Expression, Purification, and Solid-State NMR Characterization of the Membrane Binding Heme Protein Nitrophorin 7 in Two Electronic Spin States

The nitrophorins (NPs) comprise a group of NO transporting ferriheme <i>b</i> proteins found in the saliva of the blood sucking insect Rhodnius prolixus. In contrast to other nitrophorins (NP1–4), the recently identified membrane binding isoform NP7 tends to form oligomers and precipitates at higher concentrations in solution. Hence, solid-state NMR (ssNMR) was employed as an alternative method to gain structural insights on the precipitated protein. We report the expression and purification of ¹³C,¹⁵N isotopically labeled protein together with the first ssNMR characterization of NP7. Because the size of NP7 (21 kDa) still provides a challenge for ssNMR, the samples were reverse labeled with Lys and Val to reduce the number of crosspeaks in two-dimensional spectra. The two electronic spin states with <i>S</i> = 1/2 and <i>S</i> = 0 at the ferriheme iron were generated by the complexation with imidazole and NO, respectively. ssNMR spectra of both forms are well resolved, which allows for sequential resonance assignments of 22 residues. Importantly, the ssNMR spectra demonstrate that aggregation does not affect the protein fold. Comparison of the spectra of the two electronic spin states allows the determination of paramagnetically shifted cross peaks due to pseudocontact shifts, which assists the assignment of residues close to the heme center

Size and Compositional Effects on Contrast Efficiency of Functionalized Superparamagnetic Nanoparticles at Ultralow and Ultrahigh Magnetic Fields

Magnetic resonance imaging (MRI) systems at ultralow and ultrahigh field have been developed for biomedical imaging. We synthesized and functionalized superparamagnetic (SPM) nanoparticles (NPs) and studied the spin–lattice (<i>T</i>₁) and spin–spin (<i>T</i>₂) relaxation time of protons in water surrounding these NPs at ultralow and at ultrahigh magnetic fields. In these fields, size and compositional effects of SPM NPs were observed contributing to the spin–lattice (<i>r</i>₁) and spin–spin (<i>r</i>₂) relaxivities as well as <i>r</i>₂/<i>r</i>₁ ratios. These results reveal the relationship between magnetic characteristics of SPM NPs and relaxation behavior of water proton at ultralow or ultrahigh field