Electron Paramagnetic Resonance as a Tool for Studying Membrane Proteins

Sahu, Indra D.; Lorigan, Gary A.

doi:10.3390/biom10050763

Cited by 45 publications

(41 citation statements)

References 180 publications

(339 reference statements)

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“…In recent years, great efforts have been made to develop technical and methodological improvements in structural biology approaches for studying membrane proteins. However, the challenges introduced in sample preparation of membrane proteins in suitable membrane environments and their complex behavior in lipid bilayer membrane environments severely limit the application of biophysical approaches for studying membrane proteins [35][36][37][38][39][40][41]. X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy are the two most successful and widely utilized biophysical techniques for obtaining structural data on protein systems.…”

Section: Challenges and Recent Improvements Using Biophysical Techniqmentioning

confidence: 99%

“…CW-EPR spectroscopy of spin-labeled macromolecules can provide structural dynamics of nitroxide side-chain, solvent accessibility, solvent polarity, and intra-or intermolecular distances between two nitroxides [5,6,41,59,72,83]. The EPR spectral lineshape analysis of the series of spin-labeled protein sequences can be used to probe the secondary structural information of the protein systems [41,[84][85][86][87]. (B) Cartoon representation of the structure of MTSL and the resulting side-chain produced by reaction with a cysteine residue (L134C) and (C) the structure of BSL (bifunctional spin label) and the resulting side-chain produced by reaction with cysteine residues (L134C and I138C) on a KCNQ1-VSD membrane protein.…”

Section: Site Directed Spin Labeling (Sdsl) Approaches For Epr Spectrmentioning

confidence: 99%

“…Distance measurements obtained by using double SDSL EPR spectroscopy is very popular and a rapidly growing structural biology technique to probe secondary, tertiary and quaternary structures of bio-macromolecules [41,72,[88][89][90]. Distances and distance distribution can be also utilized to obtain conformational rearrangements or complex formation of membrane proteins.…”

Section: Site Directed Spin Labeling (Sdsl) Approaches For Epr Spectrmentioning

confidence: 99%

“…Nitroxide spin labeling based SDSL DEER spectroscopy is a widely used biophysical technique for studying secondary, tertiary and quaternary structures, and conformational dynamics of a numerous membrane proteins [6,11,32,41,83,98,[104][105][106][107][108][109][110]. However, other spin labels including functionalized chelators of paramagnetic lanthanides (Gd III ), carbonbased radicals ((trityl), and metals such as copper (Cu II ) have been recently applied for DEER experiments for studying membrane proteins [111][112][113][114][115][116].…”

Section: Site Directed Spin Labeling (Sdsl) Approaches For Epr Spectrmentioning

confidence: 99%

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Probing Structural Dynamics of Membrane Proteins Using Electron Paramagnetic Resonance Spectroscopic Techniques

Sahu

Lorigan

2021

Biophysica

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Membrane proteins are essential for the survival of living organisms. They are involved in important biological functions including transportation of ions and molecules across the cell membrane and triggering the signaling pathways. They are targets of more than half of the modern medical drugs. Despite their biological significance, information about the structural dynamics of membrane proteins is lagging when compared to that of globular proteins. The major challenges with these systems are low expression yields and lack of appropriate solubilizing medium required for biophysical techniques. Electron paramagnetic resonance (EPR) spectroscopy coupled with site directed spin labeling (SDSL) is a rapidly growing powerful biophysical technique that can be used to obtain pertinent structural and dynamic information on membrane proteins. In this brief review, we will focus on the overview of the widely used EPR approaches and their emerging applications to answer structural and conformational dynamics related questions on important membrane protein systems.

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Section: Challenges and Recent Improvements Using Biophysical Techniqmentioning

confidence: 99%

Section: Site Directed Spin Labeling (Sdsl) Approaches For Epr Spectrmentioning

confidence: 99%

Section: Site Directed Spin Labeling (Sdsl) Approaches For Epr Spectrmentioning

confidence: 99%

Section: Site Directed Spin Labeling (Sdsl) Approaches For Epr Spectrmentioning

confidence: 99%

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Probing Structural Dynamics of Membrane Proteins Using Electron Paramagnetic Resonance Spectroscopic Techniques

Sahu

Lorigan

2021

Biophysica

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“…Spectroscopic approaches are powerful tools for monitoring protein conformational dynamics that are not captured in static structures (Elvington and Maduke, 2008;McHaourab et al, 2011;Sekhar and Kay, 2019;Sahu and Lorigan, 2020). Single-molecule Förster resonance energy transfer (smFRET) has emerged as an important player in the spectroscopic toolbox, as a method that can monitor conformational transitions in real time (Zhao et al, 2010;Landes et al, 2011;Akyuz et al, 2015;Dolino et al, 2015;Vafabakhsh et al, 2015;Juette et al, 2016;Dyla et al, 2017;Han et al, 2017;Lerner et al, 2018;Ren et al, 2018;Wang et al, 2018;de Boer et al, 2019;MacLean et al, 2019;Mazal and Haran, 2019;Zhu et al, 2019;Carrillo et al, 2020;Durham et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

Single-molecule FRET monitors CLC transporter conformation and subunit independence

Cheng

Krishnamoorti

Berka

et al. 2020

Preprint

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CLC transporters catalyze the exchange of chloride ions for protons across cellular membranes. As secondary active transporters, CLCs must alternately allow ion permeation to the extracellular and intracellular sides of the membrane, adopting outward-facing and inward-facing conformational states. Here, we use single-molecule Forster resonance energy transfer (smFRET) to monitor the conformational state of CLC-ec1, an E. coli homolog for which high-resolution structures of occluded and outward-facing states are known. Since each subunit within the CLC homodimer contains its own transport pathways for chloride and protons, we developed a labeling strategy to follow conformational change within a subunit, without crosstalk from the second subunit of the dimer. Using this strategy, we evaluated smFRET efficiencies for labels positioned on the extracellular side of the protein, to monitor the status of the outer permeation pathway. When [H+] is increased to enrich the outward-facing state, the smFRET efficiencies for this pair decrease. In a triple-mutant CLC-ec1 that mimics the protonated state of the protein and is known to favor the outward-facing conformation, the lower smFRET efficiency is observed at both low and high [H+]. These results confirm that the smFRET assay is following the transition to the outward-facing state and demonstrate the feasibility of using smFRET to monitor the relatively small (~1 Angstrom) motions involved in CLC transporter conformational change. Using the smFRET assay, we show that the conformation of the partner subunit does not influence the conformation of the subunit being monitored by smFRET, thus providing evidence for the independence of the two subunits in the transport process.

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Deep‐UV resonance Raman spectroscopy of hydrated and dehydrated model α‐helical transmembrane peptides in liposomes

Wei

JiJi

Zare

et al. 2021

J Raman Spectroscopy

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Water hydrogen bonding (H‐bonding) to α‐helical transmembrane (TM) peptides is fundamental to better understand the behavior and function of α‐helical peptides, disease pathways, and the development of new drugs. Deep‐UV resonance Raman (dUVRR) spectroscopy is a non‐destructive technique amenable to both lipophilic and aqueous environments, which is an excellent and convenient approach for studying water H‐bonding (or water accessibility) to α‐helical TM peptides in a membrane mimicking environment. The dUVRR results indicate that water molecules can access the lipid membrane and form H‐bonds with carbonyl groups along α‐helical backbones. Raman bands at ~1,629 and ~1,672 cm−1 can be used to monitor the hydration and dehydration conditions along TM α‐helices. Two bands at ~1,300 and ~1,340 cm−1 are also potential characteristic features of the dehydration and hydration along the α‐helices in a membrane environment.

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Electron Paramagnetic Resonance as a Tool for Studying Membrane Proteins

Cited by 45 publications

References 180 publications

Probing Structural Dynamics of Membrane Proteins Using Electron Paramagnetic Resonance Spectroscopic Techniques

Probing Structural Dynamics of Membrane Proteins Using Electron Paramagnetic Resonance Spectroscopic Techniques

Single-molecule FRET monitors CLC transporter conformation and subunit independence

Deep‐UV resonance Raman spectroscopy of hydrated and dehydrated model α‐helical transmembrane peptides in liposomes

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