Understanding the origin of the cooperativity seemingly inherent in a folding or unfolding reaction has been a major challenge. In particular, the relationship between folding cooperativity and stability is poorly understood. In this study, native state hydrogen exchange in conjunction with mass spectrometry has been used to explore the free energy landscape accessible to the small protein monellin, when the stability of the protein is varied. Mass distributions obtained in the EX1 limit of exchange have allowed a direct distinction between correlated (cooperative) and uncorrelated (noncooperative) structure-opening processes. Under conditions where the native protein is maximally stable, a continuum of partially unfolded states is gradually sampled before the globally unfolded state is transiently sampled. Under conditions that stabilize the unfolded state of the protein, the slowest structure-opening reactions leading to complete unfolding become cooperative. The present study provides experimental evidence for a gradual uphill unfolding transition on a very slow time scale, in the presence of a large free energy difference between the native and unfolded states. The results suggest that the cooperativity that manifests itself in protein folding and unfolding reactions carried out in the presence of denaturant might merely be a consequence of the effect of the denaturant on the unfolded state and transition state stabilities.

The intestinal reclamation of bile acids is crucial for the maintenance of their enterohepatic circulation. The majority of bile acids are actively absorbed via specific transport proteins that are highly expressed in the distal ileum. The uptake of bile acids by intestinal epithelial cells modulates the activation of cytosolic and membrane receptors such as the farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 1 (GPBAR1), which has a profound effect on hepatic synthesis of bile acids as well as glucose and lipid metabolism. Extensive research has focused on delineating the processes of bile acid absorption and determining the contribution of dysregulated ileal signaling in the development of intestinal and hepatic disorders. For example, a decrease in the levels of the bile acid-induced ileal hormone FGF15/19 is implicated in bile acidinduced diarrhea (BAD). Conversely, the increase in bile acid absorption with subsequent overload of bile acids could be involved in the pathophysiology of liver and metabolic disorders such as fatty liver diseases and type 2 diabetes mellitus. This review article will attempt to provide a comprehensive overview of the mechanisms involved in the intestinal handling of bile acids, the pathological implications of disrupted intestinal bile acid homeostasis, and the potential therapeutic targets for the treatment of bile acid-related disorders.

A thermodynamically and kinetically simple picture of protein folding envisages only two states, native (N) and unfolded (U), separated by a single activation free energy barrier, and interconverting by cooperative two-state transitions. The folding/unfolding transitions of many proteins occur, however, in multiple discrete steps associated with the formation of intermediates, which is indicative of reduced cooperativity. Furthermore, much advancement in experimental and computational approaches has demonstrated entirely non-cooperative (gradual) transitions via a continuum of states and a multitude of small energetic barriers between the N and U states of some proteins. These findings have been instrumental towards providing a structural rationale for cooperative versus noncooperative transitions, based on the coupling between interaction networks in proteins. The cooperativity inherent in a folding/unfolding reaction appears to be context dependent, and can be tuned via experimental conditions which change the stabilities of N and U. The evolution of cooperativity in protein folding transitions is linked closely to the evolution of function as well as the aggregation propensity of the protein. A large activation energy barrier in a fully cooperative transition can provide the kinetic control required to prevent the accumulation of partially unfolded forms, which may promote aggregation. Nevertheless, increasing evidence for barrier-less "downhill" folding, as well as for continuous "uphill" unfolding transitions, indicate that gradual non-cooperative processes may be ubiquitous features on the free energy landscape of protein folding.