Effect of the N3 residue on the stability of the α‐helix

Abstract

N3 is the third position from the N terminus in the ␣-helix with helical backbone dihedral angles. All 20 amino acids have been placed in the N3 position of a synthetic helical peptide (CH 3 CO-[AAX AAAAKAAAAKAGY]-NH 2 ) 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; macrodipoleThe ␣-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 backbonebackbone 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 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 st...

Contributions of Conserved TPLH Tetrapeptides to the Conformational Stability of Ankyrin Repeat Proteins

“…As reported previously19, Ala has the highest intrinsic preference for the interior of the helix, and a neutral His residue has the lowest helix propensity at N3 position. Thus a helical His to Ala mutation does not necessarily reduce the stability in terms of the secondary conformation.…”

Extrinsic Interactions Dominate Helical Propensity in Coupled Binding and Folding of the Lactose Repressor Protein Hinge Helix

“…Note that V52A, V52F, V52H, V52L, V52S, and V52W show high affinity O sym binding in the presence of a saturating inducer. Correlation between DNA binding affinities (O 1 -squares and O sym -bowties) of V52 mutants and N3 helical propensity order (top) (69)(70)(71)(72)(73), hydropathy (middle) (75), and molar volume (bottom) (74). For helical propensity versus K d , a trend is noted with a line but has many obvious outliers.…”

The cell cycle regulator GpsB functions as cytosolic adaptor for multiple cell wall enzymes

“…The Ser7Ala and Ala10Pro mutations each reduced the affinity for Bs GpsB 1–68 by at least 6-fold ( Figure 1D, Supplementary Figure 1C, Supplementary Table 1 ) by affecting the α-helix of Bs PBP1 1–32 . Ser7 acts as the helix N-cap, a role that can also be performed by Asn and Thr 31 , and substitutions equivalent in helical positions to Ser7Ala and Ala10Pro destabilise model peptides 31,32 . Finally, the importance of PBP1 Ser7, Arg8 and Arg11 to GpsB binding is highlighted because these are the most well conserved residues in an alignment of the cytoplasmic mini-domains of Bacillaceae PBP1 PG synthases ( Supplementary Figure 1D ).…”