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-helix
1 Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology (UMIST), Manchester M60 1QD, UK
Reprint request to: Andrew J. Doig, Department of Biomolecular Sciences, UMIST, P.O. Box 88, Manchester M60 1QD, UK; e-mail: Andrew.Doig{at}umist.ac.uk; fax: 44-161-236-0409.
(RECEIVED July 30, 2003; FINAL REVISION September 16, 2003; ACCEPTED September 23, 2003)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03341804.
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Abstract |
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-helix with helical backbone dihedral angles. All 20 amino acids have been placed in the N3 position of a synthetic helical peptide (CH3CO-[AAX AAAAKAAAAKAGY]-NH2) and the helix content measured by circular dichroism spectroscopy at 273 K. The dependence of peptide helicity on N3 residue identity has been used to determine a free energy scale by analysis with a modified Lifson-Roig helix coil theory that includes a parameter for the N3 energy (n3). The most stabilizing residues at N3 in rank order are Ala, Glu, Met/Ile, Leu, Lys, Ser, Gln, Thr, Tyr, Phe, Asp, His, and Trp. Free energies for the most destabilizing residues (Cys, Gly, Asn, Arg, and Pro) could not be fitted. The results correlate with N1, N2, and helix interior energies and not at all with N-cap preferences. This completes our work on studying the structural and energetic preferences of the amino acids for the N-terminal positions of the -helix. These results can be used to rationally modify protein stability, help design helices, and improve prediction of helix location and stability.
Keywords: -helix; N3 position; circular dichroism; protein folding; protein stability; helix propensities; helix-coil theory; macrodipole
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Introduction |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsReferences |
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-helix is the most frequently observed secondary structure in proteins. N1, N2, and N3 are the first three amino acids with helical 
, 
backbone dihedral angles at the helix N terminus. They differ from interior positions as their amide NH groups do not participate in backbone–backbone i,i+4 hydrogen bonds within the helix. The presence of these otherwise unsatisfied hydrogen bond donors has profound structural effects, most often satisfied by hydrogen bonds to side chains local in sequence, such as to preceding N-cap side chains. In native proteins, Ala, Asp, Gln, and Glu have the highest propensity for the N3 position. We have made a thorough study of the structures adopted by side chains at the N-cap, N1, N2, and N3 positions in proteins (Doig et al. 1997; Penel et al. 1999). Asn, Asp, Gln, Glu, and Thr occasionally form i,i hydrogen bonds to the N3 backbone NH group, although these bonds may be weak, as they are nonlinear, particularly for the shorter side chains (Penel et al. 1999; Wan and Milner-White 1999). A second important feature of the N3 position is the capping box, where the N3 side chain accepts a hydrogen bond from the N-cap backbone NH group (Harper and Rose 1993). This is most important for Gln or Glu at N3. Helix termini are highly solvent exposed, and tertiary interactions are rare, so the environment of peptide and protein helices are very similar. The unique structural trends for N3 imply that the free energies for substituting amino acids at this position differ from all other positions in the helix. In this work, we measure these free energies using helical peptides.
-Helices in aqueous solution adopt a large number of structures, with fully helical, fully coil, and partly helical conformations all populated. For a complete understanding of helix formation and stability, all the factors contributing to this equilibrium need to be assessed thoroughly. These include the helix-forming tendencies of constituent amino acids, capping preferences at the carboxyl and amino termini, and preferences for the N1, N2, and N3 positions. Our helix-coil model N1N2N3 (Sun et al. 2000) distinguishes between each of these by assigning them unique weights. Factors such as side-chain–main-chain hydrogen bonds, electrostatic interactions with the helix dipole, solvent exposure, and conformational entropy are implicitly included in the positional preferences. Side-chain interactions between residues spaced i,i+3 and i,i+4 are also of considerable importance to helix stability, although are not included in the N1N2N3 helix/coil model or the peptides studied here.
We recently made a study of the energetics of the N1 and N2 helix position for all 20 amino acid residues (Cochran and Doig 2001; Cochran et al. 2001). Previous studies have measured position-dependent effects on helix content of several amino acid residues (Asp [Huyghues-Despointes et al. 1993], Glu [Scholtz et al. 1993], His [Armstrong and Baldwin 1993], Val, Leu, Met, Ile, Gly, Ser, Thr, Asn, Gln [Petukhov et al. 1998, 1999]), but a complete survey of all 20 residues in the same peptide is needed. The energetic contribution of the N3 residue to helix stability in the controlled environment of a synthetic peptide is of fundamental interest, with practical implications in the development of secondary structure prediction algorithms, peptide, and protein design, and development of site-directed mutagenesis strategies.
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Results and Discussion |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsReferences |
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-helix almost always terminates at, or before, this position so that problems arising from a helical Tyr affecting the CD signal are minimized (Chakrabartty et al. 1993). The acetyl group is a strong N-cap so helix formation will often initiate at the acetyl group. Ala is a poor N-cap and a relatively good N1 and N2, so conformations that initiate with the first two Ala residues at the N-cap, and hence, X at N1 or N2 will have low populations. The third residue (X) will therefore have a high probability of being at the N3 position, increasing the sensitivity of the helix content to the N3 preference.
The stability of the substituted peptides is readily evaluated by measurements of CD intensity at 222 nm, which is proportional to the average helicity of the peptide in solution. The N3 preference can thus be found by finding the value of n3 that gives predicted helix content in agreement with the experiment. No side-chain interactions are present in this sequence. The energy scales of the interior and N-cap of Rohl et al. (1996) as well as the energy scales of N1 (Cochran et al. 2001) and N2 (Cochran and Doig 2001) are used in the calculations, leaving the N3 preference (n3 in the helix-coil theory) values as the only unknown parameter.
The N termini of naturally occurring helices in proteins are very often solvent exposed (Doig et al. 1997). Thus, the AK peptide is a good model for the study of factors that stabilize the -helix in naturally occurring proteins.
Helix contents of N3 peptides
The helix content of each peptide was measured by CD at 222 nm at a concentration of 20 µM in 10 mM NaCl, 5 mM sodium phosphate pH 7.0 at 273 K. Low-temperature measurements are made because the helix-coil equilibrium favors the helical conformation at lower temperature so signal intensities are consequently larger. Molar ellipticities for these types of simple peptides are known to be concentration independent (Padmanabhan et al. 1990; Stapley and Doig 1997) indicating that aggregation does not occur. This was confirmed by checking that the helix contents of N3(Glu) and N3(Ile) were invariant between 5–200 µM. The CD signal at 222 nm was converted to [
]222 (see Materials and Methods), and the percentage helicity calculated with ±3% uncertainty as experimental error (Table 1
). N3(Cys) was measured in the presence of 1 mM dithiotreitol to keep it reduced.
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The percentage helicities for the series of homologous peptides are shown in Table 1
, and range widely from 59% for Ala to 16% for Pro. This large range indicates that the N3 residue plays a significant role in the overall stability of the peptide. However, the equilibrium that exists between the multitudes of partially helical states must be analyzed using the helix-coil theory. This makes it possible to separate all the possible effects that can arise from making the substitution, because X is at the N-cap, N1, N2, or N3 positions or in a coil state in different conformations. The contribution of the helix dipole is dealt implicitly by the helix coil theory, as its effect is subsumed within the n3 parameter.
Determination of n3 values and 
G from helix contents
The residue n3 values are presented in rank order in Table 1. The order is different when compared with the order of helicity values. This confirms that analysis using the helix-coil theory is essential for understanding the equilibrium. For example, a high helix content in one of these peptides may arise from a strong preference for N-cap, N1, N2, or N3, leading to high populations for that residue to be at each of these positions.
The n3 values were calculated from helicity measurements as described previously for the N1 and N2 positions (Cochran et al. 2001; Cochran and Doig 2001) using the N1N2N3 helix coil algorithm (Sun et al. 2000). In these sequences the only unknown parameter is the N3 preference (n3). Initially, n3 for the four sequences with an X residue matching residues of the generic peptide (Ala, Gly, Lys, and Tyr) were solved simultaneously by fitting the equilibrium equation to the observed helicities using the N1N2N3 program (see Materials and Methods). Then, n3 for other residues were found by varying them until the calculated helix content agreed with experiment.
The statistical weight for the N3 coil to helix reaction is n3w, the product of w, the intrinsic interior preference value, and n3, the intrinsic N3 preference value (Sun et al. 2000). Free energies for transfer from the coil to N3 are thus calculated as G = -RTln(n3w) and the free energy change relative to Ala is given by G = GXaa - GAla.
n3 is an adjustment to the intrinsic helix preference (w), and thus shows how the helix preference changes on moving from the helix interior to N3. Table 1 presents the free energies of each amino acid in rank order of decreasing stability. The n3 values for several N3 peptides (C-, C0, G, H+, H0, N, P, and R) could not be evaluated as positive numbers, and have been assigned a value of zero. An approximate rank order for their N3 preferences can be seen in the helix contents of the peptides, however. It is not clear why these data could not be fitted. Possibilities include errors in previously determined parameters, interactions in nonhelical conformations and interactions involving these amino acids that extend beyond the N3 position. The n3-values for W, Y, and F are unreliable because aromatic groups perturb the CD signal (Chakrabartty et al. 1993), although this is a minor effect for Phe and Tyr (Andrew et al. 2002). This aromatic effect may well differ at N3 from the helix center, due to the differing conformations between these sites. The N-cap energy of Asp0, Cys0, Glu0, and His+ have not been measured previously. The n3 values of these protonated amino acids were therefore omitted, because this requires also knowing their N-cap preferences.
The n-cap, n1, n2, and n3 parameters represent the probabilities that a particular residue will adopt an N-cap, N1, N2, or N3 conformation, respectively. These parameters are interdependent, so the n3 values could be perturbed by errors in n, n1, or n2 values. This problem is most important when X in the sequence has a high N-cap preference, such as Asp, Asn, and Cys, as conformations with these residues at the N-cap will be more populated. When the n-cap value for an amino acid is large and of similar magnitude to the acetyl group (n-cap = 5.9; Doig et al. 1994), it will spend much of its time in a nonhelical N-cap conformation. The large n-cap values for Asp (6.6), Asn (6.8), Cys (5.4), and Tyr (4.9) tend to shift the equilibrium in favor for the residue X to be at N-cap. Thus, the helices will nucleate with X as the N-cap. In contrast, when X has a low N-cap preference, the acetyl will then be the N-cap, X will be at N3. The conformation when X is the N-cap will therefore be rarely populated and an error in n(X) will be small.
All amino acids in N3 peptides destabilize the helix relative to Ala. The free energies range from 0 kcal•mole-1 for N3(Ala) to 4 kcal•mole-1 for N3(Trp). The highest stabilizing effect of N3(Ala) is not surprising because Ala is close to the center of the sequence where the preference is high. Helix breaking residues at the third position of the peptide have a larger destabilizing effect than at N1, as they are moving toward the center of the sequence. For instance, G for N1(Pro) is 0.6 kcal•mole-1 and G N1(Gly) is 1.06 kcal•mole-1. In this study the G for these residues could not be measured as their G are very high.
The AGADIR program (Muñoz and Serrano 1994) predicts the helix contents of monomeric peptides in aqueous solution so work such as this offers a valuable blind test of its accuracy. Table 1 lists predictions of helix contents for all the peptides studied here. Errors range from 0% (Ala and Met) to 31% (Arg). The AGADIR results are all quite similar, apart from the accurate low value for Pro, in contrast to the large variation in helix contents we find when varying the N3 residue.
Comparison with other reported n3 values
Free energies for N3 for nonpolar and noncharged polar residues for a series of AR peptides have been reported (Petukhov et al. 1998, 1999), and are included in Table 1 for comparison. No other G(N3) values have been reported. The free energies measured here are somewhat higher than those of Petukhov et al. This may be due to differences in their peptide sequence and computational methods of Petukhov et al. The peptides studied here are 16 mers, with a wider helicity range, from 59% for N3(Ala) to 16% for N3(Pro), while those of Petukhov et al. range from 28% of N3(Thr) to 47% of N3(Ala). Our data may therefore give more accurate N3 energies.
pH Titrations
The helicities of N3(Asp), N3(Cys), N3(Glu), and N3(His) peptides were titrated as a function of pH. pKa values of the ionizable side chain groups were evaluated between approximately pH 2–9 by curve fitting to the Henderson-Hasselbach equation (Fig. 1A–D). The upper range of accessible pH is given by Lys deprotonation, which leads to peptide aggregation.
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G = -RTln[n3 (charged)n3(uncharged)] could therefore not be calculated. The pKa of N3(Glu) is 4.48 ± 0.11, and this value is similar to model compounds (Glu in an N-acetyl acid amide has a pKa of 4.5; Nozaki and Tanford 1967). The unperturbed pKa value for residue Glu at N3 indicates that the side chain is not involved in any strong interactions, yet the helix content is strongly influenced by the ionization state.
The pKa of N3(Asp) of 3.64 ± 0.09 is below those of other reported compounds. In comparison with the pKa value of Asp in N-acetyl acid amide (pKa 4.1; Nozaki and Tanford 1967), N3(Asp) is about 0.5 units lower. The pKa of Asp has also been measured in the coil state of CH3CO-[AADAA]-NH2 and reported as 3.91 ± 0.02 (HuyghuesDespointes et al. 1993). The significant difference between the perturbations of the pKas of Asp and Glu could be attributed to the shorter side chain of Asp, interacting more strongly with the helix dipole.
The pKa of N3(Cys) of 7.73 ± 0.12 differs from other reported pKa values (Miranda 2003). However, the pKa value of Cys in a helical peptide is significantly lower than that in a coil peptide (pKa Cys 8.69 ± 0.07; Kortemme and Creighton 1995). The increase in helix content of N3(Cys) at higher pH was not determined because titration beyond pH 8.5 in this peptide begins to deprotonate the side chain of Lys residues leading to peptide aggregation.
In general, Asp, Glu, and Cys residues are helix stabilizing at N3 when in the ionized form. The stabilizing contribution might be due to the ability of these residues to form a hydrogen bond to one of unsatisfied amide groups at the N terminus (Penel et al. 1999). Another reasonable contribution to stabilization is the electrostatic interactions of the charged side chains with the positive end of the helix dipole. These two contributions cannot be separated experimentally, however.
The helicity of N3(His) is also pH dependent (Fig. 1D). The uncharged N3(His) at pH 9 is about 9% more helical than the fully protonated species at pH 3. The His+ side chain destabilizes the helix, possibly as a result of repulsion with the positive end of the helix macrodipole and free amide groups at the N terminus. The pKa for His at N3 is 6.45 ± 0.04, which is comparable to values commonly found in the range of 6.5 and 7.1 (Kyte 1995). His–dipole interactions have also been measured (Armstrong and Baldwin 1993). His+ is destabilizing to the helix, and this effect is reduced in 1 M NaCl. The pKa of His increases as the His is moved further from the N terminus. This was again interpreted as a charge–dipole effect. The titration above pH 11 is due to Tyr deprotonation.
Correlation of N3 energies with N3 propensities
Figure 2 shows the correlation between the N3 energies measured here and the N3 amino acid propensities found by surveying helices in proteins (Penel et al. 1999). There is a weak correlation observed. Deviations from the line of best fit for Glu, Gln, and Asp show that these are more favored at N3 in proteins than in this peptide. This is due to these side chains forming capping box hydrogen bonds to the backbone NH of the N-cap (Harper and Rose 1993). In the peptides the N-cap is an acetyl that lacks this NH group so this stabilizing hydrogen bond cannot form. In principle, it is possible to design a series of peptides that could form this capping box bond to measure its contribution to helix formation. In practice, however, this is difficult. If the peptides had Gly at their N terminus, they could be studied at high pH, where the N terminus is H2N-CH2—. These peptides would have low helix contents, particularly at their N termini, as Gly is a poor N-cap, making N3 energies inaccurate. They would also need to be studied at high pH where the N-terminal amine is neutral. At high pH the peptides are likely to be insoluble, as Lys side chains would lose their positive charges. In addition, the titration of Tyr would interfere with helix content measurements.
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Comparison of N3 preferences with N-cap, N1, N2, and helix interior preferences
Figure 3 shows a plot of the N-cap preference (Rohl et al. 1996) against the N3 preference (n3w). There is no correlation, showing that the amino acid preferences for the N-cap position are completely different to N3. Although both are being at the helix N terminus, they adopt different conformations as N-cap and N3 is in nonhelical and helical conformations, respectively.
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and 5 compare the N1 and N2 preferences (Cochran et al. 2001; Cochran and Doig 2001) to the N3 preference, respectively. There is a reasonable correlation showing that the forces at both sites are similar. The notable deviations from the linear correlation are Asp- and Glu- in both comparisons of which have higher N1 and N2 preferences than N3, respectively. A possible reason for this is that Asp- and Glu- at N1 and N2 can form i,i+1 hydrogen bonds with the adjacent backbone amide group (Penel et al. 1999). This is not possible when they are at N3 because the amide group at N4 is occupied hydrogen bonding to the N-cap C=O.
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compares preferences at N3 and helix interior positions. There is a good correlation showing that the N3 and interior environments are similar. Deviations from a linear correlation shown by Arg+ and Lys+ can be attributed to charge effects where both residues are disfavored at N3 compared to the helix interior. In general, residues with the ability to hydrogen bond with free amide groups at the helix N terminus (e.g., Asp0, Glu, Ser, and Thr) are more stable at N3 than at helix interior.
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-helix. The substitution of amino acids at the N3 position in an AK peptide has shown the unique structural trends for N3 and yielded the range of intrinsic preferences for residues at this position in the -helix. Although the preferences at N3 correlate well to those at the helix interior, the N3 position favors negatively charged residues due to electrostatic interactions with helix dipole. We suggest that this information can be used to rationally modify protein stability, particularly as helix N termini are solvent-exposed and very rarely form tertiary interactions. |
Materials and methods |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsReferences |
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Peptide concentration was determined by measuring tyrosine UV absorbance of aliquots of stock solution dissolved in water or 6 M guanidine hydrochloride using 
275 = 1390 M-1 • cm-1 or 275 = 1450 M-1 • cm-1, respectively (Brandts and Kaplan 1973). Concentrations of peptides containing Trp were determined in 6M GuHCl, pH 7.0, 298 K using 281 (Trp) = 5690 M-1 • cm-1 + 281 (Tyr) = 1250 M-1 • cm-1 (Edelhoch 1967).
Circular dichroism measurements
Equilibrium CD measurements were made at 222 nm with a Jasco J810 spectropolarimeter at 273 K. Ellipticity was measured in 10 mM NaCl, 5 mM sodium phosphate pH 7.0 in a quartz cell with a 0.5-cm path length. The Cys peptide was measured in the presence of 1 mM dithiothreitol. CD data in mdeg were converted to mean residue ellipticity using []222 = /(Molar conc. x 16 residues x 10), in deg•cm2•dmole-1. Helix content was calculated as []222(observed)/[]222(max). []222(max) is given by -40,000*(1 - 2.5/ n), where n is the number of amino acids in the peptide (Chen et al. 1974).
pH titrations were performed in a 3-mL 1.0-cm path length quartz cell at 273 K. The buffer used for pH titrations contained 10 mM NaCl, 1 mM sodium phosphate, 1 mM sodium borate, and 1 mM sodium citrate. The pH meter was calibrated for measurements at 273 K. The pH was adjusted during the titrations with aliquots of HCl or NaOH. Data were only used in the pH range where the equilibrium is reversible. The ellipticity data from the titrations were fitted to a Henderson-Hasselbach equation:
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[]222 high_pH, and []222 low_pH, are the molar ellipticities measured at 222 nm at the titration end points at high and low pH.
Determination of helix-coil parameters
n3-Values were determined using a statistical mechanical algorithm implemented in the program N1N2N3 (http://www.bi.umist.ac.uk/users/mjfajdg/n1n2n3.htm). N1N2N3 implements the modified Lifson-Roig helix-coil theory (Sun et al. 2000) to calculate the helix content of a given peptide sequence. The inputs to the program are the peptide sequences and corresponding experimental helix contents; a library of w, n-cap, n1, n2, n3, c1, and c-cap values for each amino acid; an n-cap value for acetyl and a c-cap value for amide; and a list of parameters to be determined. All c-values were set to 1; other parameters were taken from Rohl et al. (1996), Cochran et al. (2001), or Cochran and Doig (2001). Parameters were varied until they converged on essentially unchanging values.
Results using the AGADIR program (Muñoz and Serrano 1994), which predicts the helix content of any peptide, were obtained from the Web site http://www.embl-heidelberg.de/cgi/agadir-wrapper.pl.
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Acknowledgments |
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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References |
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TOPAbstractIntroductionResults and DiscussionMaterials and methodsReferences |
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