Dynamics of GLP-1R peptide agonist engagement are correlated with kinetics of G protein activation

The glucagon-like peptide-1 receptor (GLP-1R) has broad physiological roles and is a validated target for treatment of metabolic disorders. Despite recent advances in GLP-1R structure elucidation, detailed mechanistic understanding of how different peptides generate profound differences in G protein-mediated signalling is still lacking. Here we combine cryo-electron microscopy, molecular dynamics simulations, receptor mutagenesis and pharmacological assays, to interrogate the mechanism and consequences of GLP-1R binding to four peptide agonists; glucagon-like peptide-1, oxyntomodulin, exendin-4 and exendin-P5. These data reveal that distinctions in peptide N-terminal interactions and dynamics with the GLP-1R transmembrane domain are reciprocally associated with differences in the allosteric coupling to G proteins. In particular, transient interactions with residues at the base of the binding cavity correlate with enhanced kinetics for G protein activation, providing a rationale for differences in G protein-mediated signalling efficacy from distinct agonists

Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor

Calcitonin gene-related peptide (CGRP) is a widely expressed neuropeptide that plays a major role in sensory neurotransmission. The CGRP receptor is a heterodimer of the calcitonin receptor-like receptor (CLR) class B G-protein-coupled receptor and the type 1 transmembrane domain protein, receptor activity modifying protein (RAMP) 1. Herein, we report the 3.3 Å structure of the human CGRP receptor in complex with CGRP and the Gs40 protein heterotrimer determined by Volta phase plate cryo-electron microscopy. The RAMP transmembrane domain sits at the interface between transmembrane domains 3, 4 and 5 of CLR, and stabilises CLR extracellular loop 2. RAMP1 makes only limited direct interaction with CGRP, consistent with allosteric modulation of CLR as its key function. Molecular dynamics simulations indicate that RAMP1 provides stability to the receptor complex, particularly the location of the CLR extracellular domain. The work provides novel insight into the control of G-protein-coupled receptor function

Ag/Pd core-shell nanoparticles by a successive method: Pulsed laser ablation of Ag in water and reduction reaction of PdCl2

In this study Ag/Pd nanoparticles (NPs) have been fabricated by a successive method; first, colloids of Ag nanoparticles (NPs) have been prepared in water by pulsed laser ablation in liquid (PLAL) method. Then PdCl2 solution (up to 0.2 g/l) were added to the as-prepared or aged colloidal Ag NPs. Characterizations were done using UV-vis spectroscopy, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and transmissions electron microscopy (TEM) techniques. Spectroscopy data showed that surface plasmon resonance (SPR) peaks of as-prepared Ag NPs at about lambda = 400nm were completely extinguished after addition of PdCl2 solution while this effect was not observed when aged Ag NPs are used. XRD and XPS results revealed that by addition of the PdCl2 solution into the as-prepared Ag NPs, metallic palladium, and silver chloride composition products are generated. TEM images revealed that as a result of this reaction, single and core-shell nanoparticles are obtained and their average sizes are 2.4 nm (Ag) and 3.2 nm (Ag/Pd). The calculated d-spacing values form XRD data with observations on high magnification TEM images were able to explain the chemical nature of different parts of Ag/Pd NPs. (C) 2013 Elsevier B.V. All rights reserved

Cryo-EM structure of haemoglobin at 3.2 angstrom determined with the Volta phase plate

With the advent of direct electron detectors, the perspectives of cryo-electron microscopy (cryo-EM) have changed in a profound way. These cameras are superior to previous detectors in coping with the intrinsically low contrast and beam-induced motion of radiation-sensitive organic materials embedded in amorphous ice, and hence they have enabled the structure determination of many macromolecular assemblies to atomic or near-atomic resolution. Nevertheless, there are still limitations and one of them is the size of the target structure. Here, we report the use of a Volta phase plate in determining the structure of human haemoglobin (64 kDa) at 3.2 angstrom. Our results demonstrate that this method can be applied to complexes that are significantly smaller than those previously studied by conventional defocus-based approaches. Cryo-EM is now close to becoming a fast and cost-effective alternative to crystallography for high-resolution protein structure determination

Revisiting the Structure of Hemoglobin and Myoglobin with Cryo-Electron Microscopy

Sixty years ago, the first protein structure of myoglobin was determined by John Kendrew and his colleagues; hemoglobin followed shortly thereafter. For quite some time, it seemed that only X-ray crystallography would be capable of determining the structure of proteins to high resolution. In recent years, cryo-electron microscopy has emerged as a viable alternative and indeed in many cases the preferred approach. It is capable of studying proteins that span a size range from several megadaltons to proteins as small as myoglobin and hemoglobin. (C) 2017 Elsevier Ltd. All rights reserved

Volta potential phase plate for in-focus phase contrast transmission electron microscopy

We describe a phase plate for transmission electron microscopy taking advantage of a hitherto-unknown phenomenon, namely a beam-induced Volta potential on the surface of a continuous thin film. The Volta potential is negative, indicating that it is not caused by beam-induced electrostatic charging. The film must be heated to similar to 200 degrees C to prevent contamination and enable the Volta potential effect. The phase shift is created "on the fly" by the central diffraction beam eliminating the need for precise phase plate alignment. Images acquired with the Volta phase plate (VPP) show higher contrast and unlike Zernike phase plate images no fringing artifacts. Following installation into the microscope, the VPP has an initial settling time of about a week after which the phase shift behavior becomes stable. The VPP has a long service life and has been used for more than 6 mo without noticeable degradation in performance. The mechanism underlying the VPP is the same as the one responsible for the degradation over time of the performance of thin-film Zernike phase plates, but in the VPP it is used in a constructive way. The exact physics and/or chemistry behind the process causing the Volta potential are not fully understood, but experimental evidence suggests that radiation-induced surface modification combined with a chemical equilibrium between the surface and residual gases in the vacuum play an important role