Site Directed Spin Labeling Electron Paramagnetic Resonance Technique For Protein Structure Characterization

Site Directed Spin Labeling Electron Paramagnetic Resonance (SDSL-EPR) has emerged as an effective method to study the structure and function of bio-macromolecules, especially proteins. SDSL-EPR measurement can be performed on samples in aqueous solution at ambient temperature, a condition which is very similar as the natural environment of bio-macromolecules. Acquired EPR signals of spin labeled proteins can provide detailed information on side chain dynamics, solvent accessibility and the distances between two residues, as well as the real-time information on the conformation change of proteins at different states. Due to its high sensitivity and no limits on the size of proteins, SDSL-EPR has been proven to be an very effective tool to investigate membrane proteins. In this dissertation, we will apply SDSL-EPR to develop an inter-residue distance-measurement method which can be conducted for proteins in aqueous solution at physiological temperatures. Also, SDSL-EPR will be used to examine the structural and dynamic properties of the transmembrane domain of integrin β1a. There are five chapters in this dissertation.Chapter1contains a brief introduction to the theory of Electron Paramagnetic Resonance spectroscopy and its applications. An introduction of EPR spectrometer is also included in this part.Chapter2introduces the SDSL-EPR technique and its applications in protein studies. SDSL-EPR involves introducing a spin label into a specific site on the protein and collecting the resultant EPR signals of the spin-labeled protein. Moreover, information of side chain dynamics, solvent accessibility and inter-residue distances are encoded in the EPR spectrum. Here, methods and examples are introduced to illustrate the three kinds of information obtained by SDSL-EPR.Chapter3introduces the Continuous Wave EPR (CW-EPR) distance measurement method, which is based on analyzing the spectra broadening effect due to the dipolar interaction between interacting spins. Normally, the measurement is carried out at low temperature (below200K) to meet the "rigid lattice" condition. However, the physiological characters of proteins are lost at such low temperature. So it is very necessary to develop a method which can precisely extract the inter-residue distances at physiological condition. Here, we introduced spin labels onto two flexible sites of T4lysozyme and used CW-EPR to conduct distance measurement at ambient temperature (298K). By elevating the viscosity of protein solution, the rotational correlation time of spin labeled side chain was increased to be sufficiently long not to average the anisotropy of dipolar broadening. The distances derived at ambient temperature are in good agreement with distances obtained at low temperature.SDSL-EPR has been proven to have great advantages in studying membrane proteins. In Chapter4, we carry out an in-depth study on the structural and dynamic properties of membrane proteins using SDSL-EPR technique. Here, the structural and dynamic properties of the integrin β1a in detergent micelles or liposomes are studied by SDSL-EPR. Remarkable differences in dynamics or topology were observed between the integrin β1a in detergent micelles and in liposomes. The mechanism for the homo-oligomerization between transmembrane domains of integrin was also discussed.Chapter5introduces the recent advances in EPR spectroscopy and their applications in protein investigation. An outlook into the future developments of SDSL-EPR is also included in this chapter.