The Effects of Dissolved Oxygen upon Amide Proton Relaxation and Chemical Shift in a Perdeuterated Protein

The effects of dissolved molecular oxygen upon amide proton ( 1H N ) longitudinal and transverse relaxation rates and chemical shifts were studied for a small protein domain, the second type 2 module of fibronectin ( 2F2)—isotopically enriched to 99% 2H, 98% 15N. Longitudinal relaxation rate enhancements, R O 2 ( 1H N ), of individual backbone 1H N nuclei varied up to 14 fold between a degassed and oxygenated (1 bar) solution, indicating that the oxygen distribution within the protein is inhomogeneous. On average, smaller relaxation rate enhancements were observed for 1H N nuclei associated with the core of the protein compared to 1H N nuclei closer to the surface, suggesting restricted oxygen accessibility to some regions. In agreement with an O 2– 1H N hyperfine interaction in the extreme narrowing limit, the 1H N transverse relaxation rates showed no significant change, up to an oxygen pressure of 9.5 bar (the maximum pressure used in this study). For most 1H N resonances, small Δδ O 2 ( 1H N ) hyperfine chemical shifts could be detected between oxygen pressures of 1 bar and 9.5 bar.