Protein Denaturation with Guanidinium: A 2D-IR Study

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 dear. Here, we use two-dimensional infrared spectroscopy (2D-IR) to investigate changes in the secondary structure of two proteins with mainly beta-helical or beta-sheet content upon addition of Gdm-(CN3)-C-13-N-15 center dot Cl. We find that upon denaturation, the beta-sheet protein shows a complete loss of beta-sheet structure, whereas the alpha-helical protein maintains most of its secondary structure. These results suggest that Gdm(+) disrupts beta-sheets much more efficiently than alpha-helices, possibly because in the former, hydrophobic interactions are more important and the number of dangling hydrogen bonds is larger.