scholarly journals Conformational States of Exchange Protein Directly Activated by cAMP (EPAC1) Revealed by Ensemble Modeling and Integrative Structural Biology

Cells ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 35 ◽  
Author(s):  
Mark Andrew White ◽  
Tamara Tsalkova ◽  
Fang C. Mei ◽  
Xiaodong Cheng
Keyword(s):  
Bound States ◽  
Ternary Complex ◽  
Complex Model ◽  
Conformational States ◽  
X Ray ◽  
Solution Structures ◽  
X Ray Scattering ◽  
Ternary Complex Model ◽  
Saxs Analysis ◽  
Allosteric Regulators

Exchange proteins directly activated by cAMP (EPAC1 and EPAC2) are important allosteric regulators of cAMP-mediated signal transduction pathways. To understand the molecular mechanism of EPAC activation, we performed detailed Small-Angle X-ray Scattering (SAXS) analysis of EPAC1 in its apo (inactive), cAMP-bound, and effector (Rap1b)-bound states. Our study demonstrates that we can model the solution structures of EPAC1 in each state using ensemble analysis and homology models derived from the crystal structures of EPAC2. The N-terminal domain of EPAC1, which is not conserved between EPAC1 and EPAC2, appears folded and interacts specifically with another component of EPAC1 in each state. The apo-EPAC1 state is a dynamic mixture of a compact (Rg = 32.9 Å, 86%) and a more extended (Rg = 38.5 Å, 13%) conformation. The cAMP-bound form of EPAC1 in the absence of Rap1 forms a dimer in solution; but its molecular structure is still compatible with the active EPAC1 conformation of the ternary complex model with cAMP and Rap1. Herein, we show that SAXS can elucidate the conformational states of EPAC1 activation as it proceeds from the compact, inactive apo conformation through a previously unknown intermediate-state, to the extended cAMP-bound form, and then binds to its effector (Rap1b) in a ternary complex.

scholarly journals Pilot study on the effects of preservatives on corneal collagen parameters measured by small angle X-ray scattering analysis

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Susyn Joan Kelly ◽  
Lizette duPlessis ◽  
John Soley ◽  
Frazer Noble ◽  
Hannah Carolyn Wells ◽  
...  
Keyword(s):  
Small Angle ◽  
Collagen Fibril ◽  
Diameter Distribution ◽  
X Ray ◽  
X Ray Scattering ◽  
Saxs Analysis ◽  
Triton X ◽  
Fibril Diameter ◽  
Corneal Collagen ◽  
Ray Scattering

Abstract Objective Small angle X-ray scattering (SAXS) analysis is a sensitive way of determining the ultrastructure of collagen in tissues. Little is known about how parameters measured by SAXS are affected by preservatives commonly used to prevent autolysis. We determined the effects of formalin, glutaraldehyde, Triton X and saline on measurements of fibril diameter, fibril diameter distribution, and D-spacing of corneal collagen using SAXS analysis. Results Compared to sections of sheep and cats’ corneas stored frozen as controls, those preserved in 5% glutaraldehyde and 10% formalin had significantly larger mean collagen fibril diameters, increased fibril diameter distribution and decreased D-spacing. Sections of corneas preserved in Triton X had significantly increased collagen fibril diameters and decreased fibril diameter distribution. Those preserved in 0.9% saline had significantly increased mean collagen fibril diameters and decreased diameter distributions. Subjectively, the corneas preserved in 5% glutaraldehyde and 10% formalin maintained their transparency but those in Triton X and 0.9% saline became opaque. Subjective morphological assessment of transmission electron microscope images of corneas supported the SAXS data. Workers using SAXS analysis to characterize collagen should be alerted to changes that can be introduced by common preservatives in which their samples may have been stored.

scholarly journals Exploring the in meso crystallization mechanism by characterizing the lipid mesophase microenvironment during the growth of single transmembrane α-helical peptide crystals

2016 ◽  
Vol 374 (2072) ◽  
pp. 20150125 ◽  
Author(s):  
Leonie van 't Hag ◽  
Konstantin Knoblich ◽  
Shane A. Seabrook ◽  
Nigel M. Kirby ◽  
Stephen T. Mudie ◽  
...  
Keyword(s):  
Crystal Growth ◽  
Self Assembly ◽  
Lamellar Phase ◽  
Cubic Phase ◽  
Supporting Evidence ◽  
X Ray ◽  
X Ray Scattering ◽  
Standard Procedures ◽  
Helical Peptide ◽  
Saxs Analysis

The proposed mechanism for in meso crystallization of transmembrane proteins suggests that a protein or peptide is initially uniformly dispersed in the lipid self-assembly cubic phase but that crystals grow from a local lamellar phase, which acts as a conduit between the crystal and the bulk cubic phase. However, there is very limited experimental evidence for this theory. We have developed protocols to investigate the lipid mesophase microenvironment during crystal growth using standard procedures readily available in crystallography laboratories. This technique was used to characterize the microenvironment during crystal growth of the DAP12-TM peptide using synchrotron small angle X-ray scattering (SAXS) with a micro-sized X-ray beam. Crystal growth was found to occur from the gyroid cubic mesophase. For one in four crystals, a highly oriented local lamellar phase was observed, providing supporting evidence for the proposed mechanism for in meso crystallization. A new observation of this study was that we can differentiate diffraction peaks from crystals grown in meso , from peaks originating from the surrounding lipid matrix, potentially opening up the possibility of high-throughput SAXS analysis of in meso grown crystals. This article is part of the themed issue ‘Soft interfacial materials: from fundamentals to formulation’.

scholarly journals REGALS: a general method to deconvolve X-ray scattering data from evolving mixtures

2020 ◽  
Author(s):  
Steve P. Meisburger ◽  
Da Xu ◽  
Nozomi Ando
Keyword(s):  
A Priori ◽  
Scattering Data ◽  
Biological Macromolecules ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
Open Source Software Package ◽  
Saxs Analysis ◽  
Structural Methods ◽  
Ray Scattering

AbstractMixtures of biological macromolecules are inherently difficult to study using structural methods, as increasing complexity presents new challenges for data analysis. Recently, there has been growing interest in studying evolving mixtures using small-angle X-ray scattering (SAXS) in conjunction with time-resolved, high-throughput, or chromatography-coupled setups. Deconvolution and interpretation of the resulting datasets, however, are nontrivial when neither the scattering components nor the way in which they evolve are known a priori. To address this issue, we introduce the REGALS method (REGularized Alternating Least Squares), which incorporates simple expectations about the data as prior knowledge and utilizes parameterization and regularization to provide robust deconvolution solutions. The restraints used by REGALS are general properties such as smoothness of profiles and maximum dimensions of species, which makes it well-suited for exploring datasets with unknown species. Here we apply REGALS to analyze experimental data from four types of SAXS experiment: anion-exchange (AEX) coupled SAXS, ligand titration, time-resolved mixing, and time-resolved temperature jump. Based on its performance with these challenging datasets, we anticipate that REGALS will be a valuable addition to the SAXS analysis toolkit and enable new experiments. The software is implemented in both MATLAB and python and is available freely as an open-source software package.

scholarly journals In Situ Synchrotron X-ray Study of the Mechanical Properties of Pure Mg Produced by Powder Metallurgy

Metals ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1198
Author(s):  
Li Li ◽  
Leyun Wang ◽  
Jie Wang ◽  
Huan Zhang ◽  
Qingchun Zhu ◽  
...  
Keyword(s):  
Powder Metallurgy ◽  
Tensile Deformation ◽  
Line Broadening ◽  
X Ray ◽  
X Ray Scattering ◽  
Type Dislocation ◽  
Diffraction Patterns ◽  
Saxs Analysis ◽  
Ray Scattering

In this study, in situ synchrotron X-ray experiments with wide-angle X-ray scattering (WAXS) and small-angle X-ray scattering (SAXS) detectors were performed on two pure magnesium materials produced by powder metallurgy. According to SAXS analysis, each of the two materials has a porosity of less than 0.5%. Line broadening analysis was performed on diffraction patterns collected by WAXS to analyze the dislocation evolution during material deformation. In both materials, <a>-type dislocation activities dominate the tensile deformation. The influence of grain size and texture on the different tensile behaviors of these two materials is also discussed.

Solution Structure of Myosin-ADP-MgFn Ternary Complex by Fluorescent Probes and Small-Angle Synchrotron X-Ray Scattering

2000 ◽  
Vol 128 (4) ◽  
pp. 687-694 ◽  
Author(s):  
S. Manita ◽  
T. Aihara ◽  
Y. Uyehara ◽  
K. Homma ◽  
Y. Sugimoto ◽  
...  
Keyword(s):  
Fluorescent Probes ◽  
Small Angle ◽  
Solution Structure ◽  
Ternary Complex ◽  
X Ray ◽  
X Ray Scattering ◽  
Ray Scattering

scholarly journals Investigating allosteric effects on the functional dynamics of β2-adrenergic ternary complexes with enhanced-sampling simulations

2017 ◽  
Vol 8 (5) ◽  
pp. 4019-4026 ◽  
Author(s):  
Noureldin Saleh ◽  
Giorgio Saladino ◽  
Francesco Luigi Gervasio ◽  
Timothy Clark
Keyword(s):  
Ternary Complex ◽  
Cooperative Effect ◽  
Complex Model ◽  
Ternary Complexes ◽  
Enhanced Sampling ◽  
Binding Partner ◽  
Functional Dynamics ◽  
Ternary Complex Model ◽  
Intracellular Binding

We propose an experimentally testable extended ternary complex model, where direction of the cooperative effect between ligand and intracellular binding partner (positive or negative) and its magnitude are predicted to be a characteristic of the ligand signaling bias.

Spare receptors, partial agonists, and ternary complex model of drug action

1987 ◽  
Vol 253 (1) ◽  
pp. E114-E121
Author(s):  
L. D. Homer ◽  
T. B. Nielsen
Keyword(s):  
Ternary Complex ◽  
Drug Action ◽  
Complex Model ◽  
Published Data ◽  
Receptor Ligand ◽  
Spare Receptors ◽  
Contractile Responses ◽  
Partial Agonists ◽  
Receptor Inactivation ◽  
Ternary Complex Model

The occurrence of spare receptors and partial agonists for smooth muscle contractions mediated by alpha 1-adrenergic receptors can be accounted for with a ternary complex model of drug action presented here. In this model, receptor-ligand complexes are assumed to be inactive until the complex binds to an activating protein. Contractile responses are assumed to be proportional to the concentration of ternary complex (receptor-ligand-activator) regardless of the ligand involved. Antagonists are unable to form the ternary complex. Spare receptors are present as the inactive receptor-ligand complex. Such a model is shown to fit already published data on membrane binding of alpha 1-adrenergic agonists as well as contractile responses to the agonist. Schild plots are expected to resemble those of a single-site model of drug action. The double-reciprocal plots of receptor-inactivation studies will display only a slight curvature as may be seen in previously published articles. Partial agonists may have 50% response doses lowe or higher than full agonists. The hypothesis that ternary complexes are formed with alpha 1-receptors could be tested more critically with receptor-inactivation studies using both antagonists and agonists. Partial inactivation of receptor and activator protein should reduce the binding of antagonist without altering the concentration needed to bind to 50% of the receptors. On the other hand, the concentration of agonist required to displace 50% of a bound antagonist is expected to increase. The proposal that contractile responses are proportional to the ternary complex concentration could be tested by fitting the ternary complex model to the data from studies of contractions induced by partial agonists as well as full agonists.

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