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Riboswitch RNAs regulate gene expression by conformational changes induced by environmental conditions and specific ligand binding. The guanidine-II riboswitch is proposed to bind the small molecule guanidinium and to subsequently form a kissing loop interaction between the P1 and P2 hairpins. While an interaction was shown for isolated hairpins in crystallization and electron paramagnetic resonance experiments, an intrastrand kissing loop formation has not been demonstrated. Here, we report the first evidence of this interaction in cis in a ligand and Mg2+ dependent manner. Using single-molecule FRET spectroscopy and detailed structural information from coarse-grained simulations, we observe and characterize three interconvertible states representing an open and kissing loop conformation as well as a novel Mg2+ dependent state for the guanidine-II riboswitch from E. coli. The results further substantiate the proposed switching mechanism and provide detailed insight into the regulation mechanism for the guanidine-II riboswitch class. Combining single molecule experiments and coarse-grained simulations therefore provides a promising perspective in resolving the conformational changes induced by environmental conditions and to yield molecular insights into RNA regulation.
Maximum likelihood estimates of diffusion coefficients from single-particle tracking experiments
(2021)
Single-molecule localization microscopy allows practitioners to locate and track labeled molecules in biological systems. When extracting diffusion coefficients from the resulting trajectories, it is common practice to perform a linear fit on mean-squared-displacement curves. However, this strategy is suboptimal and prone to errors. Recently, it was shown that the increments between the observed positions provide a good estimate for the diffusion coefficient, and their statistics are well-suited for likelihood-based analysis methods. Here, we revisit the problem of extracting diffusion coefficients from single-particle tracking experiments subject to static noise and dynamic motion blur using the principle of maximum likelihood. Taking advantage of an efficient real-space formulation, we extend the model to mixtures of subpopulations differing in their diffusion coefficients, which we estimate with the help of the expectation–maximization algorithm. This formulation naturally leads to a probabilistic assignment of trajectories to subpopulations. We employ the theory to analyze experimental tracking data that cannot be explained with a single diffusion coefficient. We test how well a dataset conforms to the assumptions of a diffusion model and determine the optimal number of subpopulations with the help of a quality factor of known analytical distribution. To facilitate use by practitioners, we provide a fast open-source implementation of the theory for the efficient analysis of multiple trajectories in arbitrary dimensions simultaneously.
Nitrate is an abundant nutrient and electron acceptor throughout Earth’s biosphere. Virtually all nitrate in nature is produced by the oxidation of nitrite by the nitrite oxidoreductase (NXR) multiprotein complex. NXR is a crucial enzyme in the global biological nitrogen cycle, and is found in nitrite-oxidizing bacteria (including comammox organisms), which generate the bulk of the nitrate in the environment, and in anaerobic ammonium-oxidizing (anammox) bacteria which produce half of the dinitrogen gas in our atmosphere. However, despite its central role in biology and decades of intense study, no structural information on NXR is available. Here, we present a structural and biochemical analysis of the NXR from the anammox bacterium Kuenenia stuttgartiensis, integrating X-ray crystallography, cryo-electron tomography, helical reconstruction cryo-electron microscopy, interaction and reconstitution studies and enzyme kinetics. We find that NXR catalyses both nitrite oxidation and nitrate reduction, and show that in the cell, NXR is arranged in tubules several hundred nanometres long. We reveal the tubule architecture and show that tubule formation is induced by a previously unidentified, haem-containing subunit, NXR-T. The results also reveal unexpected features in the active site of the enzyme, an unusual cofactor coordination in the protein’s electron transport chain, and elucidate the electron transfer pathways within the complex.
Under natural conditions, the visual system often sees a given input repeatedly. This provides an opportunity to optimize processing of the repeated stimuli. Stimulus repetition has been shown to strongly modulate neuronal-gamma band synchronization, yet crucial questions remained open. Here we used magnetoencephalography in 30 human subjects and find that gamma decreases across ~10 repetitions and then increases across further repetitions, revealing plastic changes of the activated neuronal circuits. Crucially, changes induced by one stimulus did not affect responses to other stimuli, demonstrating stimulus specificity. Changes partially persisted when the inducing stimulus was repeated after 25 minutes of intervening stimuli. They were strongest in early visual cortex and increased interareal feedforward influences. Our results suggest that early visual cortex gamma synchronization enables adaptive neuronal processing of recurring stimuli. These and previously reported changes might be due to an interaction of oscillatory dynamics with established synaptic plasticity mechanisms.
Cysteine cross-linking in native membranes establishes the transmembrane architecture of Ire1
(2021)
The ER is a key organelle of membrane biogenesis and crucial for the folding of both membrane and secretory proteins. Sensors of the unfolded protein response (UPR) monitor the unfolded protein load in the ER and convey effector functions for maintaining ER homeostasis. Aberrant compositions of the ER membrane, referred to as lipid bilayer stress, are equally potent activators of the UPR. How the distinct signals from lipid bilayer stress and unfolded proteins are processed by the conserved UPR transducer Ire1 remains unknown. Here, we have generated a functional, cysteine-less variant of Ire1 and performed systematic cysteine cross-linking experiments in native membranes to establish its transmembrane architecture in signaling-active clusters. We show that the transmembrane helices of two neighboring Ire1 molecules adopt an X-shaped configuration independent of the primary cause for ER stress. This suggests that different forms of stress converge in a common, signaling-active transmembrane architecture of Ire1.
The structure and flexibility of RNA depends sensitively on the microenvironment. Using pulsed electron-electron double-resonance (PELDOR)/double electron-electron resonance (DEER) spectroscopy combined with advanced labeling techniques, we show that the structure of double-stranded RNA (dsRNA) changes upon internalization into Xenopus lævis oocytes. Compared to dilute solution, the dsRNA A-helix is more compact in cells. We recapitulate this compaction in a densely crowded protein solution. Atomic-resolution molecular dynamics simulations of dsRNA semi-quantitatively capture the compaction, and identify non-specific electrostatic interactions between proteins and dsRNA as a possible driver of this effect.
Funktionelle und strukturelle Charakterisierung von SLC-Transportern in eukaryotischen Systemen
(2018)
Die evolutionäre Voraussetzung für die Entwicklung komplexer, differenzierter Organismen bildet die Separierung der Zelle in Reaktionsräume, die so genannte Kompartimentierung. Das Prinzip der Kompartimentierung ermöglicht zahlreiche lebensnotwendige, biochemische Prozesse, wie die Konservierung von Energie durch Protonengradienten in der Atmungskette oder parallele, gegenläufige Stoffwechselwege. Zelluläre Kompartimente werden häufig durch Biomembranen gebildet, welche aus einer zweilagigen Lipidschicht bestehen. Lipidmoleküle in einer Zelle sind meistens amphipathisch, das bedeutet, sie bestehen aus einer polaren, hydrophilen Kopfgruppe und einem unpolaren, hydrophopen Ende (Abbildung 1). Die Lipidzusammensetzung in einer Biomembran ist sehr divers und unterscheidet sich in verschiedenen Organismen und Organellen. Phosphoglyceride bilden den Hauptbestandteil der Lipidschicht. Phosphoglyceride besteht aus einem Glycerin Rückgrat, welches an dem C1- und C2-Atom mit zwei Fettsäuren verestert und an dem C3-Atom mit einem Phosphorsäurediester verbunden ist. ...
High-resolution cryo-EM structures of respiratory complex I: Mechanism, assembly, and disease
(2019)
Respiratory complex I is a redox-driven proton pump, accounting for a large part of the electrochemical gradient that powers mitochondrial adenosine triphosphate synthesis. Complex I dysfunction is associated with severe human diseases. Assembly of the one-megadalton complex I in the inner mitochondrial membrane requires assembly factors and chaperones. We have determined the structure of complex I from the aerobic yeast Yarrowia lipolytica by electron cryo-microscopy at 3.2-Å resolution. A ubiquinone molecule was identified in the access path to the active site. The electron cryo-microscopy structure indicated an unusual lipid-protein arrangement at the junction of membrane and matrix arms that was confirmed by molecular simulations. The structure of a complex I mutant and an assembly intermediate provide detailed molecular insights into the cause of a hereditary complex I-linked disease and complex I assembly in the inner mitochondrial membrane.
Mechanistic understanding of dynamic membrane proteins such as transporters, receptors, and channels requires accurate depictions of conformational ensembles, and the manner in which they interchange as a function of environmental factors including substrates, lipids, and inhibitors. Spectroscopic techniques such as electron spin resonance (ESR) pulsed electron–electron double resonance (PELDOR), also known as double electron–electron resonance (DEER), provide a complement to atomistic structures obtained from x-ray crystallography or cryo-EM, since spectroscopic data reflect an ensemble and can be measured in more native solvents, unperturbed by a crystal lattice. However, attempts to interpret DEER data are frequently stymied by discrepancies with the structural data, which may arise due to differences in conditions, the dynamics of the protein, or the flexibility of the attached paramagnetic spin labels. Recently, molecular simulation techniques such as EBMetaD have been developed that create a conformational ensemble matching an experimental distance distribution while applying the minimal possible bias. Moreover, it has been proposed that the work required during an EBMetaD simulation to match an experimentally determined distribution could be used as a metric with which to assign conformational states to a given measurement. Here, we demonstrate the application of this concept for a sodium-coupled transport protein, BetP. Because the probe, protein, and lipid bilayer are all represented in atomic detail, the different contributions to the work, such as the extent of protein backbone movements, can be separated. This work therefore illustrates how ranking simulations based on EBMetaD can help to bridge the gap between structural and biophysical data and thereby enhance our understanding of membrane protein conformational mechanisms.