Functional equivalence of the nicotinic acetylcholine receptor transmitter binding sites in the open state

The subunits of the muscle-type nicotinic acetylcholine receptor (AChR) are not uniformly oriented in the resting closed conformation: the two α subunits are rotated relative to its non-α subunits. In contrast, all the subunits overlay well with one another when agonist is bound to the AChR, suggesting that they are uniformly oriented in the open receptor. This gating-dependent increase in orientational uniformity due to rotation of the α subunits might affect the relative affinities of the two transmitter binding sites, making the two affinities dissimilar (functionally non-equivalent) in the initial ligand-bound closed state but similar (functionally equivalent) in the open state. To test this hypothesis, we measured single-channel activity of the αG153S gain-of-function mutant receptor evoked by choline, and estimated the resting closed-state and open-state affinities of the two transmitter binding sites. Both model-independent analyses and maximum-likelihood estimation of microscopic rate constants indicate that channel opening makes the binding sites' affinities more similar to each other. These results support the hypothesis that open-state affinities to the transmitter binding sites are primarily determined by the α subunits

Plasticity of Acetylcholine Receptor Gating Motions via Rate-Energy Relationships

AbstractLike other protein conformational changes, ion channel gating requires the protein to achieve a high-energy transition-state structure. It is not known whether ion channel gating takes place on a broad energy landscape on which many alternative transition state structures are accessible, or on a narrow energy landscape where only a few transition-state structures are possible. To address this question, we measured how rate-equilibrium free energy relationships (REFERs) for di-liganded and unliganded acetylcholine receptor gating vary as a function of the gating equilibrium constant. A large slope for the REFER plot indicates an openlike transition state, whereas a small slope indicates a closedlike transition state. Due to this relationship between REFERs and transition-state structure, the sensitivity of the REFER slope to mutation-induced energetic perturbations allows estimation of the breadth of the energy landscape underlying gating. The relatively large sensitivity of di-liganded REFER slopes to energetic perturbations suggests that the motions underlying di-liganded gating take place on a broad, shallow energy landscape where many alternative transition-state structures are accessible

Patent: Fusion Proteins for Treating Metabolic Disorders

The invention relates to the identification of fusion proteins comprising polypeptide and protein variants of fibroblast growth factor 21 (FGF21) with improved pharmaceutical properties. Also disclosed are methods for treating FGF21 associated disorders, including metabolic conditions

Patent: Methods of Treating FGF21-Associated Disorders

The invention relates to the identification of new polypeptide and protein variants of fibroblast growth factor 21 (FGF21) that have improved pharmaceutical properties. Also disclosed are methods for treating FGF21-associated disorders, includ ing metabolic conditions