Dataset Information

Protein Denaturation with Guanidinium: A 2D-IR Study.

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 ?-helical or ?-sheet content upon addition of Gdm-(13)C(15)N3·Cl. We find that upon denaturation, the ?-sheet protein shows a complete loss of ?-sheet structure, whereas the ?-helical protein maintains most of its secondary structure. These results suggest that Gdm(+) disrupts ?-sheets much more efficiently than ?-helices, possibly because in the former, hydrophobic interactions are more important and the number of dangling hydrogen bonds is larger.

SUBMITTER: Huerta-Viga A

PROVIDER: S-EPMC3805561 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Protein Denaturation with Guanidinium: A 2D-IR Study.

Huerta-Viga Adriana A Woutersen Sander S

The journal of physical chemistry letters 20130923 20

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 fin ...[more]

PMID: 24163724

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Project description:Dynamic and structural properties of carbonmonoxy (CO)-coordinated cytochrome c(552) from Hydrogenobacter thermophilus (Ht-M61A) at different temperatures under thermal equilibrium conditions were studied with infrared absorption spectroscopy and ultrafast two-dimensional infrared (2D IR) vibrational echo experiments using the heme-bound CO as the vibrational probe. Depending on the temperature, the stretching mode of CO shows two distinct bands corresponding to the native and unfolded proteins. As the temperature is increased from low temperature, a new absorption band for the unfolded protein grows in and the native band decreases in amplitude. Both the temperature-dependent circular dichroism and the IR absorption area ratio R(A)(T), defined as the ratio of the area under the unfolded band to the sum of the areas of the native and unfolded bands, suggest a two-state transition from the native to the unfolded protein. However, it is found that the absorption spectrum of the unfolded protein increases its inhomogeneous line width and the center frequency shifts as the temperature is increased. The changes in line width and center frequency demonstrate that the unfolding does not follow simple two-state behavior. The temperature-dependent 2D IR vibrational echo experiments show that the fast dynamics of the native protein are virtually temperature independent. In contrast, the fast dynamics of the unfolded protein are slower than those of the native protein, and the unfolded protein fast dynamics and at least a portion of the slower dynamics of the unfolded protein change significantly, becoming faster as the temperature is raised. The temperature dependence of the absorption spectrum and the changes in dynamics measured with the 2D IR experiments confirm that the unfolded ensemble of conformers continuously changes its nature as unfolding proceeds, in contrast to the native state, which displays a temperature-independent distribution of structures.

Dataset Information

Protein Denaturation with Guanidinium: A 2D-IR Study.

Publications

Protein Denaturation with Guanidinium: A 2D-IR Study.

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