Magnetic susceptibility induced alignment of proteins in reverse micelles

With the completion of several grand scale genome sequencing efforts, it has become possible to undertake a comprehensive analysis of the structural basis for the function of the proteins encoded by the human and other genomes.1 The sheer number of proteins involved is daunting.2 The task is made even more difficult by the observation that a significant fraction of the proteomes of various species is somewhat ill-suited for analysis by the two main structural methods: X-ray crystallography and solution NMR spectroscopy.1–4 In particular, proteins of marginal stability in vitro are problematic for both approaches. In addition, solution NMR spectroscopy is somewhat limited by the relaxation properties of slowly tumbling macromolecules. One approach is to employ extensive deuteration and the TROSY-effect.5 Another approach actively seeks to increase the effective rate of molecular reorientation by encapsulating the protein of interest within the protective shell of a reverse micelle and dissolving the resulting particle in a low viscosity fluid.6 This method also allows the study of marginally stable proteins where the confined space of the reverse micelle is used to stabilize the compact native state.7 Human ubiquitin is the only example of a structure of an encapsulated protein determined to high resolution.8 A current significant deficiency for structure determinations of encapsulated proteins has been the absence of longer range restraints derived from residual dipolar couplings (RDCs) arising from partial alignment of the protein in the magnetic field.9,10 RDCs are an extremely powerful structural restraint.11,12 Here we report that encapsulated proteins partially align within a magnetic field. We have examined the magnetically-induced alignment of three encapsulated proteins (Table 1). The apparent splittings (J+D) in the IPAP 15N-1H HSQC spectra were measured13 at 17.6 Tesla and 11.7 Tesla. Encapsulated ubiquitin gave RDCs that were small (−0.9 to +0.5 Hz) but still ~5 fold greater than those measured in aqueous solution14. Encapsulated cytochrome c (cyt c), both the paramagnetic (oxidized) and diamagnetic (reduced) states, and oxidized (diamagnetic) encapsulated flavodoxin all showed significant RDCs (Figure 1). The RDCs for encapsulated oxidized cytochrome c are roughly five times larger than those seen in free solution15. Interestingly, the character of the alignment tensor for flavodoxin changes significantly with an increase in water loading. Figure 1 Correlation plots of predicted 15N-1H RDCs based on the determined alignment tensor and refined structure versus the experimentally observed 15N-1H RDCs for each of the encapsulated proteins examined (lower panels) The axis system for the determined alignment ... Table 1 Magnetic Susceptibility Tensors and Refinement Statistics of Encapsulated Proteina A simple strategy was used to evaluate whether the measured RDCs were structurally meaningful. Previously determined structures were used as a starting point in the evaluation (Table I). Distance restraints were generated for all heavy atoms within ~5 A and given +/− 0.20 A variance. A random selection of 15% of these distance restraints were combined with the experimental RDCs and the estimated magnitudes of the axial and rhombic components of the susceptibility anisotropy tensor (Table 1) as the total constraints list. The structures were refined using a simulated annealing protocol implemented in CNS17 and employing torsion angle dynamics for the high temperature stages and Cartesian coordinate dynamics for the final cooling and energy minimization stages. In addition to the usual restraints on covalent geometry, the amide N-H geometry was explicitly constrained to prevent deviations of the ω angle from planarity. The unusually extensive set of relatively tight heavy atom distance restraints combined with strictly enforced covalent geometry, particularly at the amide N-H, was designed to largely ameliorate concern about local structural distortion resulting from the satisfaction of a limited set of RDC-based structural restraints.20 Essentially ideal geometry was maintained in the final structures (see Supporting Information). Refined structures were selected using an acceptance criterion of a maximum of 2 RDCs > 0.2 Hz deviation and no significant violation of the imposed distance restraints. The alignment of the magnetic susceptibility tensor within the set of structures was found to be common to all members of the set (Table 1). Analysis of the refined structures with REDCAT22 gave essentially identical results. REDCAT solutions were not found for the unrefined starting structures. The final minimum energy structures showed good agreement with the starting structure with respect to overall r.m.s.d. (Table 1) and to ψ ϕ Ramachandran angles. The fit of the predicted to the experimental RDCs are shown in Figure 1. The precision is excellent. This indicates that the RDCs are structurally meaningful and that the structures of the encapsulated proteins are closely similar to the corresponding reference structure. Figure 1 indicates the orientation of the magnetic susceptibility tensor in each of the proteins. The χ33 element of the tensor in oxidized cytochrome c makes an angle of ~30° with the normal to the heme plane. This deviates significantly from the free aqueous solution of oxidized cytochrome c where the angle was ~7°.15 This suggests that an alignment mechanism that overrides the protein’s contributions to the magnetic susceptibility is operative. This is not unexpected. The magnetic susceptibility tensor of the reverse micelle assembly not only includes contributions from the protein and its bound prosthetic group but also from the surrounding surfactants, water and ions comprising the reverse micelle. Indeed, the simple addition of water to the flavodoxin sample significantly changed the alignment orientation. The individual susceptibilities combine with the three dimensional geometric constraints imposed by the organization of the assembly to result in a non-spherical distribution. Motion of charged species may also contribute. Though the details are quite involved and alignment therefore not easily predicted, the magnetic basis for the observed alignment is clear and would appear to be general. In addition, using the normalized scalar product as a measure,23 there appears to be no correlation of the observed alignment tensors with those predicted24 by steric alignment. Importantly, the proteins need not be paramagnetic in order to show significant alignment. The ability to vary the orientation of the alignment tensor by changing the water content of the reverse micelle should also provide additional independent sets of RDCs for refinement by numerical analysis. In summary, we have shown several examples of encapsulated proteins that partially align in a magnetic field. The alignment results in residual dipolar couplings of a magnitude measurable by standard methods. The RDCs are structurally meaningful and, when comprehensively utilized,20 should provide a powerful set of restraints for the determination of high resolution structures of encapsulated proteins.