Abstract
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-13C15N3·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.
Highlights
S ignificant effort has been dedicated to elucidating the molecular mechanism of the effect of chemical denaturants, the most effective one of which is the guanidinium cation (Gdm+).[1−3] There are two mechanisms by which a denaturant could destabilize proteins, indirectly by altering the solvent properties of water and directly by interacting with groups of the protein
The denaturation mechanism of Gdm+ involves interactions with the side chains of proteins, but the physical nature of these interactions is only just starting to become clear.[7−11]. It is not known whether Gdm+ disrupts certain types of protein secondary structure more efficiently than others as most studies to date focus on interactions between Gdm+ and the side chains of proteins.[7−11] Addressing this question requires techniques that are sensitive to the secondary structure of proteins
Circular dichroism (CD), a commonly used probe of structure loss upon denaturation, is not so well suited for this purpose because it has a high sensitivity to αhelical content but a low one to β-sheet secondary structure
Summary
Studying Gdm+ denaturation of proteins with IR is difficult because Gdm+ absorbs at 1600 cm−1 due to a degenerate mode involving a combined CN3 antisymmetric stretch and NH2 scissor motion.[14]. The amide I′ band of denatured lysozyme blue shifts with the respect to the spectra of the native protein, indicating increased random coil content (this shift is more seen in Figure S2 (Supporting Information), where FTIR spectra for intermediate Gdm+ concentrations are shown). As for lysozyme, the amide I′ band of native and denatured α-chymotrypsin undergoes a blue shift, indicating increased random coil conformation (see Figure S2 (Supporting Information) for intermediate Gdm+ concentrations). In the inset of this figure, we plot the amplitude of the βsheet cross peak as a function of Gdm+ concentration, and a clear decay to zero is observed This result indicates that the βsheet secondary structure of α-chymotrypsin is completely destabilized by the presence of the Gdm+ ions.
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