Thermodynamics of the Protonation Equilibria of Two Fragments of N-Terminal β-Hairpin of FPB28 WW Domain

Abstract

The pK(a) values of two peptides derived from the formin-binding protein 28 WW domain [Ac-Lys-Thr-Ala-Asp-Gly-Lys-Thr-NH(2) (D7), Ac-Tyr-Lys-Thr-Ala-Asp-Gly-Lys-Thr-Tyr-NH(2) (D9)] were determined by potentiometric titration in the temperature range from 25 to 60 °C, and their heat capacities were determined, by differential scanning calorimetry, in the temperature range from 10 to 90 °C. For both peptides, heat capacity has a maximum at t ≈ 50 °C, with height about 0.1 kcal/(mol × deg), suggesting that a modest unfolding transition occurs. The first two pK(a)'s are low at temperatures below 50 °C, suggesting that the two lysine residues are close to each other and the peptides have bent shapes at lower temperatures; this effect is greater for D7 compared with D9. With increasing temperature beyond 50 °C (i.e., that of the thermodynamic unfolding transition), pK(a1) and pK(a2) increase rapidly for D9, whereas their temperature variation is less significant for D7. This observation, and the fact that the enthalpies and entropies of the dissociation of the two first protons (determined from the temperature dependence of the respective pK(a)'s) decrease significantly near the transition temperature, suggest that the peptide undergoes a transition from a bent to an amorphous shape and that the presence of charged lysine residues stabilizes the folded state.

A Study of the Influence of Charged Residues on β-Hairpin Formation by Nuclear Magnetic Resonance and Molecular Dynamics

“…The observed long-range interactions for D9 indicates that the D9 peptide adopts a bent structure and has a U-shape, which is in good agreement with pK a values measured for these peptides in our earlier potentiometric study [ 15 ].…”

“…The β-hairpin from the FBP28 WW domain has two charged (lysine) residues on the opposite strand. According to our previous research [ 15 ], these residues might stabilize the structure. Conversely, the β-hairpin fragment of the IgG protein B3 domain has negatively charged (aspartic acid) residues on one side and a positively charged (lysine) residue on the opposite side.…”

“…In our previous work [ 15 ] we investigated two peptides: FPB28 (11–19) (D9), with sequence YKTADGKTY-NH 2 , and FPB28 (12–18) (D7), with sequence KTADGKT-NH 2 , (Fig. 1 a), by using potentiometric titration at different temperatures, circular dichroism spectroscopy (CD), and differential scanning calorimetry (DSC) measurements, respectively.…”

“…Above t = 50 °C (the melting point), pK a1 and pK a2 rise for D9, whereas for D7 the increase is less significant. This results suggests that the peptide turn bend to an shapeless structure and that the basic amino-acids (lysines) stabilizes the ordered state [ 15 ].…”

Investigations of copper(II) complexation by fragments of the FBP28 protein using isothermal titration (ITC) and differential scanning calorimetry (DSC)

“…The b-hairpin from the FBP28 WW domain has two charged (lysine) residues on opposite strands. According to our previous research [10], these residues, in addition to the charged aspartic acid group that is located in the central part of the sequence, may stabilize the shape of the structure. In order to measure the affinity of the D7 peptide to the Cu 2?…”