Modulating repeat protein stability: the effect of individual helix stability on the collective behavior of the ensemble

Abstract

Repeat proteins are tandem arrays of a small structural motif, in which tertiary structure is stabilized by interactions within a repeat and between neighboring repeats. Several studies have shown that this modular structure is manifest in modular thermodynamic properties. Specifically, the global stability of a repeat protein can be described by simple linear models, considering only two parameters: the stability of the individual repeated units (H) and the coupling interaction between the units (J). If the repeat units are identical, single values of H and J, together with the number of repeated units, is sufficient to completely describe the thermodynamic behavior of any protein within a series. In this work, we demonstrate how the global stability of a repeat protein can be changed, in a predictable fashion, by modifying only the H parameter. Taking a previously characterized series of consensus tetratricopeptide repeats (TPR) (CTPRa) proteins, we introduced mutations into the basic repeating unit, such that the stability of the individual repeat unit was increased, but its interaction with neighboring units was unchanged. In other words, we increased H but kept J constant. We demonstrated that the denaturation curves for a series of such repeat proteins can be fit and additional curves can be predicted by the one-dimensional Ising model in which only H has changed from the original fit for the CTPRa series. Our results show that we can significantly increase the stability of a repeat protein by rationally increasing the stability of the units (H), whereas the interaction between repeats (J) remains unchanged.

Figure 1

(A) A schematic of the CTPRa unit sequences indicating the amino acid positions in a TPR repeat and nomenclature of the helices. The residue identity at each position corresponds to the TPR consensus sequence (CTPRa)., The residues that are mutated in this work are highlighted and the identity of the mutated residues is shown in the lower row (G8A in yellow; E19K in red; and Y23A in orange). The i – i + 3 and i − i + 4 charge-charge interaction network introduced by the E19K mutation is shown below being the negatively charged residues colored in red and the positively charged colored in blue. (B) Ribbon representation of the structure of the parent protein, CTPRa3 (PDB ID: 1na0) depicting in space filling representation the three residues that were mutated (G8 colored in yellow, E19 colored in red, and Y23 colored in orange). The structure on the right is rotated 180° around the vertical axis relative to the structure on the left.

Figure 2

Thermodynamic stability of CTPRa2 variants. Chemical (A) and thermal (B) denaturation curves of CTPRa2 variants: CTPRa2 (filled circles), CTPRa2-G8A (empty squares), CTPRa2-Y23A (empty triangles), and CTPRa2-E19K (empty circles). The chemical denaturation studies (A) were performed by titrating a solution at 3 μM protein concentration in 50 mM phosphate pH 6.5, 150 mM NaCl and 7M GuHCl into an identical protein solution with 0M GuHCl. Thermal denaturation (B) was monitored at 12 μM protein concentration following the ellipticity at 222 nm from 15 to 95°C in 1°C steps. Only the “ramp-up” curves are displayed in the figure. The “ramp-down” curves were recorded and all the thermal denaturation curves displayed 85 to 95% reversibility (data not shown).

Figure 3

Thermodynamic stability of the parent and two redesigned CTPRa2, 3, 4 series and the fit to the 1D Ising model. The fraction unfolded (monitored by CD) is plotted versus the concentration of GuHCl for CTPRa2, CTPRa3 and CTPRa4 (dark blue); CTPRa2-E19K, CTPRa3-E19K and CTPRa4-E19K (magenta); and CTPRa2-Y23A, CTPRa3-Y23A and CTPRa4-Y23A (cyan) are shown in squares, crosses, or triangles for proteins in each series with two, three, or four repeats. The best fit of the data to the Ising model is shown in solid lines of corresponding color. The 1D-Ising model parameters for the fit are: J = 2.3 is constant value for all the curves in the plot, x_c = 3.79 ± 0.01 and m₁ = 0.37 ± 0.01 for CTPRa series, x_c = 6.14 ± 0.01 and m₁ = 0.28 ± 0.01 for Y23A series, and x_c = 4.61 ± 0.02 and m₁ = 0.42 ± 0.01 for E19K series. The goodness-of-fit parameter χ² obtained was 0.0005924 for CTPRa series, 0.0003965 for CTPRa-Y23A series, and 0.0003224 for CTPRa-E19K series. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]