Partial Orientation of Oxidized and Reduced Cytochromeb₅at High Magnetic Fields:  Magnetic Susceptibility Anisotropy Contributions and Consequences for Protein Solution Structure Determination

Abstract

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.

The Binding Mode of ATP Revealed by the Solution Structure of the N-domain of Human ATP7A

“…S1). For residual dipolar coupling restraints, the tensor parameters were obtained with FANTAORIENT, and they were optimized through iterative cycles of PSEUDOCYANA until convergence (22). For structure calculations, 500 random conformers were annealed in 13,000 steps using the program CYANA-2.1 (23).…”

Experimentally exploring the conformational space sampled by domain reorientation in calmodulin

“…Any set of meaningful rdc is always describable by a single orientation tensor, independently of the fact that they originate from a weighted average of a number of conformations. The latter tensor can be obtained by a simple fit of the tensor parameters to the experimental rdc and to the existing C-terminal Bax structure, using the FANTAORIENT program (48,52). The results of the fit are shown in Fig.…”

“…In paramagnetic metalloproteins the metal magnetic susceptibility tensor, para , is usually anisotropic, owing to orbital contributions to the electron magnetic moment. In solution, this anisotropy produces pcs of the nuclei that are dipole-dipole coupled to the paramagnetic metal ion as well as rdc attributable to partial self-orientation (47)(48)(49). The pertinent equations are published as Supporting Text on the PNAS web site.…”

Intermolecular dynamics studied by paramagnetic tagging

“…For some metalloproteins, weak molecular alignment can be achieved at high fields due to their anisotropic magnetic susceptibility (Tolman et al 1995;Banci et al 1998). For other proteins, external alignment media have to be used Bax and Grishaev 2005).…”