Cold Denaturation of Encapsulated Ubiquitin

Abstract

Theoretical considerations suggest that protein cold denaturation can potentially provide a means to explore the cooperative substructure of proteins. Protein cold denaturation is generally predicted to occur well-below the freezing point of water. Here NMR spectroscopy of ubiquitin encapsulated in reverse micelles dissolved in low viscosity alkanes is used to follow cold-induced unfolding to temperatures below −25 °C. Comparison of cold-induced structural transitions in a variety of reverse micelle-buffer systems indicate that protein-surfactant interactions are negligible and allow the direct observation of the multi-state cold-induced unfolding of the protein.It has been recognized for a very long time that proteins of even modest size have an astronomical number of potential conformations. 1 Subsequent statistical mechanical 2 and thermodynamic 3,4 treatments of this conformational landscape have emerged to illuminate roles for non-native states in phenomena ranging from protein folding to allostery. The experimental exploration of the ensemble of states accessible to protein molecules has however proven to be difficult. Recently we introduced an approach that seeks to differentiate cooperative substructure of proteins based on fundamental thermodynamic parameters and to do so with the potential of resolving them from each other. 5 The approach employs a simple prediction of the temperature dependence of the various types of interactions that stabilize protein structure. For a two-state equilibrium with a temperature independent change in heat capacity (ΔC p ), a familiar form of the Gibbs-Helmholtz equation is applicable:where T ref is an arbitrary reference temperature and the remaining symbols have their usual meanings. Proteins generally have a large positive ΔC p and are predicted to unfold at both high and low temperatures. 6-8 This two-state treatment is easily generalized to the ensemble view of proteins. 5The key is that proteins are dominated by two types of interactions that have quite distinct changes in heat capacity associated with their disruption. 5 Hydrophobic interactions generally have a large ΔC p while polar interactions generally have a ΔC p near zero associated with their disruption in water. Importantly, states with different ΔC p values underlying their stability can potentially be distinguished and significantly populated during cold-induced disassembly. 5 This is in distinct contrast to high temperature unfolding, where effective two-state behavior is generally observed and careful analysis can only infer the presence of intermediates. 9 The idea then is to employ cold-induced unfolding to dissect and characterize the cooperative substructure of proteins using NMR. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptUnfortunately, the thermodynamic parameters of most proteins are such that cold-induced unfolding is not expected to occur until well below the freezing point of water. 7 Historically, destabilizing perturbations such as mutations, chemical...

Simulations of the confinement of ubiquitin in self-assembled reverse micelles

“…Ubiquitin cold denaturation has been previously studied by the groups of Wand,66 and Flynn. 67,68 Under low excess salt concentration, which are the condition of our self-assembly simulations, it is observed that denaturation is non-cooperative, multistate, with resonances corresponding to residues that make up the I44 face are lost, while resonances corresponding to the opposite face remained. [66][67][68] In Fig.…”

“…Under slightly varying conditions of pH and temperature and in a timescale appropriate for folding/unfolding dynamics, the interactions between the protein and surfactant may lead to unfolding, as seen in the non-cooperative cold denaturation of ubiquitin in RM. [66][67][68] Our simulation studies presented here provide a detailed atomic description of the structure and dynamics of a protein-RM system. The results and findings presented can serve to guide future studies on protein-RM system.…”

The non-uniform early structural response of globular proteins to cold denaturing conditions: A case study with Yfh1

“…In order to gain preliminary insights into the higher susceptibility of Yfh1 to cold denaturing conditions, we compared its nanosecond timescale behavior with that of Ubiquitin (Ubq) at T c . Ubq is known to display high thermal stability 87 and to cold denature at temperatures as low as 238 K. 23 Visual inspection of snapshots of Ubq from simulations produced at T s (292 K) and T c (268 K), presented in Figures 8(a) and 8(b), respectively, indicate that the structural integrity of this protein is maintained at T c . Contrary to the structural effects in Yfh1, the beta strands of Ubq are mostly found to be nearly intact at T c .…”

“…19 It has been suggested that the thermodynamic advantage in terms of translational entropy gain of water molecules associated with the large decrease in solvent accessible surface area during folding is lost at lower temperatures. 20,21 Further, although cold denaturation has been largely considered to be non-cooperative, 22,23 recent NMR experiments showing similar structural loss at distal sites 24 suggest that there may be exceptions. We also mention here that cold protein denaturation is analogous to polymer unfolding resulting from hydrophobic hydration.…”

Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy

“…The populations of nonnative conformational states can be enhanced by changing the temperature, pressure, or pH, or by adding alcohol cosolvents. A cold-denaturation study by Wand et al (41,42) shows for reverse micelle-encapsulated ubiquitin that the mixed ␤-sheet is progressively destabilized from the C-terminal side in the temperature range from Ϫ20°to Ϫ30°C. In an NMR study by Cordier and Grzesiek (43), the change in hydrogen bond strength with increasing temperature was measured.…”