Pressure-Temperature Analysis of the Stability of the CTL9 Domain Reveals Hidden Intermediates

Abstract

The observation of two-state unfolding for many small single-domain proteins by denaturants has led to speculation that protein sequences may have evolved to limit the population of partially folded states that could be detrimental to fitness. How such strong cooperativity arises from a multitude of individual interactions is not well understood. Here, we investigate the stability and folding cooperativity of the C-terminal domain of the ribosomal protein L9 in the pressure-temperature plane using site-specific NMR. In contrast to apparent cooperative unfolding detected with denaturant-induced and thermal-induced unfolding experiments and stopped-flow refolding studies at ambient pressure, NMR-detected pressure unfolding revealed significant deviation from twostate behavior, with a core region that was selectively destabilized by increasing temperature. Comparison of pressure-dependent NMR signals from both the folded and unfolded states revealed the population of at least one invisible excited state at atmospheric pressure. The core destabilizing cavity-creating I98A mutation apparently increased the cooperativity of the loss of folded-state peak intensity while also increasing the population of this invisible excited state present at atmospheric pressure. These observations highlight how local stability is subtly modulated by sequence to tune protein conformational landscapes and illustrate the ability of pressure-and temperature-dependent studies to reveal otherwise hidden states.

High-Pressure NMR Reveals Volume and Compressibility Differences Between Two Stably Folded Protein Conformations

“…If the interconversion of ARNT PAS-B Y456T truly goes through a chiefly unfolded intermediate state, the activation volumes derived are expected to be comparable to the folding volumes of similar sized (10-20 kDa) proteins. Indeed, the numbers are in good agreement with several other proteins studied with pressure-dependent unfolding approaches, as summarized in Table S1 41,[53][54][55][56][57][58] . Thus, the kinetic analyses further support our hypothesis.…”

Volume and compressibility differences between protein conformations revealed by high-pressure NMR

“…We additionally compared the unfolding volumes of WT and TRIP to the unfolding volumes of similarly-sized proteins (10-20 kDa). Indeed, the numbers are in good agreement with several other proteins studied with pressure-dependent unfolding approaches, as summarized in Table S1 [42,45,[48][49][50][51][52] . A schematic figure describing the relationships among the two folded conformations, the intermediate state, and the unfolded state is shown in Fig.…”

Pressure and Chemical Unfolding of an α-Helical Bundle Protein: The GH2 Domain of the Protein Adaptor GIPC1

“…Proton, nitrogen, and carbon NMR resonances of GIPC1-GH2, renumbered 1-79 for simplicity, have been assigned and its solution structure solved using essentially [ 1 H, 15 N, 13 C] triple-resonance and [ 1 H, 15 N] double-resonance 3D NMR spectroscopy (see Section 4) with the classical sequential assignment strategy. 1 H and 15 N resonances have been assigned for all amide groups of nonproline (76) residues (Figure 1), and Cα, Cβ, C' resonances for 96.2% residues.…”

“…The construct GIPC1-GH2 domain (residues 255-333) was subcloned in pProEXHTB, allowing the expression of a 6xHis-TEV fusion protein, and was transformed into E. coli BL21-Gold (DE3) (Stratagene, Amsterdam, The Netherlands). Uniform 15 N or 15 N/ 13 C labeling was obtained by growing cells in minimal M9 medium containing 15 NH4Cl and/or 15 NH4Cl/ 13 C-u-labeled glucose as the sole nitrogen or carbon sources (Cortecnet). Protein was expressed overnight at 20 • C after induction with 0.2 mM IPTG.…”

“…High-pressure NMR unfolding studies have been applied to several single-domain proteins in order to characterize their folding landscape. Until now, these studies have concerned essentially all-β [ 10 , 11 ] or mixed-α/β [ 9 , 12 , 13 ] protein scaffolds, and similar studies are lacking for all-α structures although they represent a widespread assembly motif [ 14 ]. In the present manuscript, we report the NMR study of the folding of an α-helical bundle, the GH2 domain of the protein adaptor GIPC1 [ 15 ].…”