scholarly journals Lipid Dynamics in Diisobutylene-Maleic Acid (DIBMA) Lipid Particles in Presence of Sensory Rhodopsin II

2021 ◽  
Vol 22 (5) ◽  
pp. 2548
Author(s):  
Natalia Voskoboynikova ◽  
Philipp Orekhov ◽  
Marine Bozdaganyan ◽  
Felix Kodde ◽  
Malte Rademacher ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Maleic Acid ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Coarse Grained ◽  
Spin Labels ◽  
Resonance Spectroscopy ◽  
Sensory Rhodopsin ◽  
Lipid Dynamics ◽  
Lipid Ratio ◽  
Lipid Particles

Amphiphilic diisobutylene/maleic acid (DIBMA) copolymers extract lipid-encased membrane proteins from lipid bilayers in a detergent-free manner, yielding nanosized, discoidal DIBMA lipid particles (DIBMALPs). Depending on the DIBMA/lipid ratio, the size of DIBMALPs can be broadly varied which makes them suitable for the incorporation of proteins of different sizes. Here, we examine the influence of the DIBMALP sizes and the presence of protein on the dynamics of encased lipids. As shown by a set of biophysical methods, the stability of DIBMALPs remains unaffected at different DIBMA/lipid ratios. Coarse-grained molecular dynamics simulations confirm the formation of viable DIBMALPs with an overall size of up to 35 nm. Electron paramagnetic resonance spectroscopy of nitroxides located at the 5th, 12th or 16th carbon atom positions in phosphatidylcholine-based spin labels reveals that the dynamics of enclosed lipids are not altered by the DIBMALP size. The presence of the membrane protein sensory rhodopsin II from Natronomonas pharaonis (NpSRII) results in a slight increase in the lipid dynamics compared to empty DIBMALPs. The light-induced photocycle shows full functionality of DIBMALPs-embedded NpSRII and a significant effect of the protein-to-lipid ratio during preparation on the NpSRII dynamics. This study indicates a possible expansion of the applicability of the DIBMALP technology on studies of membrane protein–protein interaction and oligomerization in a constraining environment.

scholarly journals Protein fold prediction using simulated DEER distance distributions and decay traces

2019 ◽  
Author(s):  
Diego del Alamo ◽  
Maxx Tessmer ◽  
Richard Stein ◽  
Jimmy B. Feix ◽  
Hassane S. Mchaourab ◽  
...  
Keyword(s):  
Experimental Data ◽  
Structure Prediction ◽  
De Novo ◽  
Time Window ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Coarse Grained ◽  
Spin Labels ◽  
Resonance Spectroscopy ◽  
Software Suite ◽  
Distance Distributions

AbstractDespite advances in sampling and scoring strategies, Monte Carlo modeling methods still struggle to accurately predict de novo the structures of large proteins, membrane proteins, or proteins of complex topologies. Previous approaches have addressed these shortcomings by leveraging sparse distance data gathered using site-directed spin labeling and electron paramagnetic resonance spectroscopy (SDSL-EPR) to improve protein structure prediction and refinement outcomes. However, existing computational implementations must choose between coarse-grained models of the spin label that lower the resolution and explicit models that lead to resource-intense simulations. Existing methods are further limited by their reliance on distance distributions, which are calculated from a primary refocused echo decay signal and may contain artifacts introduced during this processing step. Here, we addressed these challenges by developing RosettaDEER, a scoring method within the Rosetta software suite capable of simulating distance distributions and echo decay traces between spin labels fast enough to fold proteins de novo. We demonstrate that the accuracy of resulting distance distributions match or exceed those generated by more computationally intensive methods. Moreover, decay traces generated from these distributions recapitulate intermolecular background coupling parameters, allowing RosettaDEER to discriminate between poorly-folded and native-like models even when the time window of EPR data collection is truncated, rendering them unsuitable for accurate transformation into distance distributions. Finally, we demonstrate that one decay trace per nine residues is sufficient to predict the folds of Bax and the C-terminus of ExoU, two soluble proteins with surface-exposed amphipathic structural features that prevent the Rosetta energy function from correctly identifying native-like models in the absence of experimental data. These benchmarking results confirm that RosettaDEER can effectively leverage sparse experimental data for a wide array of modeling applications built into the Rosetta software suite.

scholarly journals Membrane-active Polymers: NCMNP13-x, NCMNP21-x and NCMNP21b-x for Membrane Protein Structural Biology

2022 ◽  
Author(s):  
Thi Kim Hoang Trinh ◽  
Claudio Catalano ◽  
Youzhong Guo
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Structural Biology ◽  
Maleic Acid ◽  
Drug Targets ◽  
Design Synthesis ◽  
Native Cell ◽  
Wide Range ◽  
Lipid Particles ◽  
High Resolution Structure

Membrane proteins are a ubiquitous group of bio-macromolecules responsible for many crucial biological processes and serve as drug targets for a wide range of modern drugs. Detergent-free technologies such as styrene-maleic acid lipid particles (SMALP), diisobutylene-maleic acid lipid particles (DIBMALP), and native cell membrane nanoparticles (NCMN) systems have recently emerged as revolutionary alternatives to the traditional detergent-based approaches for membrane protein research. NCMN systems aim to create a membrane-active polymer library suitable for high-resolution structure determination. Herein, we report our design, synthesis, characterization and comparative application analyses of three novel classes of NCMN polymers, NCMNP13-x, NCMNP21-x and NCMNP21b-x. Although each NCMN polymer can solubilize various model membrane proteins and conserve native lipids into NCMN particles, only the NCMNP21b-x series reveals lipid-protein particles with good buffer compatibility and high homogeneity suitable for single-particle cryo-EM analysis. Consequently, the NCMNP21b-x polymers that bring out high-quality NCMN particles are particularly attractive for membrane protein structural biology.

scholarly journals Protein functional dynamics from the rigorous global analysis of DEER data: Conditions, components, and conformations

2021 ◽  
Vol 153 (11) ◽  
Author(s):  
Eric J. Hustedt ◽  
Richard A. Stein ◽  
Hassane S. Mchaourab
Keyword(s):  
Spectroscopic Data ◽  
Dipolar Coupling ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Global Analysis ◽  
Experimental Studies ◽  
Spin Labeling ◽  
Spin Labels ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
Functional Dynamics

The potential of spin labeling to reveal the dynamic dimension of macromolecules has been recognized since the dawn of the methodology in the 1960s. However, it was the development of pulsed electron paramagnetic resonance spectroscopy to detect dipolar coupling between spin labels and the availability of turnkey instrumentation in the 21st century that realized the full promise of spin labeling. Double electron-electron resonance (DEER) spectroscopy has seen widespread applications to channels, transporters, and receptors. In these studies, distance distributions between pairs of spin labels obtained under different biochemical conditions report the conformational states of macromolecules, illuminating the key movements underlying biological function. These experimental studies have spurred the development of methods for the rigorous analysis of DEER spectroscopic data along with methods for integrating these distributions into structural models. In this tutorial, we describe a model-based approach to obtaining a minimum set of components of the distance distribution that correspond to functionally relevant protein conformations with a set of fractional amplitudes that define the equilibrium between these conformations. Importantly, we review and elaborate on the error analysis reflecting the uncertainty in the various parameters, a critical step in rigorous structural interpretation of the spectroscopic data.

scholarly journals Site Selective and Efficient Spin Labeling of Proteins with a Maleimide-Functionalized Trityl Radical for Pulsed Dipolar EPR Spectroscopy

Molecules ◽  
2019 ◽  
Vol 24 (15) ◽  
pp. 2735 ◽  
Author(s):  
J. Jacques Jassoy ◽  
Caspar A. Heubach ◽  
Tobias Hett ◽  
Frédéric Bernhard ◽  
Florian R. Haege ◽  
...  
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Spin Labeling ◽  
Single Frequency ◽  
Spin Labels ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
Site Directed Spin Labeling ◽  
Site Selective ◽  
Electron Paramagnetic

Pulsed dipolar electron paramagnetic resonance spectroscopy (PDS) in combination with site-directed spin labeling (SDSL) of proteins and oligonucleotides is a powerful tool in structural biology. Instead of using the commonly employed gem-dimethyl-nitroxide labels, triarylmethyl (trityl) spin labels enable such studies at room temperature, within the cells and with single-frequency electron paramagnetic resonance (EPR) experiments. However, it has been repeatedly reported that labeling of proteins with trityl radicals led to low labeling efficiencies, unspecific labeling and label aggregation. Therefore, this work introduces the synthesis and characterization of a maleimide-functionalized trityl spin label and its corresponding labeling protocol for cysteine residues in proteins. The label is highly cysteine-selective, provides high labeling efficiencies and outperforms the previously employed methanethiosulfonate-functionalized trityl label. Finally, the new label is successfully tested in PDS measurements on a set of doubly labeled Yersinia outer protein O (YopO) mutants.

scholarly journals Styrene maleic-acid lipid particles (SMALPs) into detergent or amphipols: An exchange protocol for membrane protein characterisation

2020 ◽  
Vol 1862 (5) ◽  
pp. 183192 ◽  
Author(s):  
Sophie J. Hesketh ◽  
David P. Klebl ◽  
Anna J. Higgins ◽  
Maren Thomsen ◽  
Isabelle B. Pickles ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Maleic Acid ◽  
Lipid Particles ◽  
Protein Characterisation

A radical approach to radicals

2022 ◽  
Vol 78 (1) ◽  
Author(s):  
Youjia Liu ◽  
Malgorzata Biczysko ◽  
Nigel W. Moriarty
Keyword(s):  
Protein Structure ◽  
Structural Information ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Molecular Model ◽  
Structural Parameters ◽  
Spin Labels ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
Widespread Application ◽  
Electron Paramagnetic

Nitroxide radicals are characterized by a long-lived spin-unpaired electronic ground state and are strongly sensitive to their chemical surroundings. Combined with electron paramagnetic resonance spectroscopy, these electronic features have led to the widespread application of nitroxide derivatives as spin labels for use in studying protein structure and dynamics. Site-directed spin labelling requires the incorporation of nitroxides into the protein structure, leading to a new protein–ligand molecular model. However, in protein crystallographic refinement nitroxides are highly unusual molecules with an atypical chemical composition. Because macromolecular crystallography is almost entirely agnostic to chemical radicals, their structural information is generally less accurate or even erroneous. In this work, proteins that contain an example of a radical compound (Chemical Component Dictionary ID MTN) from the nitroxide family were re-refined by defining its ideal structural parameters based on quantum-chemical calculations. The refinement results show that this procedure improves the MTN ligand geometries, while at the same time retaining higher agreement with experimental data.

Sign in / Sign up

Export Citation Format

Share Document