The backbone 15 N-1 H 1 J values have been measured for oxidized and reduced cytochrome b 5 at 500 and 800 MHz. Their field dependence, due to increasing partial orientation of the molecule in solution at high magnetic fields, provides structural constraints relative to the orientation of the NH bond vector with respect to the principal directions of the molecular susceptibility tensor. The constraints have been used in a distance geometry algorithm together with the NOE constraints and with or without pseudocontact shifts constraints. The three sets of constraints are found to be consistent with one another, and their relative contribution to the definition of the structure depends on the number of constraints and their assigned weight. The orientation-dependent 15 N-1 H 1 J values provide the principal directions and anisotropies of the molecular or overall magnetic susceptibility tensor, χ mol . The χ mol parameters obtained for the oxidized form differ from those for the diamagnetic reduced form essentially by the magnetic susceptibility of the paramagnetic ion, whose anisotropy is responsible for the occurrence of pseudocontact shifts. The χ mol tensor of the paramagnetic form displays a sizable rhombic anisotropy, thus permitting the full assessment of the orientation of individual amide vectors in the molecular axes frame.

No abstract

The hydrophobic cavity of Lipid Transfer Protein 1 from Nicotiana tabacum is investigated in detail by NMR using xenon as a spy. The analysis of the (129)Xe chemical shifts and self-relaxation times gives evidence of protein-xenon interaction. Thermodynamics of the binding is characterized through the study of aliphatic (1)H and (13)C chemical shift variation as a function of xenon pressure. The binding constant is evaluated to 75.5 +/- 1.0 M(-1) at 293 K. The location of xenon inside the cavity is deduced from SPINOE experiments. The noble gas appears to occupy four sites, and xenon self-relaxation experiments indicate that it quickly jumps between different sites. The chemical shifts of amide protons and nitrogens also depend on the xenon concentration, either specifically or nonspecifically for atoms at the external surface of the protein. Yet, contrary to aliphatic atoms, they do not correspond to short-range interactions as confirmed by magnetization transfer experiments between laser-polarized xenon and protons in H(2)O. These (15)N chemical shift variations, used in combination with (15)N transverse self-relaxation rates to determine the lower limit of the binding rate, consequently reveal subtle changes in the structure of the protein upon binding.