Novel heteronuclear methods of assignment transfer from a diamagnetic to a paramagnetic protein: application to rat cytochrome b5

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Abstract
15N and 1H resonance assignments for backbone and side-chain resonances of both equilibrium forms of rat ferricytochrome b5 have been obtained, using a combination of novel heteronuclear assignment transfer methods from the known assignments of the diamagnetic protein [Guiles, R. D., Basus, V. J., Kuntz, I. D., & Waskell, L. A. (1992) Biochemistry 31, 11365-11375] and computational methods which depend on an accurate determination of the orientation of the components of the susceptibility tensor. The transfer of amide proton resonance assignments takes advantage of the apparent insensitivity of amide 15N resonances to pseudocontact effects, evident in overlays of 15N-1H heteronuclear correlation spectra. Amide-proton resonance assignments tentatively transferred from the known diamagnetic assignments to the paramagnetic form of the protein were confirmed using conventional assignment strategies employing 600-MHz COSY, HOHAHA, and NOESY spectra of the oxidized protein. As was observed in rat ferrocytochrome b5, more than 40% of all residues exhibited NMR detectable heterogeneity due to the two different orientations of the heme. Complete assignment of both forms enabled accurate determination of the orientation of the susceptibility tensor for both conformations of the heme. The orientation of the z-component of the susceptibility tensors for the two forms are indistinguishable, while the in-plane components appear to differ by about 6 degrees. Differences in the orientation of the in-plane susceptibility components are undoubtedly due dominantly to the relative axial rotation of the heme of between 5 degrees and 10 degrees indicated by the NOESY contacts to the protein observed in the spectra of the ferrocytochrome [Guiles, R. D., Basus, V. J., Kuntz, I. D., & Waskell, L. A. (1992) Biochemistry 31, 11365-11375; Pochapsky, T. C., Sligar, S. G., McLachlan, S. J., & LaMar, G. N. (1990) J. Am. Chem. Soc. 112, 5258-5263].