The modern view of protein thermodynamics predicts that proteins undergo cold-induced unfolding. Unfortunately, the properties of proteins and water conspire to prevent the detailed observation of this fundamental process. Here we use protein encapsulation to allow cold denaturation of the protein ubiquitin to be monitored by high-resolution NMR at temperatures approaching -35 degrees C. The cold-induced unfolding of ubiquitin is found to be highly noncooperative, in distinct contrast to the thermal melting of this and other proteins. These results demonstrate the potential of cold denaturation as a means to dissect the cooperative substructures of proteins and to provide a rigorous framework for testing statistical thermodynamic treatments of protein stability, dynamics and function.

Integrins are a family of ␣͞␤ heterodimeric membrane proteins, which mediate cell-cell and cell-matrix interactions. The molecular mechanisms by which integrins are activated and cluster are currently poorly understood. One hypothesis posits that the cytoplasmic tails of the ␣ and ␤ subunits interact strongly with one another in a 1:1 interaction, and that this interaction is modulated in the course of the activation of ␣IIb␤3 [Hughes, P. E., et al. (1996) J. Biol. Chem. 271, 6571-6574]. To examine the structural basis for this interaction, protein fragments encompassing the transmembrane helix plus cytoplasmic tails of the ␣ and ␤ subunits of ␣IIb␤3 were expressed and studied in phospholipid micelles at physiological salt concentrations. Analyses of these fragments by analytical ultracentrifugation, NMR, circular dichroism, and electrophoresis indicated that they had very little or no tendency to interact with one another. Instead, they formed homomeric interactions, with the ␣-and ␤-fragments forming dimers and trimers, respectively. Thus, these regions of the protein structure may contribute to the clustering of integrins that accompanies cellular adhesion.I ntegrins, a family of ␣͞␤ heterodimers, mediate essential cell-cell and cell-matrix interactions (1). Each subunit of the integrin heterodimer is composed of a large extracellular domain, a transmembrane (TM) helix, and a short cytoplasmic (CYTO) tail. Heterodimer formation results from interactions between sequences located in the extracellular domain of each subunit (2). Many cells actively regulate integrin ligand-binding activity (3). The prototypic example of integrin regulation is the platelet integrin ␣IIb␤3 (4). In unstimulated platelets, ␣IIb␤3 is inactive, whereas exposing platelets to agonists such as ADP and thrombin enables ␣IIb␤3 to bind ligands such as fibrinogen and von Willebrand factor. The integrin is activated in a bidirectional manner, in which intracellular events can trigger a conformational change in the extracellular ligand-binding domains (inside-out signaling) or vice versa. In one proposed mechanism for this process, the CYTO tails of ␣IIb and ␤3 interact in the inactive state through the formation of a salt bridge (5). This interaction is broken and the CYTO domains separate when the integrin is activated. Evidence for this hypothesis came from mutational studies (5), as well as biochemical studies that seemed to show a weak but divalent cation-dependent interaction between peptides corresponding to the CYTO tails of ␣IIb and ␤3 (6, 7). Further, elegant protein engineering studies by Springer and coworkers (8,9) unambiguously demonstrated that when the CYTO domains or the C termini of the extracellular domains were forced to interact, the integrin ␣L␤2 or ␣5␤1 was inactivated. However, a very recent and carefully executed NMR study indicated that the ␣IIb and ␤3 CYTO tails were unable to interact, even when tethered in close proximity from the same end of a heterodimeric coiled coil (10). Further, the observation that replaci...

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...