Application Of Electron Spin Resonance In Protein Structure Determination And Function Analysis

In recent years, the application of ESR (electron spin resonance) to the studies of structural biology showed great success to various problems on resolving structure of protein and conformational change of protein molecules.A domain is the basic building block of the structure of a protein and an evolutionarily independent structural unit of a functional protein. Certain protein domains have clearly defined functions and act as cornerstones in a variety of cellular processes. Genomic analyses have shown that over70%of eukaryotic proteins are multi-domain proteins. The modular nature of a multi-domain protein provides stability and new cooperative functions. The domains of these proteins are normally connected by flexible linkers. Therefore, determining the relative orientation of these domains is critical to our understanding of domain interactions and the functional mechanisms of a multi-domain protein. Combining distance restraints from NMR-PRE(paramagnetic resonance enhancement) and ESR-DEER(double electron electron resonance), the two domain structure of Rv0899was determined, showing potential for further applcation for structure determination of multi-domain proteins.DEER was performed on two site directed spin labed mutant of DAGK protein. The labeled calculation of two obtained structures of DAGK from crystallography and solution NMR was conducted. Results from crystal structure of DAGK was found to fit the experiment better.ESRcould also be applied for studies of magnetic properties. Antiferromagnetism is a short-distance exchange interaction between anti-parallel spins normally observed in compounds containing transition metal elements (with3d/4f orbital). Antiferromagnetism was observed from Fe-S clusters in complex I respiratory pathway. Ndil is a type-II complex I enzyme and our previous structure illustrated no presence of Fe-S clusters but two bound ubiquinones. Here, antiferromagnetism was observed in Ndil protein using temperature dependent electron spin resonance experiments and the negative Heisenberg exchange J constants were quantified using van Vleck formula. Quantum chemical calculations based on Ndil structural coordinates demonstrated that the observed two antiferromagnetic interactions originated from p-orbital based electron-transfer processes and was in the through-space mechanism. Temperature dependent ESR experiment on mutant of Ndil further confirmed the antiferromagnetic interactions came from the electron-transfer processes rather than the dipole-dipole coupling between two spins. The first-time observed antiferromagnetism from p-orbital based mixed-stack electron transfer in proteins expanded our understanding of antiferromagnetism in nature, and provided insights for fabrications of materials with special magnetic properties using biological samples.