Lipid-Protein Interactions Defined by Multi-Frequency EPR

Electron paramagnetic resonance (EPR) spectroscopy is a powerful tool to elucidate the structure and dynamics of membrane proteins and protein-lipid interactions. High magnetic fields increase EPR sensitivity and broaden its practice through increased time scale and g-tensor resolution. However, the application of high-field EPR is hindered by the lack of suitable techniques and methodologies. The focus of this work is to develop high-field and multi-frequency EPR methods for characterizing membrane proteins, with specific aims to investigate lipid-protein interactions of an antibacterial peptide (AA1) and HIV-1 envelope protein gp41. These methods include: i). A thin-layer cylindrical sample holder designed for studying low-yield biological samples at 94 GHz (W-band). The holder is non-resonant and operating in induction-mode for continuous wave EPR analyses. A concentration sensitivity of 2 µM was achieved for a spin-label standard and 20-30 µM for the biological aqueous samples, representing a ~10-fold enhancement compared to a cylindrical TE₀₁₁ resonator on a commercial Bruker W-band spectrometer (Chapter 3). ii). Magnetic alignment of cholesterol-containing bicelles at high fields. Membranes with high cholesterol content are typical in mammalian cells and are important for biophysical studies mimicking native conditions, such as using aligned bicelles to study protein orientation in membranes. In this study, field dependence of magnetic alignment of cholesterol-containing bicelles was investigated and enhanced bicelle alignment at high fields were demonstrated (Chapter 5). Other EPR techniques developed here include: 1) Membrane permeability analysis to assess membrane disruption by proteins; 2) Lipid lateral ordering induced by protein binding defined using EPR at 94 GHz; 3) A new application of EPR power saturation methods to determine membrane thinning. These EPR techniques were applied to define the molecular basis of the antimicrobial activity of AA1. The results show that the peptide selectively permeates and structurally modifies negatively charged bacterial-mimic membranes. In contrast, cholesterol-containing neutral membranes mimicking mammalian cells were minimally affected by the molecule. Based on combined EPR analyses, we proposed a “carpet-like” mechanism for the antimicrobial activities of AA1. The results provide implications for the development of effective AMPs with robust antibacterial activities against antibiotic-resistant microbes (Chapter 4). Additionally, magnetic alignment of bicelles was used to study the membrane bound region (MPERTM) of HIV-1 envelope protein gp41. Using a rigid spin-label–TOAC, the structural information of the MPERTM in membranes was determined by studying backbone dynamics, immersion depths and helical tilt in bicelles. The results indicate the existence of three separated helical segments of the MPERTM with different motional time-scales. The effect of lipid composition and cholesterol content on the conformation of the MPERTM in the membrane were also investigated (Chapter 6). In summary, we have demonstrated and further developed the applications of multi-frequency and high-field EPR in studying membrane proteins and lipid-protein interactions.