Evidence from EPR that nitroxide spin labels attached to human hemoglobin alter their conformation upon freezing

Abstract

Human oxy-hemoglobin was spin labelled at the two -93 cysteine positions with three different spin labels. Continuous wave electron paramagnetic resonance (CW EPR) was used to assess the dynamics of the attached spin labels in solution at room temperature. Solutions were frozen to 50 K and the distance between the spin labels was measured using the double electron electron resonance (DEER) pulsed EPR technique. The two sets of results were compared to the crystal structure of the protein and were found to be consistent with a model where the spin labels tend to adopt a less rigid surface exposed conformation in solution, but are positioned in a protein pocket in the frozen state.