Time-resolved cryo-EM of G-protein activation by a GPCR.

G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating guanine nucleotide exchange in the Gα subunit. To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR-G-protein complex. By monitoring the transitions of the stimulatory G protein in complex with the β-adrenergic receptor at short sequential time points after GTP addition, we identified the conformational trajectory underlying G-protein activation and functional dissociation from the receptor.

Time-resolved cryo-EM of G protein activation by a GPCR.

G protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating the exchange of guanine nucleotide in the Gα subunit. To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR-G protein complex. Using variability analysis to monitor the transitions of the stimulatory Gs protein in complex with the β -adrenergic receptor (β AR) at short sequential time points after GTP addition, we identified the conformational trajectory underlying G protein activation and functional dissociation from the receptor.

Structure of the Visual Signaling Complex between Transducin and Phosphodiesterase 6.

Heterotrimeric G proteins communicate signals from activated G protein-coupled receptors to downstream effector proteins. In the phototransduction pathway responsible for vertebrate vision, the G protein-effector complex is composed of the GTP-bound transducin α subunit (Gα·GTP) and the cyclic GMP (cGMP) phosphodiesterase 6 (PDE6), which stimulates cGMP hydrolysis, leading to hyperpolarization of the photoreceptor cell. Here we report a cryo-electron microscopy (cryoEM) structure of PDE6 complexed to GTP-bound Gα.

Structural insights into differences in G protein activation by family A and family B GPCRs.

Family B heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) play important roles in carbohydrate metabolism. Recent structures of family B GPCR-G protein complexes reveal a disruption in the α-helix of transmembrane segment 6 (TM6) not observed in family A GPCRs. To investigate the functional impact of this structural difference, we compared the structure and function of the glucagon receptor (GCGR; family B) with the β adrenergic receptor (βAR; family A).

Non-Equilibrium Mass Exchange in AOT Reverse Micelles.

Reverse micelles (RMs) composed of water and sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane have a remarkably narrow size distribution around a mean value determined by the water loading ratio of the system. It has been proposed that RMs establish this equilibrium size distribution either by the diffusion of individual components through the isooctane phase or by cycles of fusion and fission. To examine these mechanisms, a 24 μs all-atom molecular dynamics simulation of a system containing one small RM and one large RM was performed.

Mass Exchange and Equilibration Processes in AOT Reverse Micelles.

Reverse micelles (RMs) made with sodium bis(2-ethylhexyl)sulfosuccinate suspended in isooctane are commonly used experimental models of aqueous microenvironments. However, there are important unanswered questions about the very characteristic that makes them of interest, namely their size. To explore the factors that determine the size of RMs, all-atom molecular dynamics simulations of RMs with different sizes but the same water-loading ratio were performed.

Amyloid Beta Peptide Folding in Reverse Micelles.

Previously published experimental studies have suggested that when the 40-residue amyloid beta peptide is encapsulated in a reverse micelle, it folds into a structure that may nucleate amyloid fibril formation (Yeung, P. S.-W.

The Size of AOT Reverse Micelles.

Reverse micelles (RMs) made from water and sodium bis(2-ethylhexyl) sulfosuccinate (AOT) are commonly studied experimentally as models of aqueous microenvironments. They are small enough for individual RMs to also be studied by molecular dynamics (MD) simulation, which yields detailed insight into their structure and properties. Although RM size is determined by the water loading ratio (i.

Computational design of new Peptide inhibitors for amyloid Beta (aβ) aggregation in Alzheimer's disease: application of a novel methodology.

Alzheimer's disease is the most common form of dementia. It is a neurodegenerative and incurable disease that is associated with the tight packing of amyloid fibrils. This packing is facilitated by the compatibility of the ridges and grooves on the amyloid surface.

Infrared spectroscopy of proteins in reverse micelles.

Reverse micelles are a versatile model system for the study of crowded microenvironments containing limited water, such as those found in various tissue spaces or endosomes. They also preclude protein aggregation. Reverse micelles are amenable to study by linear and nonlinear infrared spectroscopies, which have demonstrated that the encapsulation of polypeptides and enzymatically active proteins into reverse micelles leads to conformational changes not seen in bulk solution.

Copper and oxidative stress in the pathogenesis of Alzheimer's disease.

Copper is a redox-active metal with many important biological roles. Consequently, its distribution and oxidation state are subject to stringent regulation. A large body of clinicopathological, circumstantial, and epidemiological evidence suggests that the dysregulation of copper is intimately involved in the pathogenesis of Alzheimer's disease.