Complete sequence-specific 1H NMR resonance assignment of hyperfine-shifted residues in the active site of a paramagnetic protein: application to Aplysia cyano-metmyoglobin.
Two-dimensional sequence-specific 1H NMR resonance assignment methodology (Wüthrich, 1986) has been applied for the first time to a 18-kDa paramagnetic hemoprotein (cyano-met Aplysia Mb) to identify all the hyperfine-shifted residues. The assignment was greatly facilitated by the fact that hyperfine shifts of residues impart a strong temperature dependence to the cross peaks, which aids location and identification, and provides improved spectral dispersion, particularly in the fingerprint region. 2D COSY and TOCSY were found to be surprisingly effective in locating the complete spin connectivities of all of the hyperfine-shifted residues, with the exception of the axially coordinated His95 imidazole ring, whose proton resonances were found to exhibit severe line broadening (> 400 Hz). Conventional 1D NOE and NOESY with short mixing times, combined with paramagnetic-induced relaxation effects, led to the successful assignment of even extremely broad proton signals. Three helical stretches and two loop regions were identified as the source of all hyperfine-shifted residues: the F helical residues 3-9, the E-helix residues 6-14, the G-helix residues 5-9, the FG-loop residues 1-4 and the CD-loop residues 1-4. These segments comprise all the residues that make contact with the heme and modulate the reactivity of the prosthetic group. The sequence-specific identifications of the active-site residues revealed that the solution structure of Aplysia metMbCN is fully consistent with that observed by X-ray diffraction in single crystals for a variety of other derivatives, except for the distal Arg66 (E10), which is turned into the heme pocket, as found only in the metMbF crystal structure (Bolognesi et al., 1990). The ready identification, by their temperature sensitivity, and the complete assignments of all hyperfine-shifted residues of Aplysia metMbCN demonstrate that sequence-specific assignment can be profitably applied to paramagnetic proteins, and that it should be possible to determine the solution structures of paramagnetic proteins, at least for low-spin complexes, by using NMR techniques used for diamagnetic proteins.
Qin
J
J
Department of Chemistry, University of California, Davis 95616.
La Mar
G N
GN
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001048:Aplysia / Q000737:chemistry*; D001665:Binding Sites; D006418:Heme / Q000737:chemistry; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy; D008280:Magnetics; D008786:Metmyoglobin / Q000031:analogs & derivatives* / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D000596:Amino Acids; C007177:cyanometmyoglobin; D008786:Metmyoglobin; D006418:Heme; D006859:Hydrogen