Crystal-state 3D-structural characterization of novel 3(10)-helical peptides.

The crystal-state conformations of two octapeptides, pBrBz-(D-Iva)8-OtBu (8I) and Ac-[L-(alphaMe)Val]8-OH (8II), the heptapeptide Z-[L-(alphaMe)Val]7-OH (7), the hexapeptide Z-[L-(alphaMe)Leu]6-OtBu (6) and the tetrapeptide alkylamide Z-(Aib)2-L-Glu(OMe)-L-Ala-L-Lol (5) were assessed by x-ray diffraction analyses. Two independent molecules are observed in the asymmetric unit of each L-(alphaMe)Val homo-peptide. All four homo-peptides are folded in a regular 3(10)-helical structure (only the C-terminal H-bonded conformation of the D-Iva octapeptide is distorted to a type-I beta-turn). The hydroxyl groups of the C-terminal carboxyl moieties of the two L-(alphaMe)Val homo-peptides participate in an oxy-analogue of the type-III beta-turn conformation. While the two L-(alphaMe)Val 3(10)-helices are right-handed, the D-Iva and L-(alphaMe)Leu helices are left-handed. The tetrapeptide alkylamide is 3(10)-helical at the N-terminus, but it is mixed 3(10)/alpha-helical at the C-terminus.     

Factors governing 3(10)-helix vs alpha-helix formation in peptides: percentage of C(alpha)-tetrasubstituted alpha-amino acid residues and sequence dependence

As an additional step toward the dissection of the factors responsible for the onset of 3(10)-helix vs alpha-helix in peptides, in this paper we describe the results of a three-dimensional (3D) structural analysis by x-ray diffraction of the N(alpha)-acylated heptapeptide alkylamide mBrBz-L-Iva-L-(alphaMe)Val-L-Abu-L-(alphaMe)Val-L-(alphaMe)Phe-L-(alphaMe)Val-L-Iva-NHMe characterized by a single (L-Abu3) C(alpha)-trisubstituted and six C(alpha)-tetrasubstituted alpha-amino acids. We find that in the crystal state this peptide is folded in a mixed helical structure with short elements of 3(10)-helix at either terminus and a central region of alpha-helix. This finding, taken together with the published NMR and x-ray diffraction data on the all C(alpha)-methylated parent sequence and its L-Val2 analog (also the latter heptapeptide has a single C(alpha)-trisubstituted alpha-amino acid) strongly supports the view that one C(alpha)-trisubstituted alpha-amino acid inserted near the N-terminus of an N(alpha)-acylated heptapeptide alkylamide sequence may be enough to switch a regular 3(10)-helix into an essentially alpha-helical conformation. As a corollary of this work, the x-ray diffraction structure of the N(alpha)-protected, C-terminal tetrapeptide alkylamide Z-L-(alphaMe)Val-L-(alphaMe)Phe-L-(alphaMe)Val-L-Iva-NHMe, also reported here, is clearly indicative of the preference of this fully C(alpha)-methylated, short peptide for the 3(10)-helix. As the same terminally blocked sequence is mixed 3(10)/alpha-helical in the L-Abu3 heptapeptide amide but regular 3(10)-helical in the tetrapeptide amide and in the parent heptapeptide amide, these results point to an evident plasticity even of a fully C(alpha)-methylated short peptide.     

Conformational change from antiparallel beta-sheet to alpha-helix in a series of depsipeptide, -(Leu-Leu-Lac)(n)-: syntheses, spectroscopic studies, and crystal structures of Boc-Leu-Lac-OEt and Boc-(Leu-Leu-Lac)(n)-OEt (n = 1, 2)

The depsipeptides Boc-Leu-Lac-OEt (1) and Boc-(Leu-Leu-Lac)(n)-OEt (n = 1, 2) (2 and 3, respectively) (Boc = tert-butyloxycarbonyl, Lac = L-lactic acid residue) has been synthesized and studied by crystallographic, CD spectroscopic, and ESI-MS analyses. In the packing cells, those three compounds adopt beta-strand conformations. Each molecule is linked into a dimer (1) or an infinite assembly (2 and 3) by tight hydrogen bonds of the type NH...O==C. Interestingly, the hexamer, 3 shows the first example of antiparallel pleated beta-sheet crystal structure for a depsipeptide molecule. In the packing cells, especially for 3, the ester groups O--C==O are perpendicularly oriented to the amide groups NH--C==O and beta-sheet planes to avoid the interaction between --O--(ester) and O==C. Therefore, when the chain length become longer, the O...O==C repulsion interaction works as a beta-sheet breaker and hence promotes an alpha-helical structure as observed for Boc-(Leu-Leu-Lac)(3)-Leu-Leu-OEt (4) (Oku et al. Biopolymers 2004, 75, 242-254) and Boc-(Leu-Leu-Lac)(n)-OEt (n = 4-6) (5-7) (Katakai et al., Biopolymers 1996, 38, 285-290), in which the O...O==C repulsion does not cause significant structural changes in alpha-helical main chains. Therefore from the structural and spectroscopic analyses, we have found governing factors for the specificity in the beta-sheet and alpha-helix decision in this series of depsipeptides, -(Leu-Leu-Lac)(n)-.     

Variants of 3(10)-helices in proteins

An analysis of the shortest 3(10)-helices, containing three helical residues and two flanking capping residues that participate in two consecutive i + 3 --> i hydrogen bonds, shows that not all helices belong to the classic 3(10)-helix, where the three central residues adopt the right-handed helical conformation (alpha(R)). Three variants identified are: 3L10-helix with all residues in the left-handed helical region (alpha(L)), 3EL10-helix where the first residue is in the extended region followed by two residues in the alpha(L) conformation, and its mirror-image, the 3E'R10-helix. In the context of these helices, as well as the equivalent variants of alpha-helices, the length dependence of the handedness of secondary structures in protein structure is discussed. There are considerable differences in the amino acid preferences at different positions in the various types of 3(10)-helices. Each type of 3(10)-helix can be thought to be made up of an extension of a particular type of beta-turn (made up of residues i to i + 3) such that the (i + 3)th residue assumes the same conformation as the preceding residue. Distinct residue preferences at i and i + 3 positions seem to decide whether a particular stretch of four residues will be a beta-turn or a 3(10)-helix in the folded structure.     

Crystal-state 3D-structural characterization of novel, Aib-based, turn and helical peptides.

The crystal-state conformations of the hexapeptide amide Pht-(Aib)(6)-NH-C(CH(3))(2)-O-OtBu (7), the hexapeptide Ac-L-aIle-(Aib)(5)-OtBu (6), the pentapeptide Z-(Aib)(3)-L-Glu(OtBu)-Aib-O-(CH(2))(2)-(1)Nap (5), the tetrapeptides Z-(Aib)(2)-L-His(N(tau)-Trt)-Aib-OMe (4 I) and Z-(Aib)(2)-L-Nva-Aib-OtBu (4 II), the tripeptide Pyr-(Aib)(3)-OtBu (3 I), the dipeptide amides Pyr-(Aib)(2)-(4)NH-TEMPO (3 II) and Piv-(Aib)(2)-NH-C(CH(3))(2)-O-OtBu (3 III), and the dipeptides Pht-Aib-betaAc(6)c-OtBu (2 I), Pht-Aib-NH-C(CH(3))(2)-O-OtBu (2 II) and Boc-gGly-mAib-OH (2 III) have been determined by X-ray diffraction analyses. All peptides investigated are characterized by one or more turn/helix forming Aib residues. Except the three short dipeptides, all are folded into C==O...H--N intramolecularly H-bonded 3(10)-helices, or into various types of beta-turns. In the structure of 6, two independent molecules of opposite screw sense were observed in the asymmetric unit, generating diastereomeric 3(10)-helices.     

Addition of a peptide fragment on an alpha-helical depsipeptide induces alpha/3(10)-conjugated helix: synthesis, crystal structure, and CD spectra of Boc-Leu-Leu-Ala-(Leu-Leu-Lac)3-Leu-Leu-OEt

The depsipeptide Boc(1)-Leu(2)-Leu(3)-Ala(4)-Leu(5)-Leu(6)-Lac(7)-Leu(8)-Leu(9)-Lac(10)-Leu(11)-Leu(12)-Lac(13)-Leu(14)-Leu(15)-OEt(16) (1) (Boc = tert-butyloxycarbonyl, Lac = L-lactic acid residue) has been synthesized from the peptide Boc-Leu-Leu-Ala-OEt (2) and a depsipeptide, Boc-(Leu-Leu-Lac)(3)-Leu-Leu-OEt (3). Single crystals of 1 were successfully obtained and the structure has been solved by direct methods (such as Sir2002 and Shake-and-Bake). Interestingly, 1 adopts an alpha/3(10)-conjugated helix containing a kink at the junction of peptide and depsipeptide segments, Leu3-Lac7. This is significantly different from the conformation of 3, which has a straight alpha-helical structure with standard phi and psi angles. Microcrystalline CD spectra were also studied to compare structural properties of 1 and 3. The differences between alpha/3(10)- and alpha-helices appear in these CD spectra.     

Helix packing of leucine-rich peptides: a parallel leucine ladder in the structure of Boc-Aib-Leu-Aib-Aib-Leu-Leu-Leu-Aib-Leu-Aib-OMe.

The packing of peptide helices in crystals of the leucine-rich decapeptide Boc-Aib-Leu-Aib-Aib-Leu-Leu-Leu-Aib-Leu-Aib-OMe provides an example of ladder-like leucylleucyl interactions between neighboring molecules. The peptide molecule forms a helix with five 5----1 hydrogen bonds and two 4----1 hydrogen bonds near the C terminus. Three head-to-tail NH ... O = C hydrogen bonds between helices form continuous columns of helices in the crystal. The helicial columns associate in an antiparallel fashion, except for the association of Leu ... Leu side chains, which occurs along the diagonal of the cell where the peptide helices are parallel. The peptide, with formula C56H102N10O13, crystallizes in space group P2(1)2(1)2(1) with Z = 4 and cell parameters a = 16.774(3) A, b = 20.032(3) A and c = 20.117(3) A; overall agreement factor R = 10.7% for 2014 data with magnitude of F(obs) greater than 3 sigma (F); resolution 1.0 A.     

The effects of alpha-helix on the stability of Asn residues: deamidation rates in peptides of varying helicity

Asn deamidation was monitored in Ala-based octadecapeptides of varying alpha-helicity. Gly was substituted for Ala residues at positions 6 and 16 to create a peptide with less helicity. Ala --> Gly substitutions were made at three or more residues from the Asn to negate known primary sequence effects on deamidation rates. The extent of helicity and rate of Asn deamidation for alkaline aqueous solutions of each peptide was measured as a function of temperature by circular dichroism and reversed-phase high-performance liquid chromatography, respectively. The rate of deamidation in the peptides was inversely proportional to the extent of alpha-helicity. The results support the conclusion that Asn deamidation only occurs in the nonhelical population of conformers.     

Crystal-state conformation of Calpha,alpha-dialkylated peptides containing chiral beta-homo-residues.

Secondary structure formation and stability are essential features in the knowledge of complex folding topology of biomolecules. To better understand the relationships between preferred conformations and functional properties of beta-homo-amino acids, the synthesis and conformational characterization by X-ray diffraction analysis of peptides containing conformationally constrained Calpha,alpha-dialkylated amino acid residues, such as alpha-aminoisobutyric acid or 1-aminocyclohexane-1-carboxylic acid and a single beta-homoamino acid, differently displaced along the peptide sequence have been carried out. The peptides investigated are: Boc-betaHLeu-(Ac6c)2-OMe, Boc-Ac6c-betaHLeu-(Ac6c)2-OMe and Boc-betaHVal-(Aib)5-OtBu, together with the C-protected beta-homo-residue HCl.H-betaHVal-OMe. The results indicate that the insertion of a betaH-residue at position 1 or 2 of peptides containing strong helix-inducing, bulky Calpha,alpha-disubstituted amino acid residues does not induce any specific conformational preferences. In the crystal state, most of the NH groups of beta-homo residues of tri- and tetrapeptides are not involved in intramolecular hydrogen bonds, thus failing to achieve helical structures similar to those of peptides exclusively constituted of Calpha,alpha-disubstituted amino acid residues. However, by repeating the structural motifs observed in the molecules investigated, a beta-pleated sheet secondary structure, and a new helical structure, named (14/15)-helix, were generated, corresponding to calculated minimum-energy conformations. Our findings, as well as literature data, strongly indicate that conformations of betaH-residues, with the micro torsion angle equal to -60 degrees, are very unlikely.     

Proline in alpha-helix: stability and conformation studied by dynamics simulation

Free-energy simulations have been used to estimate the change in the conformational stability of short polyalanine alpha-helices when one of the alanines is replaced by a proline residue. For substituting proline in the middle of the helix the change in free energy of folding (delta delta G degrees) was calculated as 14 kJ/mol (3.4 kcal/mol), in excellent agreement with the one available experimental value. The helix containing proline was found to be strongly kinked; the free energy for reducing the angle of the kink from 40 degrees to 15 degrees was calculated, and found to be small. A tendency to alternate hydrogen bonding schemes was observed in the proline-containing helix. These observations for the oligopeptide agree well with the observation of a range of kink angles (18-35 degrees) and variety of hydrogen bonding schemes, in the rare instances where proline occurs in helices in globular proteins. For substituting proline at the N-terminus of the helix the change in free energy of folding (delta delta G degrees) was calculated as -4 kJ/mol in the first helical position (N1) and +6 kJ/mol in the second helical position (N2). The observed frequent occurrence of proline in position N1 in alpha-helices in proteins therefore has its origin in stability differences of secondary structure. The conclusion reached here that proline may be a better helix former in position N1 than (even) alanine, and thus be a helix initiator may be testable experimentally by measurements of fraction helical conformation of individual residues in oligopeptides of appropriate sequence. The relevance of these results in regards to the frequent occurrence of proline-containing helices in certain membrane proteins is discussed.     

Determination of alpha-helix and beta-sheet stability in the solid state: a solid-state NMR investigation of poly(L-alanine)

The relative stability of alpha-helix and beta-sheet secondary structure in the solid state was investigated using poly(L-alanine) (PLA) as a model system. Protein folding and stability has been well studied in solution, but little is known about solid-state environments, such as the core of a folded protein, where peptide packing interactions are the dominant factor in determining structural stability. (13)C cross-polarization with magic angle spinning (CPMAS) NMR spectroscopy was used to determine the backbone conformation of solid powder samples of 15-kDa and 21.4-kDa PLA before and after various sample treatments. Reprecipitation from helix-inducing solvents traps the alpha-helical conformation of PLA, although the method of reprecipitation also affects the conformational distribution. Grinding converts the secondary structure of PLA to a final steady-state mixture of 55% beta-sheet and 45% alpha-helix at room temperature regardless of the initial secondary structure. Grinding PLA at liquid nitrogen temperatures leads to a similar steady-state mixture with 60% beta-sheet and 40% alpha-helix, indicating that mechanical shear force is sufficient to induce secondary structure interconversion. Cooling the sample in liquid nitrogen or subjecting it to high pressure has no effect on secondary structure. Heating the sample without grinding results in equilibration of secondary structure to 50% alpha-helix/50% beta-sheet at 100 degrees C when starting from a mostly alpha-helical state. No change was observed upon heating a beta-sheet sample, perhaps due to kinetic effects and the different heating rate used in the experiments. These results are consistent with beta-sheet approximately 260 J/mol more stable than alpha-helix in solid-state PLA.     

Conformation of N-terminal HIV-1 Tat (fragment 1-9) peptide by NMR and MD simulations.

The N-terminal portion of HIV-1 Tat covering residues 1-9 is a competitive inhibitor of dipeptidyl peptidase IV (DP IV). We have used 1H NMR techniques, coupled with molecular dynamics methods, to determine the conformation of this peptide in the three diverse media: DMSO-d6, water (pH 2.7) and 40% HFA solution. The results indicate that in both DMSO-d6 and HFA the peptide has a tendency to acquire a type I beta-turn around the segment Asp5-Pro6-Asn7-IIe8. The N-terminal end is seen to be as a random coil. In water, the structure is best described as a left-handed polyproline type II (PPII) helix for the mid segment region Asp2 to Pro6. The structures obtained in this study have been compared with an earlier report on Tat (1-9).     

The role of context on alpha-helix stabilization: host-guest analysis in a mixed background peptide model.

The helix content of a series of peptides containing single substitutions of the 20 natural amino acids in a new designed host sequence, succinyl-YSEEEEKAKKAXAEEAEKKKK-NH2, has been determined using CD spectroscopy. This host is related to one previously studied, in which triple amino acid substitutions were introduced into a background of Glu-Lys blocks completely lacking alanine. The resulting free energies show that only Ala and Glu- prove to be helix stabilizing, while all other side chains are neutral or destabilizing. This agrees with results from studies of alanine-rich peptide modela, but not the previous Glu-Lys block oligomers in which Leu and Met also stabilize helix. The helix propensity scale derived from the previous block oligomers correlated well with the frequencies of occurrence of different side chains in helical sequences of proteins, whereas the values from the present series do not. The role of context in determining scales of helix propensity values is discussed, and the ability of algorithms designed to predict helix structure from sequence is compared.     

Synthetic formyl tripeptide chemoattractants: a C(alpha,alpha)-dialkylated, amphiphilic glycyl residue at position 1.

The two diastereomeric tripeptides f-(S)-HmMet-Leu-Phe-OMe and f-(R)-HmMet-Leu-Phe-OMe, analogues of the prototypical chemoattractant f-Met-Leu-Phe-OH, were synthesized in solution by classical methods and fully characterized. A conformational study was performed in solution by 1H-NMR. Concomitantly, the two peptides were tested for their ability to induce chemotaxis, superoxide anion production and lysozyme secretion from human neutrophils. The conformational and biological data are discussed with regard to the proposed model of the chemotactic receptor on neutrophils.     

Synthesis and biological activity profile of new analogues of the cyclic opioid peptide H-Tyr-c[D-Cys-Gly-Phe(pNO2)-D-Cys]-NH2 containing (S)-alpha-hydroxymethylcysteine in place of cysteine.

To examine the effect on biological activity of replacing D-Cys in the opioid peptide H-Tyr-c[D-Cys-Gly-Phe(pNO(2))-D-Cys]-NH(2) in position 2 or/and 5 with alpha-hydroxymethylcysteine (alpha-Hmc), three analogues were synthesized. These compounds exhibit agonist activity at both mu and delta receptors. However, the most active analogue, with (S)-alpha-Hmc residue in position 5, was 3360- and 2190-fold less active than the parent peptide in the GPI and MVD assays, respectively.     

Probing structural requirements of fMLP receptor: on the size of the hydrophobic pocket corresponding to residue 2 of the tripeptide.

The conformationally constrained f-L-Met-Ac(n)c-L-Phe-OMe (n = 4,9-12) tripeptides, analogues of the chemoattractant f-L-Met-L-Leu-L-Phe-OH, were synthesized in solution by classical methods and fully characterized. These compounds and the published f-L-Met-Xxx-L-Phe-OMe (Xxx = Aib and Ac(n)c where n = 3, 5-8) analogues were compared to determine the combined effect of backbone preferred conformation and side-chain bulkiness at position 2 on the relation of 3D-structure to biological activity. A conformational study of all the analogues was performed in solution by FT-IR absorption and 1H-NMR techniques. In parallel, each peptide was tested for its ability to induce chemotaxis, superoxide anion production and lysozyme secretion from human neutrophils. The biological and conformational data are discussed in relation to the proposed model of the chemotactic receptor on neutrophils, in particular of the hydrophobic pocket accommodating residue 2 of the tripeptide.     

Hybrid alpha/beta3-peptides with proteinogenic side chains. Monosubstituted analogues of the chemotactic tripeptide For-Met-Leu-Phe-OMe.

The alpha/beta3-mixed tripeptides R-CO-beta3-HMet-Leu-Phe-OMe (1a,b), R-CO-Met-beta3-HLeu-Phe-OMe (2a,b) and R-CO-Met-Leu-beta3-HPhe-OMe (3a,b) (a, R = tert-butyloxy-; b, R = H-), analogues of the potent chemoattractant For-Met-Leu-Phe-OMe, have been synthesized by classical solution methods and fully characterized. The activities of the new analogues as chemoattractants, superoxide anion producers and lysozyme releasers have been determined on human neutrophils. Whereas all of the three N-formyl derivatives are significantly less active than the parent tripeptide as chemoattractants, compound 1b has been found to be highly active as a superoxide anion producer and 3b as a lysozyme releaser. The results show that the replacement of the native Leu residue at the central position is, in each of the examined cases, the least favourable modification. The three N-Boc derivatives are, as expected, devoid of activity as agonists, but they are all good inhibitors of chemotaxis. Information on the solution conformation has been obtained by examining the involvement of the NH groups in intramolecular H-bonds using 1H NMR. The conformation of the N-Boc analogue 1a has also been determined in the crystal state by x-ray diffraction analysis. The molecule is extended at the beta3-HMet residue (phi1 = -87 degrees; theta1 = 172 degrees; psi1 = 126 degrees) and no intramolecular H-bond is present.     

Addition of side-chain interactions to 3(10)-helix/coil and alpha-helix/3(10)-helix/coil theory.

An increasing number of experimental and theoretical studies have demonstrated the importance of the 3(10)-helix/ alpha-helix/coil equilibrium for the structure and folding of peptides and proteins. One way to perturb this equilibrium is to introduce side-chain interactions that stabilize or destabilize one helix. For example, an attractive i, i + 4 interaction, present only in the alpha-helix, will favor the alpha-helix over 3(10), while an i, i + 4 repulsion will favor the 3(10)-helix over alpha. To quantify the 3(10)/alpha/coil equilibrium, it is essential to use a helix/coil theory that considers the stability of every possible conformation of a peptide. We have previously developed models for the 3(10)-helix/coil and 3(10)-helix/alpha-helix/ coil equilibria. Here we extend this work by adding i, i + 3 and i, i + 4 side-chain interaction energies to the models. The theory is based on classifying residues into alpha-helical, 3(10)-helical, or nonhelical (coil) conformations. Statistical weights are assigned to residues in a helical conformation with an associated helical hydrogen bond, a helical conformation with no hydrogen bond, an N-cap position, a C-cap position, or the reference coil conformation plus i, i + 3 and i, i + 4 side-chain interactions. This work may provide a framework for quantitatively rationalizing experimental work on isolated 3(10)-helices and mixed 3(10)-/alpha-helices and for predicting the locations and stabilities of these structures in peptides and proteins. We conclude that strong i, i + 4 side-chain interactions favor alpha-helix formation, while the 3(10)-helix population is maximized when weaker i, i + 4 side-chain interactions are present.     

Differences in the amino acid distributions of 3(10)-helices and alpha-helices.

Local determinants of 3(10)-helix stabilization have been ascertained from the analysis of the crystal structure data base. We have clustered all 5-length substructures from 51 nonhomologous proteins into classes based on the conformational similarity of their backbone dihedral angles. Several clusters, derived from 3(10)-helices and multiple-turn conformations, had strong amino acid sequence patterns not evident among alpha-helices. Aspartate occurred over twice as frequently in the N-cap position of 3(10)-helices as in the N-cap position of alpha-helices. Unlike alpha-helices, 3(10)-helices had few C-termini ending in a left-handed alpha conformation; most 3(10) C-caps adopted an extended conformation. Differences in the distribution of hydrophobic residues among 3(10)- and alpha-helices were also apparent, producing amphipathic 3(10)-helices. Local interactions that stabilize 3(10)-helices can be inferred both from the strong amino acid preferences found for these short helices, as well as from the existence of substructures in which tertiary interactions replace consensus local interactions. Because the folding and unfolding of alpha-helices have been postulated to proceed through reverse-turn and 3(10)-helix intermediates, sequence differences between 3(10)- and alpha-helices can also lend insight into factors influencing alpha-helix initiation and propagation.     

Determination of alpha-helix N1 energies after addition of N1, N2, and N3 preferences to helix/coil theory

Surveys of protein crystal structures have revealed that amino acids show unique structural preferences for the N1, N2, and N3 positions in the first turn of the alpha-helix. We have therefore extended helix-coil theory to include statistical weights for these locations. The helix content of a peptide in this model is a function of N-cap, C-cap, N1, N2, N3, C1, and helix interior (N4 to C2) preferences. The partition function for the system is calculated using a matrix incorporating the weights of the fourth residue in a hexamer of amino acids and is implemented using a FORTRAN program. We have applied the model to calculate the N1 preferences of Gln, Val, Ile, Ala, Met, Pro, Leu, Thr, Gly, Ser, and Asn, using our previous data on helix contents of peptides Ac-XAKAAAAKAAGY-CONH2. We find that Ala has the highest preference for the N1 position. Asn is the most unfavorable, destabilizing a helix at N1 by at least 1.4 kcal mol(-1) compared to Ala. The remaining amino acids all have similar preferences, 0.5 kcal mol(-1) less than Ala. Gln, Asn, and Ser, therefore, do not stabilize the helix when at N1.