and PC9 rather than the all alanine-containing peptide PA. In Figure 1, the contributions from the sum of DDEes and DDEnb for some dominant interresidue interactions, which include all backbone–backbone, backbone–side chain, and side chain–side chain interactions, to the DDEtot are shown. The higher propensities of the peptides PN1, PNcap, PN9, and PC9 than the peptides PN2 to PCcap are brought by the favored interresidue interactions between Ala ~ p23! and Ala ~ p11! residues and between Ala ~ p22! and Ala ~ p12! residues, as well as those between Ala ~ p22! and Ala ~ p11! residues. The first two interactions for the peptides PN2 to PC1 are ascribed mainly to the breaking in hydrogen bonds between corresponding residues by introducing the proline in-between them. The third term is due to the fact that the distance between the carbonyl oxygen of Ala ~ p22! residue and the amido group of Ala ~ p11! residue for each of the

peptides PN1, PNcap, PN9, and PC9 is similar to that of the peptide PA, whereas the corresponding distance for the peptides PN2 to PCcap becomes shorter by about 1 Å because of the kink of a-helix. For the peptide PCcap, these three interactions are also favored. However, the low propensity of the peptide PCcap is mainly brought by the unfavored interresidue interaction between Ala ~ p21! and Pro residues. From analyzing the hydrogen-bonding pattern of each helical peptide PRi compared to that of the peptide PA, it was noted that the peptides PN1, PNcap, PN9, and PC9 have no different patterns in hydrogen bonds from the all alanine-containing peptide PA, which appears to be the main factor to allow these peptides to be stabilized. The peptide PCcap has also the same pattern of hydrogen bonds except for no hydrogen bond between Ala ~ p24! and Pro residues. As expected, it is confirmed that a hydrogen bond between Ala ~ p24! and Pro residues does not exist for a-helical conformations of the peptides PN2 to PC1 due to the lack of amido hydrogen for proline. For the peptides PN2 to PC1, the substitution of Ala by Pro in a a-helix results in destabilizing the helical conformation by ;6.5 kcal0mol in the unhydrated state. The higher propensity of the peptide PN1 to form a a-helical conformation than the peptides PNcap, PN9, and PC9 is caused by the favored interresidue interactions between Ala ~ p22! and Ala ~ p21! residues and the intrinsic stability of a-helical conformation of the proline residue itself, of which the latter also holds for Ac-Pro-NHMe ~Kang, 1996! and proline-containing dipeptides ~Han et al., 1997; Kang et al., 1999!. However, the peptide PN1 has the unfavored interresidue interaction between Ala ~ p21! and Pro residues and the unfavored inter-

Table 1. Relative stabilization energies and their components of peptides in the unhydrated state a Peptide b

Etot ~h! c

Etot ~e! d

DEtot e

DDEtot f

DDEes g

DDEnb g

DDEtor g

DEpro h

PAi PN9 PNcap~E! PN1~F! PN2~F! PN3~F! PN4~D! PN5~D! PM~D! PC5~D! PC4~D! PC3~D! PC2~D! PC1~D! PCcap~A! PC9~E!

215.17 230.20 230.79 231.33 225.80 226.47 224.77 221.37 221.07 221.16 222.93 224.86 224.87 224.28 219.02 230.17

34.17 19.65 19.15 19.15 19.12 19.13 19.12 19.12 19.12 19.12 19.11 19.12 19.11 19.11 19.09 19.30

249.33 249.85 249.95 250.47 244.93 245.60 243.89 240.49 240.19 240.28 242.04 243.98 243.98 243.39 238.11 249.47

0.00 20.52 20.62 21.14 4.40 3.73 5.44 8.85 9.14 9.06 7.29 5.35 5.36 5.94 11.23 20.13

0.00 20.61 20.35 20.96 0.22 3.05 2.32 2.77 2.92 2.81 4.36 2.84 2.13 0.63 1.22 0.00

0.00 0.05 20.81 20.74 3.64 20.96 2.52 5.08 5.26 5.18 1.66 1.34 2.11 4.51 8.23 20.23

0.00 0.04 0.55 0.08 0.07 1.17 0.12 0.51 0.48 0.59 0.80 0.70 0.65 0.33 1.78 0.10

0.00 0.00 0.48 0.48 0.48 0.48 0.48 0.48 0.48 0.48 0.48 0.48 0.48 0.00 0.00

a

Energies in kcal0mol. The name of each peptide is designated according to the position of Pro in the sequence. The conformational letter code of Ala preceding proline for a-helical conformation is shown in parenthesis. c,d Total energies for helical and extended conformations, respectively. e DEtot 5 Etot ~h! 2 Etot ~e!. f i 0 i 0 DDEtot 5 DEtot 2 DEtot , where DEtot and DEtot are relative conformational energies for the peptide with Pro at the i th residue and the peptide PA, respectively. DDEtot 5 DDEes 1 DDEnb 1 DDEtor 1 DEpro . g Relative electrostatic, nonbonded, and torsional energies, respectively. h Relative internal energy of the trans proline with up-puckering to that with down-puckering. i This means no Pro residues in the peptide, i.e., the peptide with only alanine residues. b


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Fig. 1. Contributions of DDEes and DDEnb for some residues neighboring proline to DDEtot to form a a-helix in the unhydrated state. The total sum of DDEes and DDEnb is indicated by “total” in the legend. The interaction between two residues A and B is designated by A0B in the legend. Each pair of residues is represented by their positions relative to proline, e.g., ~ p 2 3!0~ p 1 1! stands for a pair interaction of Ala ~ p23! and Ala ~ p11! residues, etc.

nal conformational energy for the up-puckered proline by 0.48 kcal0mol than the down-puckered one ~Némethy et al., 1992!. When proline forms a a-helical conformation from an extended one, there is the decrease in energy by ;0.9 kcal0mol for the peptides PN1 to PC1 ~i.e., estimated by the sum of DDEes and DDEnb for the proline residue itself shown in Fig. 1!, which is mainly due to the intrinsic lower energy of the conformation A of proline than the conformation F, as predicted lower by ;0.5 kcal0 mol for Ac-Pro-NHMe ~Némethy et al., 1992!. This intrinsic lower energy of the conformation A of proline appears to be due to the favorable interaction between two peptide dipoles located at both sides of proline. The peptide PCcap has the more favored interaction between Ala ~ p22! and Ala ~ p11! residues by 20.2 kcal0mol than the peptide PN1, but the unfavored interaction between Ala~ p21! and Pro residues by 3.8 kcal0mol contributes to destabilizing the a-helical conformation of the peptide PCcap. Conformational properties in the hydrated state Table 2 lists the energetics for each of extended and helical peptides in the hydrated state. There are no significant changes in conformational energies of helical peptides compared to those in the unhydrated state. However, it should be noted that the conformational energies are influenced mainly by the breaking in hydrogen bonds due to the lack of amido hydrogen for proline and the kink of the peptide by proline, and by favored hydration of nonhydrogen-bonded polar groups of the peptide backbone in the hydrated state. In Figure 2, minimized a-helical conformations of model peptides in the hydrated state are shown. For the peptides PN2 to PC2, there are kinks in a-helices. However, the kink angle is calculated only for the peptide PM that has at least five or more residues in each half of the helix and its value is 438. Our calculated value is close to the values of 418 and 498 for glutathione peroxidase ~Sankararamakrishnan & Vishveshwara, 1992! and melittin ~Barlow & Thornton, 1988!, respectively, but larger than mean values of 258 ~Sankararamakrishnan & Vishveshwara, 1992! and 268 ~Barlow & Thornton, 1988! estimated by analyzing X-ray structures of prolinecontaining a-helices in proteins. A larger computed value of the

kink angle than the mean values from X-ray structures appears to be due to the relatively shorter lengths of half helices for the peptide PM. In general, the lack of two hydrogen bonds between O ~ p24! and N ~ p! atoms and between O ~ p23! and N ~ p11! atoms due to the imide nitrogen of proline and the kink caused by the conformational constraint of proline is known for proline-containing a-helices from X-ray structures of proteins ~Barlow & Thornton, 1988!. Our calculations confirm the lack of these two hydrogen bonds and another hydrogen bond between C5O ~ p22! and N–H ~ p12! groups for the peptides PN2 to PC1. The lack of the third hydrogen bond is ascribed to the nonhelical conformation of the residue preceding Pro. To figure out the effects of hydration on the amide and carbonyl groups of residues neighboring proline due to the helix formation, the changes in hydration free energies of those groups, especially due to the breaking in hydrogen bonds, are shown in Figure 3. The imide nitrogen of proline and the N–H and C5O groups, liable to form hydrogen bonds between Ala ~ p23! and Ala ~ p11! residues and between Ala ~ p22! and Ala ~ p12! residues, become more favorable for hydration due to the breaking in hydrogen bonds, which have the contributions of av. 21.3, 21.7, and 21.4 kcal0mol to DDGhyd for each of the peptides PN2 to PC1, respectively. However, the unfavorable hydration of the N–H and C5O groups of Ala ~ p21! and other residues contributes to DDGhyd by 0.4 and 2.2 kcal0mol, respectively. As a result, there is the decrease in free energy by ;1.9 kcal0mol for each peptide by hydration. Discussion Substitution of Ala by Pro in a-helices Because of no significant changes in a-helical conformations of peptides by hydration, the destabilization due to the substitution of Ala by Pro results in the increase of energy by 6.5 kcal0mol as the almost same as that in the unhydrated state, as seen in Tables 1 and 2. However, the hydration lowers the total free energy for each a-helical peptide by 1.9 kcal0mol and the free energy change due to the replacement of Ala by Pro in a a-helix is estimated to be

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M.K. Kim and Y.K. Kang Table 2. Relative stabilization free energies and their components of peptides in the hydrated state a Peptide b

Gtot ~h! c

Gtot ~e! d

DGtot e

DDGtot f

DDEtot g

DDGhyd h

Propensity i

PAj PN9 PNcap~E! PN1~F! PN2~F! PN3~F! PN4~D! PN5~D! PM~D! PC5~D! PC4~D! PC3~D! PC2~D! PC1~D! PCcap~A! PC9~E!

261.19 273.12 274.46 274.82 271.92 269.39 268.01 267.48 267.33 267.38 266.31 269.25 268.97 271.52 264.00 273.33

240.93 252.47 253.01 253.04 253.06 253.05 253.06 253.06 253.06 253.06 253.06 253.06 253.07 253.07 253.10 253.03

220.26 220.65 221.45 221.79 218.87 216.34 214.95 214.42 214.27 214.32 213.24 216.19 215.90 218.45 210.91 220.29

0.00 20.39 21.18 21.52 1.40 3.93 5.31 5.84 6.00 5.95 7.02 4.07 4.37 1.82 9.36 20.03

0.00 20.50 20.62 21.14 4.40 3.74 5.44 8.85 9.15 9.06 7.31 5.35 5.36 5.94 11.23 20.13

0.00 0.11 20.57 20.38 23.01 0.19 20.13 23.00 23.15 23.11 20.29 21.28 20.99 24.13 21.87 0.11

0.15 0.30 1.13 2.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.16

a

Energies in kcal0mol. See footnote b of Table 1. c,d Total free energies for helical and extended conformations, respectively; each free energy was computed by the sum of Etot and Ghyd . e DGtot 5 Gtot ~h! 2 Gtot ~e!. f i 0 DDGtot 5 DEtot 2 DEtot . See footnote f of Table 1. g DDEtot 5 DDEes 1 DDEnb 1 DDEtor 1 DEpro . See footnote f of Table 1. h i 0 DDGhyd 5 DGhyd ~extrhlx! 2 DGhyd ~extrhlx!, relative to the peptide PA. i Normalized a-helical propensity. See Materials and methods for the procedure to calculate normalized propensities. j See footnote i of Table 1. b

4.6 kcal0mol. This is in good agreement with the experimental value of ;4 kcal0mol estimated from a noncovalent a-helical oligopeptide dimer ~O’Neil & DeGrado, 1990! and site-directed mutations in a-helices of barnase ~Horvovitz et al., 1992! and T4 lysozyme ~Blaber et al., 1994!. Although Hurley et al. ~1992! suggested that the conformational energy of ;7 kcal0mol is obtainable from the rigid geometry calculations on the Ala r Pro mutations in a-helices, the present calculations indicate that the hydration may compensate the higher energy and give a reasonable value comparable to observed estimates. Positional propensity of Pro in a-helices The favored hydration of imide nitrogens of the helical peptides PN9, PNcap, PN1, and PC9 contributes dominantly to lowering their total conformational free energies, whereas the breaking in hydrogen bonds by introducing proline does not result in a significant contribution to the favored hydration of each helix, as seen in Figure 3. This is because there are no changes in the pattern of hydrogen bonds for a-helical conformations of these prolinecontaining peptides compared to that of peptide PA, as mentioned in Results. As a result, the peptides PNcap and PN1 have favorable hydration after the helix formation, which, however, is less effective than the peptides with proline in the middle of helix. The favored hydration of the peptide PCcap is also ascribed to the hydration of the N ~ p! , N–H ~ p11! , and C5O ~ p23! groups, as seen for the peptides PN2 to PC1.

However, the positional propensity of Pro in a-helices is calculated to be the order of N1 . Ncap . N9 . C9 estimated by the values of DDGtot in the hydrated state, as shown in Table 2. As noted in Materials and methods, these propensities were normalized so as to make the propensity of the peptide PN1 be 2.00 that is almost the average value of propensities estimated by analyzing X-ray structures of a-helices in proteins ~Richardson & Richardson, 1988; Aurora & Rose, 1998; Kumar & Bansal, 1998!. The comparison of calculated and observed propensities is shown in Figure 4. Our calculated positional propensity is reasonably consistent with the results of N1 . C9 . N9 ' Ncap ~Richardson & Richardson, 1988!, N1 . N9 ' Ncap . C9 ~Aurora & Rose, 1998!, and N1 . C9 . Ncap ' N9 ~Kumar & Bansal, 1998! obtained from the analysis of X-ray structures of proteins. Conclusions The positional propensity of proline in a-helices seems to be determined remarkably by the local electrostatic and nonbonded interactions between residues around proline. In particular, the breaking in hydrogen bonds due to the imide nitrogen of proline and the kink of the peptide with proline in the middle of helix appear to be significant factors to destabilize the a-helical conformation, although they lead to the favored hydration of helices that lowers the relative total free energy of each helix in the hydrated state. The higher propensity of the peptide PN1 with proline at the N-terminus ~i.e., Ncap 1 1! position appears to be due to ~1! that proline does not prohibit the formation of hydrogen bonds of a-helix,


Fig. 2. Minimized a-helical conformations of model peptides in the hydrated state. All hydrogen atoms not shown for clarity.

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Fig. 3. Contributions of DDGhyd for N–H and C5O groups neighboring proline due to the breaking in hydrogen bonds. The total sum of DDGhyd is indicated by “total” in the legend. Each residue is represented by its position relative to proline, e.g., p 2 3, p 2 2, p 2 1, p, p 1 1, and p 1 2 stand for the residues Ala ~ p23! , Ala ~ p22! , Ala ~ p21! , Pro, Ala ~ p11! , and Ala ~ p12! , respectively. Contributions are represented by “p” for the N of Pro, “~ p 1 1! & ~ p 2 3!” for the N–H of Ala ~ p11! and C5O of Ala ~ p23! , “~ p 1 2! & ~ p 2 2!” for the N–H of Ala ~ p12! and C5O of Ala ~ p22! , and “~ p 2 1!” for the N–H and C5O of Ala ~ p21! .

~2! that proline has the intrinsic stability of a-helical conformation, and ~3! that there is favorable interaction between two ~ p 2 2! th and ~ p 2 1! th residues preceding proline. In addition, the higher propensities of the peptides PNcap, PN9, and PC9 may be also ascribed to no perturbations in hydrogen bonds of a-helix by proline, as seen for the peptide PN1.

Supplementary material in Electronic Appendix A set of 16 files containing the minimized helical conformations of the proline0alanine helical peptides, shown in Figure 2, is provided as electronic supplementary material. The files, in the PDB format, are named ProHlx00.xyz through ProHlx15.xyz.

Acknowledgments This work was supported by the Basic Science Research Institute Program, the Ministry of Education of Korea ~BSRI-96-3448!, and partially by the STEPI, Korea ~1998!.

References Aurora R, Rose GD. 1998. Helix capping. Protein Sci 7:21–38. Barlow DJ, Thornton JM. 1988. Helix geometry in proteins. J Mol Biol 201:601– 619. Blaber M, Zhang X, Lindstrom JD, Pepiot SD, Baase WA, Matthews BW. 1994. Determination of a–helix propensity within the context of a folded protein. Sites 44 and 131 in bacteriophage T4 lysozyme. J Mol Biol 235:600– 624. Chou KC, Némethy G, Scheraga HA. 1984. Energetic approach to the packing of a-helices. 2. General treatment of nonequivalent and nonregular helices. J Am Chem Soc 106:3161–3170.

Fig. 4. Comparison of calculated and observed positional preferences of proline along the a-helices: this work ~d!; X-ray structures ~Richardson & Richardson, 1988! ~▫ !; Aurora & Rose ~1998! ~1!; Kumar & Bansal ~1998! ~ n !. See Materials and methods for the procedure to calculate our normalized propensities.

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