Characterization of the Human KCNQ1 Voltage Sensing Domain (VSD) in Lipodisq Nanoparticles for Electron Paramagnetic Resonance (EPR) Spectroscopic Studies of Membrane Proteins

Sahu, Indra D.; Dixit, Gunjan; Reynolds, Warren; Kaplevatsky, Ryan; Harding, Benjamin D.; Jaycox, Colleen K.; McCarrick, Robert M.; Lorigan, Gary A.

doi:10.1021/acs.jpcb.9b11506

Cited by 15 publications

(12 citation statements)

References 118 publications

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“…Pulsed electron-electron double resonance (PELDOR) or double electron-electron resonance (DEER) spectroscopy has been proven to be a powerful method for investigating protein structure and dynamics ( 60 , 61 , 62 , 63 ). In particular, the method has been successful in assigning conformation, oligomerization, and folding under physiological conditions or within lipid environment for a variety of membrane proteins ( 14 , 37 , 38 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 ) and is capable of offering subangstrom accuracy ( 39 , 60 ). Electron spin envelope echo modulation (ESEEM) spectroscopy has been used for measuring site-specific deuterium (solvent) accessibility in membrane proteins ( 14 , 73 , 74 , 75 ), and the method is a valuable tool for identifying lipid/detergent buried or exposed membrane protein sites.…”

Section: Introductionmentioning

confidence: 99%

In-Lipid Structure of Pressure-Sensitive Domains Hints Mechanosensitive Channel Functional Diversity

Kapsalis

Bode

et al. 2020

Biophysical Journal

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The mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis has been used as a structural model for rationalizing functional observations in multiple MscL orthologs. Although these orthologs adopt similar structural architectures, they reportedly present significant functional differences. Subtle structural discrepancies on mechanosensitive channel nanopockets are known to affect mechanical gating and may be linked to large variability in tension sensitivity among these membrane channels. Here, we modify the nanopocket regions of MscL from Escherichia coli and M. tuberculosis and employ PELDOR/DEER distance and 3pESEEM deuterium accessibility measurements to interrogate channel structure within lipids, in which both channels adopt a closed conformation. Significant in-lipid structural differences between the two constructs suggest a more compact E. coli MscL at the membrane inner-leaflet, as a consequence of a rotated TM2 helix. Observed differences within lipids could explain E. coli MscL’s higher tension sensitivity and should be taken into account in extrapolated models used for MscL gating rationalization.

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Section: Introductionmentioning

confidence: 99%

In-Lipid Structure of Pressure-Sensitive Domains Hints Mechanosensitive Channel Functional Diversity

Kapsalis

Bode

et al. 2020

Biophysical Journal

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“…Recent studies have successfully demonstrated the use of synthetic polymers to directly extract membrane proteins and reconstitute them in near-native lipid bilayer nanodiscs without using a detergent. [1][2][3][4][5][6][7] However, the presence of charge on the currently known polymers drastically limits their applications. [8,9] The high charge density of the polymer interferes with the purification by ion-exchange chromatography and also detrimental to study oppositely charged membrane proteins due to charge-charge interactions.…”

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confidence: 99%

Synthesis, Characterization, and Nanodisc Formation of Non‐ionic Polymers**

Ravula

Ramamoorthy

2021

Angewandte Chemie

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Although lipid nanodiscs are increasingly used in the structural studies of membrane proteins, drug delivery and other applications, the interaction between the nanodisc belt and the protein to be reconstituted is a major limitation. To overcome this limitation and to further broaden the scope of nanodiscs, a family of non-ionic amphiphilic polymers synthesized by hydrophobic functionalization of fructo-oligosaccharides/inulin is reported. We show the stability of lipid nanodiscs formed by these polymers against pH and divalent metal ions, and their magnetic-alignment properties. The reported results also demonstrate that the non-ionic polymers extract membrane proteins with unprecedented efficiency.

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“…However, no membrane mimetic systems are universally compatible to all membrane proteins requiring rigorous time-consuming optimization processes for their incorporation in a suitable membrane environment. Currently available and widely used membrane mimetic systems are detergent micelles, bicelles, liposomes, lipodiscs, and lipodisq nanoparticles/SMALPs (styrene maleic acid lipid particles) [12][13][14][15][16]. These membrane mimetic systems have their own benefits and limitations.…”

Section: Membrane Proteinsmentioning

confidence: 99%

“…Another recent example of the application of nitroxide based DEER spectroscopy is the study of the Human KCNQ1 voltage sensing domain (Q1-VSD) in lipodisq nanoparticles [14]. Human KCNQ1 is a voltage-gated potassium channel modulated by members of the KCNE protein family.…”

Section: Distance Measurement On Membrane Proteins Using Dual Sdsl Epmentioning

confidence: 99%

“…Q1-VSD is a four-transmembrane protein consisting of 149 amino acids representing the first four helices (S1−S4) of KCNQ1. Sahu et al performed four pulsed Q-band DEER experiments on Q1-VSD mutants (F123C−S143C) incorporated into various membrane mimetics including DPC micelles, LMPG micelles, DMPC/DPC bicelles, POPC/POPG lipid bilayers, and POPC/POPG lipodisq nanoparticles for distance measurements [14]. These distances on Q1-VSD were closely matching for each membrane environment within the experimental error suggesting that the secondary structural conformation of Q1-VSD is closely matching in all of these membrane environments.…”

Section: Distance Measurement On Membrane Proteins Using Dual Sdsl Epmentioning

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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Characterization of the Human KCNQ1 Voltage Sensing Domain (VSD) in Lipodisq Nanoparticles for Electron Paramagnetic Resonance (EPR) Spectroscopic Studies of Membrane Proteins

Cited by 15 publications

References 118 publications

In-Lipid Structure of Pressure-Sensitive Domains Hints Mechanosensitive Channel Functional Diversity

In-Lipid Structure of Pressure-Sensitive Domains Hints Mechanosensitive Channel Functional Diversity

Synthesis, Characterization, and Nanodisc Formation of Non‐ionic Polymers**

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

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