Unique features of the folding landscape of a repeat protein revealed by pressure perturbation.
The volumetric properties of proteins yield information about the changes in packing and hydration between various states along the folding reaction coordinate and are also intimately linked to the energetics and dynamics of these conformations. These volumetric characteristics can be accessed via pressure perturbation methods. In this work, we report high-pressure unfolding studies of the ankyrin domain of the Notch receptor (Nank1-7) using fluorescence, small-angle x-ray scattering, and Fourier transform infrared spectroscopy. Both equilibrium and pressure-jump kinetic fluorescence experiments were consistent with a simple two-state folding/unfolding transition under pressure, with a rather small volume change for unfolding compared to proteins of similar molecular weight. High-pressure fluorescence, Fourier transform infrared spectroscopy, and small-angle x-ray scattering measurements revealed that increasing urea over a very small range leads to a more expanded pressure unfolded state with a significant decrease in helical content. These observations underscore the conformational diversity of the unfolded-state basin. The temperature dependence of pressure-jump fluorescence relaxation measurements demonstrated that at low temperatures, the folding transition state ensemble (TSE) lies close in volume to the folded state, consistent with significant dehydration at the barrier. In contrast, the thermal expansivity of the TSE was found to be equivalent to that of the unfolded state, indicating that the interactions that constrain the folded-state thermal expansivity have not been established at the folding barrier. This behavior reveals a high degree of plasticity of the TSE of Nank1-7.
Centre de Biochimie Structurale, Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Université Montpellier, Montpellier, France.
Jean-Baptiste
JB
Rouget
Martin A
MA
Schroer
Christoph
C
Jeworrek
Matthias
M
Pühse
Jean-Louis
JL
Saldana
Yannick
Y
Bessin
Metin
M
Tolan
Doug
D
Barrick
Roland
R
Winter
Catherine A
CA
Royer
D004926:Escherichia coli; D005453:Fluorescence; D007700:Kinetics; D008958:Models, Molecular; D011312:Pressure; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D051880:Receptors, Notch / Q000737:chemistry*; D053838:Scattering, Small Angle; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D013816:Thermodynamics; D014961:X-Ray Diffraction
D051880:Receptors, Notch