Chemphyschem : a European journal of chemical physics and physical chemistry 2016 7 21() doi 10.1002/cphc.201600726
A simple method, based on inversion modulated DEER EPR spectroscopy, is presented for determining populations of monomer and dimer in proteins (as well as any other biological macromolecules). The method is based on analysis of modulation depth versus ELDOR pulse flip angle. High accuracy is achieved by complete deuteration, extensive sampling of a large number of ELDOR pulse flip angle values, and combined analysis of differently labeled spin samples. We demonstrate the method using two different proteins: an obligate monomer exemplified by the small immunoglobulin binding B domain of protein A, and the p66 subunit of HIV-1 reverse transcriptase which exists as an equilibrium mixture of monomer and dimer species whose relative populations are affected by glycerol content. This information is crucial for quantitative analysis of distance distributions involving proteins that may exist as mixtures of monomer, dimer and high order multimers under the conditions of the DEER EPR experiment.