Structure and dynamics of spin label side chains in membrane protein using molecular dynamics simulations

Abstract

Site-directed spin labeling (SDSL) combined with electron paramagnetic resonance (EPR) spectroscopy is a powerful approach to explore the structure and dynamics of biological complex systems. Nitroxide spin label is the most widely used probe for SDSL in the study of structure and function of biomolecules. However, the behavior of the nitroxide spin label is not well understood. This study aims to characterize the structural and dynamical properties of the spin label in the voltage-sensing domain (VSD) of KvAP, a potassium channel from the thermophilic archaea Aeropyrum pernix by mean of molecular dynamics (MD) studies. MD simulations for unlabeled and a total of 132 spin labeled KvAP-VSD models (on attachment for the residue position from 20 to 151) were carried out in a phospholipid bilayer to evaluate the dynamics of the nitroxide spin label side chain in membrane proteins. Structural flexibility, conformational freedom of rotatable bonds, and orientation of the nitroxide side chain were investigated in relation to the spin label internal motion in different microenvironments. The analysis of MD data showed that the backbone dynamics between the unlabeled and spin-labeled proteins were similar. The conformational freedoms of the nitroxide side chain vary with the surrounding environments. The spin label motion is more restricted inside the membrane than the outside membrane. From the simulations, the rotatable bonds within the nitroxide side chain adopt distinct values of dihedral angles indicating different dynamics of the spin label in different environments. The radial distribution function plots between the spin label and the nearest-neighbor surroundings allow to categorize the spin labels into water exposure, lipid-exposure, and protein burial. The dynamics of the spin label side-chains decreases in the following order: water-exposed > lipid-exposed > buried type. The MD results were compared with available experimental EPR data. The study provides a detailed understanding of the structure, conformation, and dynamics of the spin label.