The α-Helix as the Simplest Protein Model: Helix — Coil Theory, Stability, and Design

ANDREW JAMES DOIG

Faculty of Life Sciences, The University of Manchester, Jackson's Mill, PO Box 88, Sackville Street, Manchester M60 1QD, UK

1.1 Introduction

Proteins are built of regular local folds of the polypeptide chain called secondary structure. α-Helices are present in nearly all globular proteins, with ≈ 30% of residues found in α-helices. It is such ubiquity and its structural simplicity that makes the α-helix an ideal candidate for detailed quantitative studies of the complex energetic factors involved in protein folding and stability. Here, we discuss structural features of the helix and their contributions to helix stability from studies in peptides. Some earlier reviews in this field are references 2–10.

1.2 Structure of the α-Helix

A helix combines a linear translation with an orthogonal circular rotation. In the α-helix the linear translation is a rise of 5.4 Å per turn of the helix and a circular rotation is 3.6 residues per turn. Side chains spaced i,i + 3, i,i + 4, and i,i + 7 are therefore close in space and interactions between them can affect helix stability. Spacings of i,i + 2, i,i + 5, and i,i + 6 place the side chain pairs on opposite faces of the helix avoiding any interaction. The helix is primarily stabilized by i,i + 4 hydrogen bonds between backbone amide groups.

The conformation of a polypeptide can be described by the backbone dihedral angles Φ and ψ. Most Φ, ψ combinations are sterically excluded, leaving only the broad β region and narrower α region. The residues at the N-terminus of the α-helix are called N'-N-cap-N1-N2-N3-N4 etc., where the N-cap is the residue with non-helical Φ, ψ angles immediately preceding the N-terminus of an α-helix and N1 is the first residue with helical Φ, ψ angles. The C-terminal residues are similarly called C4-C3-C2-C1-C-cap-C etc. The N1, N2, N3, C1, C2, and C3 residues are unique because their amide groups participate in i,i + 4 backbone–backbone hydrogen bonds using either only their CO (at the N-terminus) or NH (at the C-terminus) groups. The need for these groups to form hydrogen bonds has powerful effects on helix structure and stability.

1.2.1 Capping Motifs

The amide NH groups at the helix N-terminus are satisfied predominantly by side-chain H-bond acceptors. In contrast, carbonyl CO groups at the C-terminus are satisfied primarily by backbone NH groups from the sequence following the helix. The presence of such interactions would therefore stabilize helices. These interactions can be identified as specific patterns found at or near the ends of helices and are generally termed capping motifs.

A common pattern of capping at the helix N-terminus is the capping box. Here, the side chain of the N-cap forms a hydrogen bond with the backbone of N3 and, reciprocally, the side chain of N3 forms a hydrogen bond with the backbone of the N-cap. The definition of the capping box was expanded by Seale et al. to include an associated hydrophobic interaction between residues N' and N4 and is also known as a 'hydrophobic staple'. A variant of the capping box motif is termed the "big" box with an observed hydrophobic interaction between non-polar side-chain groups in residues N4 and N" (not N'). The Pro-box motif involves three hydrophobic residues and a Pro residue at the N-cap.

The two primary capping motifs found at helix C-termini are the Schellman and the al motifs. The Schellman motif is defined by a doubly hydrogen-bonded pattern between backbone partners, consisting of hydrogen bonds between the amide NH at C" and the carbonyl CO at C3 and between the amide NH at C' and the carbonyl CO at C2, respectively. The associated hydrophobic interaction is between C3 and C". In a Schellman motif, polar residues are highly favoured at the C1 position and the C' residue is typically glycine. If C" is polar, the alternative αL motif is observed, defined by a hydrogen bond between the amide NH at C' and the carbonyl CO at C3. As in the Schellman motif, the C' residue is typically glycine, which adopts a positive value of Φ. However, the hydrophobic interaction in an αL is heterogeneous, occurring between C3 and any of several residues external to the helix (C3', C4', or C5').

A notable difference between the N- and C-terminal motifs is that at the N-terminus, helix geometry favors side-chain-to-backbone hydrogen bonding and selects for compatible polar residues. Accordingly, the N-terminus promotes selectivity in all polar positions, especially N-cap and N3 in the capping box. In contrast, at the C-terminus, side-chain-to-backbone hydrogen bonding is disfavored. Backbone hydrogen bonds are satisfied instead by post-helical backbone groups. The C-terminus need only select for C' residues that can adopt positive values of the backbone dihedral angle Φ, most notably Gly.

1.2.2 Metal Binding

One way to stabilize helix conformations, especially in short peptides, is to introduce an artificial nucleation site composed of a few residues fixed in a helical conformation. For example, the calcium-binding loop from EF-hand proteins saturated with a lanthanide ion promotes a rigid short helical conformation at its C-terminus region. This system has been used to measure enthalpic terms contributing to helical preferences of the amino acids. In the presence of Cd ions, a synthetic peptide containing Cys-His ligands i,i + 4 apart at the C-terminal region increased helicity (that is the average probability of finding dihedral angle pairs in values typical of α-helix) from 54% to 90%. The helicity of a similar peptide containing...