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 over-expression systems are discussed.

Topology of active, membrane-embedded Bax in the context of a toroidal pore

“…4 and Supplementary Figure 3). ESEEM is a pulse-EPR technique, which detects interactions between the unpaired electron of a protein-attached spin label and nearby deuterons, which can be added via deuterated glycerol in the buffer [77][78][79][80]. When deuterium nuclei are close to the unpaired electron ( < 0.8 nm), a deuterium frequency modulation (around 2.3 MHz) is present in the ESEEM traces with an amplitude proportional to the concentration of nearby deuterons.…”

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

“…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.…”

Utilization of 13C-labeled amino acids to probe the α-helical local secondary structure of a membrane peptide using electron spin echo envelope modulation (ESEEM) spectroscopy

“…The data were then processed further using Hamming apodization and zero filling [29]. A cross-term averaged Fourier Transformation (FT) was then used on the resulting spectrum to generate the corresponding frequency domain with minimized dead time artifacts as has been previously established [14]. The 13 C peaks were observed at 3.5 MHz representing the 13 C Larmor frequency.…”

“…Previously, we utilized this pulsed EPR method to identify site-specific secondary structural motifs such as α-helices [1,4,11], β-sheets [12], and more recently, 310-helices [13]. Site-specific secondary structural information provides a better understanding of the functions, dynamics, and protein-lipid interactions of membrane proteins [4,14]. Not only has this ESEEM approach been successful for model peptides, it has also been utilized in over-expression systems to probe α-helical secondary structural motifs in both a water-soluble protein and a membrane protein, demonstrating the versatility of this technique [15,16].…”