Protein Denaturation with Guanidinium: A 2D-IR Study

[Image: see text] Guanidinium (Gdm(+)) is a widely used denaturant, but it is still largely unknown how it operates at the molecular level. In particular, the effect of guanidinium on the different types of secondary structure motifs of proteins is at present not clear. Here, we use two-dimensional infrared spectroscopy (2D-IR) to investigate changes in the secondary structure of two proteins with mainly α-helical or β-sheet content upon addition of Gdm-(13)C(15)N(3)·Cl. We find that upon denaturation, the β-sheet protein shows a complete loss of β-sheet structure, whereas the α-helical protein maintains most of its secondary structure. These results suggest that Gdm(+) disrupts β-sheets much more efficiently than α-helices, possibly because in the former, hydrophobic interactions are more important and the number of dangling hydrogen bonds is larger.