Dataset Information

Determining the Secondary Structure of Membrane Proteins and Peptides Via Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy.

ABSTRACT: Revealing detailed structural and dynamic information of membrane embedded or associated proteins is challenging due to their hydrophobic nature which makes NMR and X-ray crystallographic studies challenging or impossible. Electron paramagnetic resonance (EPR) has emerged as a powerful technique to provide essential structural and dynamic information for membrane proteins with no size limitations in membrane systems which mimic their natural lipid bilayer environment. Therefore, tremendous efforts have been devoted toward the development and application of EPR spectroscopic techniques to study the structure of biological systems such as membrane proteins and peptides. This chapter introduces a novel approach established and developed in the Lorigan lab to investigate membrane protein and peptide local secondary structures utilizing the pulsed EPR technique electron spin echo envelope modulation (ESEEM) spectroscopy. Detailed sample preparation strategies in model membrane protein systems and the experimental setup are described. Also, the ability of this approach to identify local secondary structure of membrane proteins and peptides with unprecedented efficiency is demonstrated in model systems. Finally, applications and further developments of this ESEEM approach for probing larger size membrane proteins produced by overexpression systems are discussed.

SUBMITTER: Liu L

PROVIDER: S-EPMC4814931 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Determining the Secondary Structure of Membrane Proteins and Peptides Via Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy.

Liu Lishan L Mayo Daniel J DJ Sahu Indra D ID Zhou Andy A Zhang Rongfu R McCarrick Robert M RM Lorigan Gary A GA

Methods in enzymology 20150801

Revealing detailed structural and dynamic information of membrane embedded or associated proteins is challenging due to their hydrophobic nature which makes NMR and X-ray crystallographic studies challenging or impossible. Electron paramagnetic resonance (EPR) has emerged as a powerful technique to provide essential structural and dynamic information for membrane proteins with no size limitations in membrane systems which mimic their natural lipid bilayer environment. Therefore, tremendous effor ...[more]

PMID: 26477255

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Project description:This paper reports on a significant improvement of a new structural biology approach designed to probe the secondary structure of membrane proteins using the pulsed EPR technique of electron spin echo envelope modulation (ESEEM) spectroscopy. Previously, we showed that we could characterize an α-helical secondary structure with ESEEM spectroscopy using a (2)H-labeled Val side chain coupled with site-directed spin-labeling (SDSL). In order to further develop this new approach, molecular dynamic (MD) simulations were conducted on several different hydrophobic residues that are commonly found in membrane proteins. (2)H-SL distance distributions from the MD results indicated that (2)H-labeled Leu was a very strong candidate to significantly improve this ESEEM approach. In order to test this hypothesis, the secondary structure of the α-helical M2δ peptide of the acetylcholine receptor (AChR) incorporated into a bicelle was investigated with (2)H-labeled Leu d(10) at position 10 (i) and nitroxide spin labels positioned 1, 2, 3, and 4 residues away (denoted i+1 to i+4) with ESEEM spectroscopy. The ESEEM data reveal a unique pattern that is characteristic of an α-helix (3.6 residues per turn). Strong (2)H modulation was detected for the i+3 and i+4 samples, but not for the i+2 sample. The (2)H modulation depth observed for (2)H-labeled d(10) Leu was significantly enhanced (×4) when compared to previous ESEEM measurements that used (2)H-labeled d(8) Val. Computational studies indicate that deuterium nuclei on the Leu side chain are closer to the spin label when compared to Val. The enhancement of (2)H modulation and the corresponding Fourier Transform (FT) peak intensity for (2)H-labeled Leu significantly reduces the ESEEM data acquisition time for Leu when compared to Val. This research demonstrates that a different (2)H-labeled amino acid residue can be used as an efficient ESEEM probe further substantiating this important biophysical technique. Finally, this new method can provide pertinent qualitative structural information on membrane proteins in a short time (few minutes) at low sample concentrations (~50 μM).

Dataset Information

Determining the Secondary Structure of Membrane Proteins and Peptides Via Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy.

Publications

Determining the Secondary Structure of Membrane Proteins and Peptides Via Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy.

Similar Datasets

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