GPCRs function as receptors for various neurotransmitters and hormones, and >30% of current drugs target GPCRs. Although substantial number of GPCR crystal structures have become available, information about GPCR features can only be partially derived from the crystallography data. NMR methods provide information about dynamics of GPCRs over a wide range of frequencies in aqueous media at near-physiological temperature, with minimal modification of the wild-type GPCR covalent structures. Here we review our recent NMR studies of the function-related conformational equilibria of GPCRs, including β2 adrenergic receptor and μ opioid receptor.
Functional roles of the membrane heterogeneity such as rafts are one of the fundamental questions in cell biology. Model membranes are emerging as an important tool for understanding the biological membrane, providing quantitative information of membrane proteins and lipids. We developed a patterned bilayer composed of liquid-ordered (Lo) and liquid-disordered (Ld) phases as a model of raft, and evaluated the partition coefficients of membrane proteins between Lo and Ld phases to gauge their affinities to lipid rafts (raftophilicity). Quantitative information attained by the model membranes is critical for elucidating the membrane functions including raft, the ubiquitous yet enigmatic membrane heterogeneity.
Latrunculin A (LatA) is the widely used reagent to depolymerize actin filaments. Its mechanism has been thought that LatA sequesters actin monomers from polymerization. Recent observation of single actin filaments by TIRF microscopy found that the binding affinity of LatA to actin monomers depends on the nucleotide status on actin. The observation of actin filaments also showed that LatA binds to actin filaments. LatA increased the depolymerization rate at ends of filaments assembled from ATP-actin to the rates of ADP-actin, but did not change these rates of ADP- or ADP-Pi-bound actin filaments. LatA also severed actin filaments. Thermodynamic analysis proposes that LatA accelerates phosphate release only at ends of actin filaments. Rapid depolymerization, severing and sequestering monomers are mechanisms of LatA to inhibit cellular actin cytoskeleton.
During protein synthesis, folding, and translocation, proteins called molecular chaperones play key roles through the mechanisms that are not well understood. The scarcity of the high-resolution structural information for the interaction between chaperones and the client proteins, which are typically in an unfolded state, had prevented the elucidation of the detailed mechanisms. However, recent NMR studies have determined several key structures of chaperones in complex with client proteins. Here in this review, we summarize the recent structural studies on molecular chaperones that shed light on how the chaperones regulate the folding and translocation of the client proteins.