Probing the Structure of Membrane Proteins Using Pulsed EPR ESEEM Spectroscopy

Abstract

Current methods used for determining the secondary structure of membrane proteins require large sample sizes and have long data acquisition times. Therefore using pulsed EPR spectroscopic technique of Electron Spin Echo Envelope Modulation (ESEEM) is advantageous due to the requirement of less sample, short data acquisition, and high sensitivity. Using this technique, we can determine short to medium range distances (up to 8 A) between a site-specific nitroxide spin label (MTSL) and a nearby NMR-active isotopic labeled residue for a variety of peptides and proteins which ultimately determine the difference between an α-helical and β-sheet secondary structure. The information can be obtained using a three-pulse ESEEM sequence which allows for the detection of the fundamental nuclear spin transitions. It is possible to calculate the radial distance between a paramagnetic electron and a weakly coupled nucleus because the modulation depth produced by a weakly dipolar-coupled nucleus is inversely proportional to the radial distance. This new method is applied to two different membrane peptides, M2δ AChR, and KIGAKI, which have α-helical and β-sheet structural components respectively. A nitroxide spin probe is attached to a specific Cys residue at a given position I, and a 2H isotopic labeled residue is attached to a nearby residue (i + 3). The corresponding data shows that an α-helical structure will yield a very large 2H peak, whereas a β-sheet will yield a much smaller 2H peak, thus a difference can be observed between the two secondary structures. The ESEEM technique requires less protein sample (about 75 μg) and data acquisition only takes about 1 hour which makes it a simple, powerful, and effective technique in the determination of the secondary structure of a membrane protein.