Nuclear magnetic dipole interactions in field-oriented proteins: information for structure determination in solution.

Abstract

The measurement ofdipolar contributions to the splitting of 15N resonances of 1H-15N amide pairs in multidimensional high-field NMR spectra of field-oriented cyanometmyoglobin is reported. The splittings appear as small field-dependent perturbations of normal scalar couplings. Assignment of more than 90 resonances to specific sequential sites in the protein allows correlation of the dipolar contributions with predictions based on the known susceptibility and known structure of the protein. Implications as an additional source of information for protein structure determination in solution are discussed.Within the past 15 years, NMR has emerged as a powerful approach to the study of protein structure in solution (1, 2). The approach relies on distance constraints extracted from nuclear Overhauser effects (NOEs) and typically proceeds in three stages: assignment of backbone resonances to specific sequential sites, identification of secondary structure elements, and determination of a tertiary fold. The latter stage is particularly demanding because it requires that a large number of long-range distance constraints be extracted from NOE data and assigned to specific proton pairs. Because NOEs drop off with the inverse 6th power of the internuclear distance, the pairs tend to arise from direct side-chain-side-chain contacts, contacts involving protons which are among the most difficult to assign. Opportunities to supplement long-range distance constraints with other types of structural data would, therefore, be welcome. We demonstrate here that NMR spectra of certain proteins, taken at very high field, may contain data that can usefully complement NOEs in determining a tertiary fold. The data come from residual dipolar contributions to the scalar couplings normally seen in high-resolution spectra. These appear when the protein has a slightly preferred orientation in a magnetic field. The contributions are angle dependent and can yield constraints for the orientation of one structural element relative to another structural element. In our case, we use a 17.5-kDa protein, cyanometmyoglobin, which has a very highly anisotropic paramagnetic susceptibility to achieve preferred orientation. Myoglobin crystals have been previously shown to orient in a magnetic field because of their anisotropy (3). Bothner-By and co-workers (4) also showed years ago that orientational effects on NMR spectra of single molecules in solution could be observed if fields and resolution were high enough. That these effects can be observed in an isolated protein molecule, and that the effects can provide useful structural constraints, has awaited higher fields (17.5 T) and multidimensional NMR experiments for the detection and assignment of 15N resonances in an isotopically labeled protein.Theory. The dipolar interaction between two spin 1/2 nuclei in the high-field limit is given by the formulawhere the -y values are the gyromagnetic ratios for the nuclei, h is Plank's constant, r is the distance between the nuclei, 0 is the angle between...

Protein dynamics and function from solution state NMR spectroscopy

“…This suggests that ubiquitin's ability to bind to a very wide range of proteins is partly due to an appreciable degree of structural plasticity in its apo state. RDCs Tolman et al 1995) are sensitive to motions that occur over ps to ms (Meiler et al 2001(Meiler et al , 2001(Meiler et al , 1997Peti et al 2002) and thus fill a gap between the time windows that can be studied using Lipari-Szabo order parameters (Lipari & Szabo, 1982a, b) (sub-τ c motion) and relaxation dispersion experiments (Loria et al 1999;Tollinger et al 2001) (between 50 µs and 100 ms). In an extensive RDC-based study, the laboratories of Griesinger and de Groot developed a method (denoted EROS) that enabled a precise determination of a structural ensemble of apo ubiquitin that is sensitive to motions in the ps-to ms-regime (Lakomek et al 2005(Lakomek et al , 2008b; this regime is henceforth referred to as the supra-τ c regime.…”

De novo determination of protein structure by NMR using orientational and long-range order restraints

“…Weak alignment of proteins, which can exist naturally due to the paramagnetic properties of the molecule (Tolman et al, 1995) or be induced by solvation in liquid crystal media (Tjandra & Bax, 1997), lipid bicelles (Sanders et al, 1994) or a suspension of ®la-mentous bacteriophage (Hansen et al, 1998), prevents complete averaging of the dipolar interaction while retaining the solution properties necessary for high resolution NMR. The measurement of dipolar couplings under these conditions provides long-range order constraints which, in combination with classical NOE data, have been shown to improve structure determination in multidomain systems and protein-ligand complexes (Tjandra, 1999;Fischer et al, 1999;Olejniczak et al, 1999;Clore & Garrett et al, 1999;Bolon et al, 1999).…”

Modélisation de la dynamique de la chaîne peptidique des protéines en solution par RMN à travers les couplages dipolaires

“…Récemment, l'utilisation de cristaux liquides dilués induisant un léger alignement des protéines par rapport au champ magnétique et introduisant, ainsi, une faible anisotropie dans l'espace orientationel de la moléculeétudiée a suscité un vif intérêt [6,7]. Les couplages dipolaires résiduels alors mesurés donnent une information très précise de l'orientation des liaisons internucléaires relativementà un repère attachéà la molécule.…”