Thermodynamic analysis of wild type hPin1 WW and variants thereof. (A) Monitoring thermal denaturation of hPin1 WW by far-UV CD spectroscopy: Spectra of folded wild type hPin1 WW domain (2°C, solid black line), denatured wild type hPin1 WW domain (98°C, dashed black line), intrinsically unstructured Trp11Ala hPin1 WW domain (2°C, solid green line) and partially folded Pro37Ala hPin1 WW domain (2°C, solid red line) are shown. (B) Raw thermal denaturation data from wild type hPin1 WW domain (filled black symbols), and the intrinsically unstructured Trp11Ala (filled green symbols) and the partially folded Phe37Ala (filled red symbols) hPin1 WW variants. The hPin1 WW domain concentration in (A) and (B) was ∼15 μM. Measurements were performed in 20 mM sodium phosphate, pH 7.0 using a 2 mm quartz cuvette. The solid line connecting the black symbols is a fit of the data to a two-state model [Eqs. and in Materials and Methods]. The solid lines connecting the red and green symbols are simply guides to the eye and have no theoretical foundation. (C) Representative normalized equilibrium thermal denaturation curves [Eq. , Materials and Methods] of hPin 1 WW variants. Shown are data for Y23A-, L7A-, T29A-, P8A-, N30A-, and wild type hPin1 WW domains, in increasing order of stability. (D) Plot of the folding free energy (ΔG, in kJ/mol) at 40°C versus the midpoint of unfolding (T_M) for the hPin1 WW point variants listed in Table . For clarity, the data point obtained for wildtype hPin1 WW is color coded red.

Quantitative analysis of the complete Ala- and partial Gly-scans of the hPin WW domain. (A) Effects of the Ala mutations on the equilibrium stability of hPin1 WW. A positive value indicates that the Ala mutation is destabilizing relative to the wild type hPin1 WW domain. Mutations that are destabilizing by more than 4 kJ/mol are labeled (single letter code). Mutations of residues Leu7, Arg14, Tyr23, Phe25, and Thr29 result in destabilized variants that are only fully folded at low temperature (2°C) (blue bars). Accurate free energies cannot be reported for Ala-mutations at residues Trp11, Tyr24, Asn26, and Pro37, as the corresponding Ala-mutations resulted in partially or fully denatured WW domains, even at 2°C. The energies reported therefore are lower estimates (red bars). Residues that contribute to β-strands 1–3 are indicated. (B) Effects of Gly substitutions at 13 positions on the equilibrium stability of hPin1 WW. A positive value indicates that the Gly mutation is destabilizing relative to the wild type protein. Mutations that are destabilizing by more than 2 kJ/mol, as well as mutations that are more stable than wild type, are labeled (single letter code). Mutations of residues Thr29 and Ala31 result in destabilized variants that are only fully folded at low temperature (2°C) (blue bars). Residues that contribute to the loop 1 and loop 2 substructures are indicated. (C) Structural depiction of the hPin1 WW domain. The side chains of residues Trp11, Tyr24, and Pro37 that constitute the hydrophobic stability minicore are shown explicitly in ball-and-stick mode and are color-coded red. (Leu7, colored blue, bottom right, also is part of this stability minicore.) The side chains of residues Arg14, Tyr23, and Phe25 that contribute to the second hydrophobic core involved in ligand binding are shown explicitly in ball-and-stick mode and are color-coded blue (top left). (D) Structure of 3,4-dehydroproline (Δ_3,4P).