Acid-induced equilibrium folding intermediate of human platelet profilin

The acid-induced unfolding of human platelet profilin (HPP) can be minimally modeled as a three-state process. Equilibrium unfolding studies have been performed on human platelet profilin1 (HPP) and monitored by far-UV circular dichroism, tryptophan fluorescence, ANS binding, and NMR spectroscopy. Far-UV CD measurements obtained by acid titration demonstrate that HPP unfolds via a three-state mechanism (N → I → U), with a highly populated intermediate between pH 4 and 5. Approximately 80% of native helical secondary structural content remains at pH 4, as indicated by monitoring the CD signal at 222 nm. The stability (ΔG_H2O) of the native conformation at pH 7.0 (obtained by monitoring the change in tryptophan signal as a function of urea concentration) is 5.56 ± 0.51 kcal mol^-1; however, the ΔG_H2O for the intermediate species at pH 4 is 2.01 ± 0.47 kcal mol^-1. The calculated m-values for the pH 7.0 and pH 4.0 species were 1.64 ± 0.15 and 1.34 ± 0.17 kcal mol^-1 M^-1, respectively, which is an indication that the native and intermediate species are similarly compact. Additionally, translational diffusion measurements obtained by NMR spectroscopy and ANS binding studies are consistent with a globular and compact conformation at both pH 7.0 and 4.0. The pK_a values for the two histidine (His) residues located on helix 4 of HPP were determined to be 5.6 and 5.7 pH units. These pK_a values coincide with the midpoint of the far-UV CD acid titration curve and suggest that the protonation of one or both His residues may play a role in the formation of the unfolding intermediate. Stable intermediate species populate the 2D ¹H-¹⁵N HSQC NMR spectra between pH 4 and 5. A number of backbone and side-chain resonances show significant perturbations relative to the native spectrum; however, considerable nativelike tertiary contacts remain. Interestingly, the residues on HPP that are significantly altered at low pH coincide with segments of the G-actin binding surface and poly-L-proline binding interface. The earlier reports that a decrease in pH below 6.0 induces structural alterations in profilin, favoring dissociation of the profilin-actin complex, corresponds with the structural alterations observed in the partially unfolded species. Our findings suggest that a novel mechanism for pH induced disruption of the profilin-G-actin complex involve a nativelike unfolding intermediate of profilin.