Use of Electron Paramagnetic Resonance To Solve Biochemical Problems

Sahu, Indra D.; McCarrick, Robert M.; Lorigan, Gary A.

doi:10.1021/bi400834a

Cited by 82 publications

(117 citation statements)

References 171 publications

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“…Nuclear Magnetic Resonance (NMR) is typically limited by sample size, but for large assemblies it can provide information such as direct interactions between monomers and relative orientations [4]. Electron Paramagnetic Resonance (EPR) allows determination of distance distributions (typically 1–10nm) that are generally more precise compared to fluorescence based methods [5]. …”

Section: Sources Of Spatial Informationmentioning

confidence: 99%

Integrative modelling of cellular assemblies

Joseph

Polles

Alber

et al. 2017

Current Opinion in Structural Biology

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A wide variety of experimental techniques can be used for understanding the precise molecular mechanisms underlying the activities of cellular assemblies. The inherent limitations of a single experimental technique often requires integration of data from complementary approaches, to gain sufficient insights into the assembly structure and function. Here, we review popular computational approaches for integrative modelling of cellular assemblies, including protein complexes and genomic assemblies. We provide recent examples of integrative models generated for such assemblies by different experimental techniques, especially including 3D electron microscopy (3D-EM) and HiC data, respectively. We highlight general concepts in integrative modelling and discuss the need for careful formulation and merging of different types of information.

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Section: Sources Of Spatial Informationmentioning

confidence: 99%

Integrative modelling of cellular assemblies

Joseph

Polles

Alber

et al. 2017

Current Opinion in Structural Biology

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“…The use of EPR spectroscopy in combination with SDSL was pioneered by Hubbell and colleagues and has been adopted for studying the molecular architecture and protein motions in a number of proteins (Altenbach, Marti, Khorana, & Hubbell, 1990; Bordignon, 2012; Columbus & Hubbell, 2002; Drescher, 2012; Fanucci & Cafiso, 2006; Hubbell et al, 2000; Hubbell, Lopez, Altenbach, & Yang, 2013; Hubbell, McHaourab, Altenbach, & Lietzow, 1996; Klare & Steinhoff, 2009; McHaourab et al, 1996; McHaourab, Steed, & Kazmier, 2011; Perozo, Cuello, Cortes, Liu, & Sompornpisut, 2002; Sahu et al, 2013). These techniques are best suited for studying the conformational dynamics of membrane proteins, especially in a native membrane environment.…”

Section: Sdsl and Epr Spectroscopy To Study Gating Mechanisms In Imentioning

confidence: 99%

“…This necessitates strategies that allow structural and functional measurements under similar conditions. In this aspect, site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy offer a unique perspective to multistep gating schemes (Columbus & Hubbell, 2002; Fanucci & Cafiso, 2006; Hubbell, Cafiso, & Altenbach, 2000; McHaourab, Lietzow, Hideg, & Hubbell, 1996; Sahu, McCarrick, & Lorigan, 2013). In comparison, comparison to X-ray and nuclear magnetic resonance, information from EPR is of relatively low resolution, however there are several advantages of this technique that surpass its limitations and make it highly complementary to other structural methods.…”

mentioning

confidence: 99%

EPR Studies of Gating Mechanisms in Ion Channels

Chakrapani

2015

Methods in Enzymology

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Ion channels open and close in response to diverse stimuli, and the molecular events underlying these processes are extensively modulated by ligands of both endogenous and exogenous origin. In the past decade, high-resolution structures of several channel types have been solved, providing unprecedented details of the molecular architecture of these membrane proteins. Intrinsic conformational flexibility of ion channels critically governs their functions. However, the dynamics underlying gating mechanisms and modulations are obscured in the information from crystal structures. While nuclear magnetic resonance spectroscopic methods allow direct measurements of protein dynamics, they are limited by the large size of these membrane protein assemblies in detergent micelles or lipid membranes. Electron paramagnetic resonance (EPR) spectroscopy has emerged as a key biophysical tool to characterize structural dynamics of ion channels and to determine stimulus-driven conformational transition between functional states in a physiological environment. This review will provide an overview of the recent advances in the field of voltage- and ligand-gated channels and highlight some of the challenges and controversies surrounding the structural information available. It will discuss general methods used in site-directed spin labeling and EPR spectroscopy and illustrate how findings from these studies have narrowed the gap between high-resolution structures and gating mechanisms in membranes, and have thereby helped reconcile seemingly disparate models of ion channel function.

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“…[12][13][14][15][16][17][18][19][20][21][22][23][24] EPR data have been used to investigate secondary structures, changes in the protein conformation, membrane-insertion depths, and protein-protein/protein-ligand interactions.…”

Section: Introductionmentioning

confidence: 99%

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Basak

Chatterjee

Chakrapani

2016

JoVE

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Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics. Video LinkThe video component of this article can be found at

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Use of Electron Paramagnetic Resonance To Solve Biochemical Problems

Cited by 82 publications

References 171 publications

Integrative modelling of cellular assemblies

Integrative modelling of cellular assemblies

EPR Studies of Gating Mechanisms in Ion Channels

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

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