Noncharged amino acid residues at the solvent-exposed positions in the middle and at the C terminus of the alpha-helix have the same helical propensity

Abstract

It was established previously that helical propensities of different amino acid residues in the middle of alpha-helix in peptides and in proteins are very similar. The statistical analysis of the protein helices from the known three-dimensional structures shows no difference in the frequency of noncharged residues in the middle and at the C terminus. Yet, experimental studies show distinctive differences for the helical propensities of noncharged residues in the middle and in the C terminus in model peptides. Is this a general effect, and is it applicable to protein helices or is it specific to the model alanine-based peptides? To answer this question, the effects of substitutions at positions 28 (middle residue) and 32 (C2 position at the C terminus) of the alpha-helix of ubiquitin on the stability of this protein are measured by using differential scanning calorimetry. The two data sets produce similar values for intrinsic helix propensity, leading to a conclusion that noncharged amino acid residues at the solvent-exposed positions in the middle and at the C terminus of the alpha-helix have the same helical propensity. This conclusion is further supported with an excellent correlation between the helix propensity scale obtained for the two positions in ubiquitin with the experimental helix propensity scale established previously and with the statistical distribution of the residues in protein helices.

Figure 1.

Representation of the three-dimensional structure of the ubiquitin molecule, showing the location of the position 28 (middle of α-helix) and position 32 (C2 position at the C terminus of the α-helix).

Figure 2.

Temperature dependence of the enthalpy of unfolding for the studied ubiquitin variants. (A) (Squares) WT, (open circles) all but G32 substitutions in WT^AAA background, and (filled circles) G32 substitution in WT^AAA background. (B) (Squares) WT^#, (filled circles) position 28 substitutions in the WT^#4A background, and (open circles) position 28 substitutions in the WT^#4AV5A background. Solid lines represent the linear fit with the slope that represents the heat capacity change upon unfolding, ΔC_p = 3.2 ± 0.3 kJ/(K•mole); dashed lines show errors at the 95% confidence.

Figure 3.

Correlation between experimentally measured propensities of noncharged residues in the C2 position at the C terminus of the α-helix of ubiquitin, ΔΔG(X32). (A) With the propensity in the middle of the α-helix of ubiquitin, ΔΔG(X28) in two different backgrounds WT^4A (triangles) and WT^4AV5A (circles). (B) With the experimentally derived thermodynamic propensity scale of Pace and Scholtz (1998), ΔΔG(P&S). (C) With the propensity of residues at the C2 position of α-helices derived from the statistical analysis of Penel et al. (1999b), ΔΔG(PHD). The solid lines show linear fits, and the dashed lines represent the perfect correlation with the slope of one. The calculated correlation coefficients and the slopes are, respectively, 0.95 and 0.99 for (A), 0.93 and 1.05 for (B), and 0.98 and 1.05 for (C).

Figure 4.

Correlation between experimentally measured propensities of noncharged residues in the C2 position at the C terminus of the α-helix of ubiquitin, ΔΔG(X32), with the experimentally derived thermodynamic propensity scale at the three C-terminal residues of alanine-based peptide by Petukhov et al (2002): position C3 (A), position C2 (B), and position C1 (C). The solid lines show linear fits, and the dashed lines represent the perfect correlation with the slope of one. The calculated correlation coefficients and the slopes are, respectively, 0.82 and 0.43 for (A), 0.45 and 0.91 for (B), and 0.29 and 0.65 for (C).