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Effect of the N2 residue on the stability of the -helix for all 20 amino acids

Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, Manchester M60 1QD, UK

Reprint requests to: Dr. Andrew J. Doig, Department of Biomolecular Sciences, The University of Science and Technology in Manchester, P.O. Box 88, Manchester M60 1QD, UK; e-mail: Andrew.Doig{at}umist.ac.uk; fax: 44-161-236-0409.

(RECEIVED November 27, 2000; FINAL REVISION March 9, 2001; ACCEPTED April 5, 2001)

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1101/

	Abstract

TOP Abstract Introduction Results and Discussion Conclusion Materials and methods References

 
N2 is the second position in the -helix. All 20 amino acids were placed in the N2 position of a synthetic helical peptide (CH₃CO-[AXAAAAKAAAAKAAGY]-NH₂) and the helix content was measured by circular dichroism spectroscopy at 273K. The dependence of peptide helicity on N2 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 N2 energy (n2). The rank order of G_{(relative to Ala)} is Glu^-, Asp^- > Ala > Glu⁰, Leu, Val, Gln, Thr, Ile, Ser, Met, Asp⁰, His⁰, Arg, Cys, Lys, Phe > Asn, > Gly, His⁺, Pro, Tyr. The results correlate very well with N2 propensities in proteins, moderately well with N1 and helix interior preferences, and not at all with N-cap preferences. The strongest energetic effects result from interactions with the helix dipole, which favors negative charges at the helix N terminus. Hydrogen bonds to side chains at N2, such as Gln, Ser, and Thr, are weak, despite occurring frequently in protein crystal structures, in contrast to the N-cap position. This is because N-cap hydrogen bonds are close to linear, whereas N2 hydrogen bonds have poor geometry. These results can be used to modify protein stability rationally, help design helices, and improve prediction of helix location and stability.

Keywords: -Helix; N2 position; circular dichroism; protein folding; protein stability; helix propensities; helix-coil theory; macrodipole

	Introduction

TOP Abstract Introduction Results and Discussion Conclusion Materials and methods References

 
The -helix is the most frequently observed secondary structural element 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 because 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 that are local in sequence, such as to those preceding N-cap side chains. In native proteins, Ala, Asp, Gln, and Glu have the highest propensity for the N2 position. Gln, Glu, Asp, Asn, Ser, Thr, and His often form i,i or i,i + 1 hydrogen bonds to the N2 NH group, though these bonds may be weak because they are nonlinear, particularly for the shorter side chains (Penel et al. 1999; Wan and Milner-White 1999). Similar bonds are found at N1 and N3, but are much more frequent at N2 and are almost unknown at helix interior positions (N4 onward). 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). Helix termini are highly solvent exposed, and tertiary interactions are rare; therefore the environments of peptide and protein helices are similar. The unique structural trends for N2 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 by 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 of the factors contributing to this equilibrium need to be assessed thoroughly. These factors include the helix-forming tendencies of constituent amino acids, capping preferences at the C and N termini, and preferences for the N1, N2, and N3 positions. Our helix-coil model N1N2N3 (Sun et al. 2000) considers the stability of every possible conformation by taking all of these factors into account. 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 important to helix stability, though are not present in the peptides studied here.

We recently made a study of the energetics of the N1 helix position for all 20 amino-acid residues (Cochran et al. 2001). Previous studies have measured position-dependent effects on the 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 N2 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.

	Results and Discussion

TOP Abstract Introduction Results and Discussion Conclusion Materials and methods References

 
Design of the N2 peptide sequence
In this study an intrinsically helical peptide was used to measure the energetic contribution (the n2 parameter) of all 20 amino acids in the N2 position in the same way that the n1 parameter has recently been investigated (Cochran et al. 2001). In our N1 study, we used an AQ-based peptide that was neutrally charged. However, when the homologous peptide CH₃CO-[AAAAAAQAAAAQAAAAQGY]-NH₂ was synthesized to study the N2 position, it tended to aggregate. The peptide CH₃CO-[AXAAAAKAAAAKAAGY]-NH₂ was therefore used instead so that the energetics of the N2 residue could be studied in relative isolation from neighboring charges; that is, the N terminus is acetylated and the positively charged lysine residues included for solubility are spaced i,i + 5 to each other and the substitution site. The acetyl group is a strong N-cap and this means that the second residue (X) will have a high probability of being at the N2 position. Ala is a poor N-cap; thus conformations that initiate with the first Ala at N-cap, and hence X at N1, will have low populations. The helix content is therefore largely sensing the N2 preference of X.

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. The stability of the substituted peptides is readily evaluated by measurements of circular dichroism (CD) intensity at 222 nm, which is proportional to the average helicity of the peptide in solution. It should be noted that interactions between distant side chains are possible in unfolded states and are not considered in the model.

Helix contents of N2 peptides
The helix content of each peptide was measured by CD at 222 nm at a concentration of 60 µM in 10 mM NaCl and 5 mM sodium phosphate (pH 7.01) at 273K. Low-temperature measurements are made because signal intensities are larger, and helix and coil parameters are known best at this temperature. 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 N2(Ala) and N2(Leu) were invariant between 23 and 116 µM and 31 and 160 µM, respectively. 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). N2(Cys) was measured in the presence of 1 mM dithiothreitol to keep it reduced. The percentage helicities for the series of homologous peptides are shown in Table 1 and range from 65% for Glu and Ala to 30% for His⁺. This large range indicates that the N2 residue plays a significant role in the overall stability of the peptide. However, the equilibrium that exists between the multitude of partially helical states must be analyzed by using helix-coil theory. This makes it possible to separate all of the possible effects that can arise from making the substitution because X is at the N-cap, N1, or N2 positions or in a coil state in different conformations. The contribution of the helix dipole is dealt with implicitly by helix-coil theory because its effect is subsumed within the n2 parameter.

Determination of n2 values and G from helix contents

Comparison with other reported n2 values
Free energies for N2 have been reported for a series of AR peptides (Petukhov et al. 1998, 1999) and are included in Table 1 for comparison. The free energies measured here are in excellent agreement with those of Petukhov et al. for Leu, Thr, Ile, and Ser, with the values for Val, Gln, and Met probably equivalent within experimental error. This agreement gives confidence in both sets of results because the peptide sequence and computational methods of Petukhov et al. are significantly different from ours. The only significant difference in results is for the Asn N2 energy. This difference is likely to result from the different Asn N-cap preferences used in the two models. Lacroix et al. (1998) found evidence for a position dependence of Lys at the helix N terminus, although this evidence was not quantified. No other G(N2) values have been reported.

pH titrations
The helicities of N2(Asp), N2(Glu), and N2(His) peptides were titrated as a function of pH. Solutions of peptide at 60 µM were equilibrated at 273K in 10 mM NaCl, 1 mM sodium phosphate, 1 mM sodium borate, and 1 mM sodium citrate. The CD signal at 222 nm was monitored after pH adjustments were made by the addition of either NaOH or HCl. pK_a values of the ionizable side-chain groups were evaluated between approximately pH 2 and pH 9 by curve fitting to the Henderson-Hasselbach equation (Fig. 1A–C).

View larger version (36K): [in this window] [in a new window]   Fig. 1. pH titrations for N2 peptides. (A) N2 Asp. (B) N2 Glu. (C) N2 His.

The helicity of N2(His) is also pH dependent (Fig. 1C). The essentially uncharged N2(His) at pH 9 is 21% more helical than the fully protonated species at pH 2. The His⁺ side chain greatly destabilizes the helix, possibly as a result of repulsion with the helix macrodipole. The pK_a for His at N2 is 6.74 ± 0.04, which is comparable to values commonly cited in the range 6.5 and 7.1 (Kyte 1995). These unperturbed pK_a values for residues at N2 indicate that the side chains are not involved in any strong interactions, yet the helix content is strongly influenced by the ionization state. These values may also be perturbed slightly by the Lys side chains.

Glu at N2 has previously been studied in an N-terminally acetylated AQ peptide (Scholtz et al. 1993). The peptide with Glu⁰ at N2 was found to be less helical compared with Ala when uncharged but the same when charged at pH 7. The effect of Glu^- was neutralized in 2.5 M NaCl. This charge-screening effect is indicative of a charge–dipole interaction. Similar results were obtained with Asp at N2 (Huyghues-Despointes et al. 1993). 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 pK_a of His at N2 in an Ac-(AQH) peptide is 6.6–6.8 and increases as the His is moved farther from the N terminus. This was again interpreted as a charge–dipole effect.

For the majority of residues, n2 > 1, indicating that most residues are more stable at the N2 position than at the helix interior. A possible explanation for this general trend is that side chains will have a greater conformational freedom at N2 than at interior positions and therefore will be favored entropically (Petukhov et al. 1998, 1999). Comparisons of rotamer populations at N2 (Penel et al. 1999) with the interior (MacGregor et al. 1987) show that a wider range of rotamers is found at N2, supporting this hypothesis. Notable exceptions to this general trend are His⁺, Lys, and Arg, whose positive charges interact unfavorably with the helix dipole at N2 and hence have n2 < 1.

Correlation of N2 energies with N2 propensities
Figure 2 shows the correlation between the N2 energies measured here and the N2 amino-acid propensities found by surveying helices in proteins (Penel et al. 1999). The correlation is excellent, showing that the environments in peptide and protein helices are similar and confirming that using peptides is a valid way to analyze helices in proteins. Note that the statistical propensities observed in proteins are not free energies, although they correlate with them. Surveying protein structures is thus no substitute for experimental measurements of free energies from amino-acid substitutions, whether by peptide synthesis or site-directed mutagenesis in proteins.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Correlation between N2 propensity and N2 statistical weight (n2w).

View larger version (11K): [in this window] [in a new window]   Fig. 3. Correlation between N-cap statistical weight (n) and N2 statistical weight (n2w).

View larger version (11K): [in this window] [in a new window]   Fig. 4 . Correlation between N1 statistical weight (n1) and N2 statistical weight (n2w).

View larger version (10K): [in this window] [in a new window]   Fig. 5. Correlation between helix interior statistical weight (w) and N2 statistical weight (n2w).

	Conclusion

TOP Abstract Introduction Results and Discussion Conclusion Materials and methods References

 
The substitution of all 20 amino acids at the N2 position in an AK peptide has yielded the range of intrinsic preferences for residues at this position in the -helix. We suggest that this information can be used to modify protein stability rationally. The difference in stability between the charged and uncharged basic and acidic side chains shows the effect of placing charged groups proximal to the peptide N terminus via interactions with the helix dipole.

	Materials and methods

TOP Abstract Introduction Results and Discussion Conclusion Materials and methods References

 
Peptide synthesis
Peptides were synthesized by the solid-phase method. C-terminal amides were made by using Rink Amide resin (CN Biosciences). N termini were acetylated with 4% (v/v) acetic anhydride/ 5% (v/v) pyridinein dimethylformamide for 30 min at room temperature. Peptides were cleaved from the resin by using 95% trifluoroacetic acid, 2.5% triisopropylsilane, and 2.5% water. Peptides were purified by C₁₈ reverse-phase high-pressure liquid chromatography (HPLC) and their molecular weights were checked by electrospray or matrix-assisted laser desorption ionization mass spectrometry. Final purity was checked by analytical C₁₈ HPLC. Solutions of peptides containing Cys were maintained in a reduced state with 1 mM dithiothreitol.

Peptide concentration was determined by measuring tyrosine ultraviolet absorbance of aliquots of stock solution dissolved in water or 6 M guanidine hydrochloride by 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) at 298K by using ₂₈₁ (Trp) = 5690 M^-1 • cm^-1 + ₂₈₁ (Tyr) = 1250 M^-1 • cm^-1 (Edelhoch 1967).

CD measurements
Equilibrium CD measurements were made at 222 nm with a Jasco J810 spectropolarimeter at 273K. Ellipticity was measured in 10 mM NaCl and 5 mM sodium phosphate (pH 7.0) in a quartz cell with a 0.1-cm path length. The Cys peptide was measured in the presence of 1 mM dithiothreitol. CD data in millidegrees was converted to mean residue ellipticity by using []₂₂₂ = /(molar concentration x 15 residues), in deg•cm²•dmole^-1. Helix content was calculated as 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 273K. 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 273K. 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:

[]_{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. The free energies for protonation were calculated by using .

Determination of helix-coil parameters
n2-values were determined by using a statistical mechanics 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 as follows: 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 and n3 values were set to 1; other parameters were taken from Rohl et al. (1996) or Cochran et al. (2001). Parameters were varied until they converged on essentially unchanging values.

	Acknowledgments

 
We thank the Michael Barber Mass Spectrometry Facility at UMIST for verifying peptide identity. This work was supported by the BBSRC (Grant 36/B09794). We thank Kate Wickson for synthesis and analysis of the CH₃CO-[AAAAAAQAAAAQAAAAQGY]-NH₂ peptides.

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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