Conformational transitions of the sodium-dependent sugar transporter, vSGLT.

Sodium-dependent transporters couple the flow of Na+ ions down their electrochemical potential gradient to the uphill transport of various ligands. Many of these transporters share a common core structure composed of a five-helix inverted repeat and deliver their cargo utilizing an alternating-access mechanism. A detailed characterization of inward-facing conformations of the Na+-dependent sugar transporter from Vibrio parahaemolyticus (vSGLT) has previously been reported, but structural details on additional conformations and on how Na+ and ligand influence the equilibrium between other states remains unknown. Here, double electron-electron resonance spectroscopy, structural modeling, and molecular dynamics are utilized to deduce ligand-dependent equilibria shifts of vSGLT in micelles. In the absence and presence of saturating amounts of Na+, vSGLT favors an inward-facing conformation. Upon binding both Na+ and sugar, the equilibrium shifts toward either an outward-facing or occluded conformation. While Na+ alone does not stabilize the outward-facing state, gating charge calculations together with a kinetic model of transport suggest that the resting negative membrane potential of the cell, absent in detergent-solubilized samples, may stabilize vSGLT in an outward-open conformation where it is poised for binding external sugars. In total, these findings provide insights into ligand-induced conformational selection and delineate the transport cycle of vSGLT

Disulfide cross-links in the interaction of a cataract-linked αA-crystallin mutant with βB1-crystallin

AbstractA number of αA-crystallin mutants are associated with hereditary cataract including cysteine substitution at arginine 49. We report the formation of affinity-driven disulfide bonds in the interaction of αA-R49C with βB1-crystallin. To mimic cysteine thiolation in the lens, βB1-crystallin was modified by a bimane probe through a disulfide linkage. Our data suggest a mechanism whereby a transient disulfide bond occurs between αA- and βB1-crystallin followed by a disulfide exchange with cysteine 49 of a neighboring αA-crystallin subunit. This is the first investigation of disulfide bonds in the confine of the chaperone/substrate complex where reaction rates are favored by orders of magnitude. Covalent protein cross-links are a hallmark of age-related cataract and may be a factor in its inherited form

A Network of Transdisciplinary Observation Mechanisms as a Digital Source of Knowledge on Rangeland, to Communicate and Exchange at Local, Regional and Global Scales

For several decades, interventions geared towards the development of drylands have been the catalysts of much change in a rapidly evolving world, and learning how to build sustainable trajectories that take into account both cultural and contextual variations is becoming of increasingly great import. As local problems become intertwined, and given the difficulty of large-scale collective action, understanding these dynamics requires cognizance of all levels of knowledge governance systems and their interactions. So far as rangelands are concerned, the lack of easily accessible documentation encompassing all knowledge to date is a major impediment to their sustainable development. With this in mind, polycentric governance would allow for centralized decision-making, which would then give rise to solutions that could be adapted to local conditions. Recent advances in technology and the proliferation of data are creating new opportunities for monitoring the progress and performance of multi-scale development efforts, and indeed new and non-traditional data sources will be paramount to the success of such endeavours. For instance, participatory observation is an emerging example of a non-traditional data source that is already making a significant contribution, and has fostered engagement at the community level. We seek to demonstrate the value of implementing transdisciplinary observation mechanisms—here, in relation to Southern Countries’ pastoral systems—and to provide concrete examples of how such mechanisms can be adopted for mainstreaming the use of data from a variety of sources, thereby facilitating the implementation of a sustainable development agenda as part of a continuous learning process. This project has been managed within the framework of the Agadir Platform, infrastructure supported and implemented by Ibn-Zohr University, Morocco

Chapter 9

Niger is a landlocked country in West Africa located between 11°37 ´ and 23°23 ´ north latitude and between 00°10 ´ and 16°00 ´ east longitude, with an area of 1,267,000 square kilometers. Niger shares borders wit

Structure and Dynamics of AMPA Receptor GluA2 in Resting, Pre-Open, and Desensitized States

SummaryIonotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory signaling in the nervous system. Despite the profound importance of iGluRs to neurotransmission, little is known about the structures and dynamics of intact receptors in distinct functional states. Here, we elucidate the structures of the intact GluA2 AMPA receptor in an apo resting/closed state, in an activated/pre-open state bound with partial agonists and a positive allosteric modulator, and in a desensitized/closed state in complex with fluorowilliardiine. To probe the conformational properties of these states, we carried out double electron-electron resonance experiments on cysteine mutants and cryoelectron microscopy studies. We show how agonist binding modulates the conformation of the ligand-binding domain “layer” of the intact receptors and how, upon desensitization, the receptor undergoes large conformational rearrangements of the amino-terminal and ligand-binding domains. We define mechanistic principles by which to understand antagonism, activation, and desensitization in AMPA iGluRs

Room-Temperature Distance Measurements of Immobilized Spin-Labeled Protein by DEER/PELDOR

Nitroxide spin labels are used for double electron-electron resonance (DEER) measurements of distances between sites in biomolecules. Rotation of gem-dimethyls in commonly used nitroxides causes spin echo dephasing times (Tm) to be too short to perform DEER measurements at temperatures between ∼80 and 295 K, even in immobilized samples. A spirocyclohexyl spin label has been prepared that has longer Tm between 80 and 295 K in immobilized samples than conventional labels. Two of the spirocyclohexyl labels were attached to sites on T4 lysozyme introduced by site-directed spin labeling. Interspin distances up to ∼4 nm were measured by DEER at temperatures up to 160 K in water/glycerol glasses. In a glassy trehalose matrix the Tm for the doubly labeled T4 lysozyme was long enough to measure an interspin distance of 3.2 nm at 295 K, which could not be measured for the same protein labeled with the conventional 1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3- (methyl)methanethio-sulfonate label

Room-Temperature Distance Measurements of Immobilized Spin-Labeled Protein by DEER/PELDOR

Nitroxide spin labels are used for double electron-electron resonance (DEER) measurements of distances between sites in biomolecules. Rotation of gem-dimethyls in commonly used nitroxides causes spin echo dephasing times (Tm) to be too short to perform DEER measurements at temperatures between ∼80 and 295 K, even in immobilized samples. A spirocyclohexyl spin label has been prepared that has longer Tm between 80 and 295 K in immobilized samples than conventional labels. Two of the spirocyclohexyl labels were attached to sites on T4 lysozyme introduced by site-directed spin labeling. Interspin distances up to ∼4 nm were measured by DEER at temperatures up to 160 K in water/glycerol glasses. In a glassy trehalose matrix the Tm for the doubly labeled T4 lysozyme was long enough to measure an interspin distance of 3.2 nm at 295 K, which could not be measured for the same protein labeled with the conventional 1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3- (methyl)methanethio-sulfonate label

New Material of the Earliest Hominid from the Upper Miocene of Chad

Anthropolog