Abstract
When combined with NMR spectroscopy, high hydrostatic pressure is an alternative perturbation method used to destabilize globular proteins that has proven to be particularly well suited for exploring the unfolding energy landscape of small single-domain proteins. To date, investigations of the unfolding landscape of all-β or mixed-α/β protein scaffolds are well documented, whereas such data are lacking for all-α protein domains. Here we report the NMR study of the unfolding pathways of GIPC1-GH2, a small α-helical bundle domain made of four antiparallel α-helices. High-pressure perturbation was combined with NMR spectroscopy to unravel the unfolding landscape at three different temperatures. The results were compared to those obtained from classical chemical denaturation. Whatever the perturbation used, the loss of secondary and tertiary contacts within the protein scaffold is almost simultaneous. The unfolding transition appeared very cooperative when using high pressure at high temperature, as was the case for chemical denaturation, whereas it was found more progressive at low temperature, suggesting the existence of a complex folding pathway.
Highlights
Small single-domain proteins are generally found to exhibit highly cooperative two-state unfolding transitions [1,2], the thousands of interactions that stabilize their 3D structure are unlikely to form simultaneously and folding intermediates should exist along their folding pathway
GIPC1-GH2 domain, we studied its folding/unfolding pathway at a residue-specific level using 2D NMR spectroscopy combined with high-pressure perturbation at three different temperatures, and with chemical perturbation
GIPC1-GH2 is very sensitive to high hydrostatic pressure, since it unfolds at 20 ◦ C in the 1–2500 bar range without adding any sub-denaturant concentration of chaotropic reagents, as was usually observed for high-pressure denaturation of all-β or mixed-α/β structures in our previous study: sub-denaturant concentration ranging from 0.5 M [25] to about 2 M [8,10] of guanidinium chloride was used to tune the stability of these proteins into the pressure range allowed by the experimental set-up
Summary
Small single-domain proteins are generally found to exhibit highly cooperative two-state unfolding transitions [1,2], the thousands of interactions that stabilize their 3D structure are unlikely to form simultaneously and folding intermediates should exist along their folding pathway. Especially in the case of small, fast-folding single-domain proteins, folding intermediates are generally low populated at equilibrium, and cannot be identified when using classical thermal or chemical perturbation in association with methods applied to a single probe in the 3D structure of the protein (for instance, intrinsic fluorescence of a tryptophan residue) or with methods giving global structural information (for instance, molar ellipticity in the case of circular dichroism (CD) study). Multidimensional NMR spectroscopy is a powerful tool to obtain highresolution structural information about protein folding events because an abundance of site-specific probes can be studied simultaneously in a single spectrum. Because the distribution of solvent-excluded voids depends on the protein structure, the pressure-induced unfolding originates from unique properties of the folded state
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