Albany 2001

category image Biomolecular
SUNY at Albany
June 19-23, 2001

Increased resolution in NMR structure determination from experiments in dilute liquid crystalline media.

To date, macromolecular structure determination by NMR has relied almost exclusively on measurement of local distance restraints. These include interproton distances derived from 1H-1 H NOEs, torsion angle restraints derived from J couplings or cross-correlated relaxation, and local conformational information derived from 1 H, 13C and 31P chemical shifts. Although this approach has been proven to be quite robust for globular, compact systems such as proteins, application to nucleic acids has been more challenging because they usually do not have a compact globular structure. Also, they have a much lower proton density than proteins, and they have five instead of two variable backbone angles per structural unit. Moreover, important questions frequently focus on relatively subtle details regarding both local and global conformation. As will be discussed, these can be addressed by measurement of dipolar couplings when weakly aligning the macromolecule with the magnetic field.

Weak alignment can be obtained for a macromolecule by dissolving it in a dilute, aqueous lyotropic nematic liquid crystalline phase. Steric obstruction and electrostatic interactions between the highly aligned liquid crystal and the solute macromolecule induce a tunable degree of alignment on the solute. As a result, rotational diffusion no longer averages dipolar interactions to zero, and their averaged values directly report on the average orientation of internuclear vectors relative to the magnetic field. The dipolar coupling is a very steep function of orientation, and structural information is therefore exceptionally precise. As all dipolar interactions are relative to a single frame, they are intrinsically global in nature and provide an ideal complement to traditional NMR measurements.

The approach will be demonstrated for the DNA dodecamer d(CGCGAATTCGCG)2. With the aid of selective isotopic labeling, a large set of one-bond 13 C-1 H and 15 N-1 H was initially measured, constraining the orientations of the corresponding bonds. In addition, new methods have been developed which permit straightforward and accurate measurement of 1H-1H dipolar couplings from regular COSY spectra, making it possible to obtain sufficient dipolar couplings even in the absence of isotopic labeling. Structures calculated using these restraints show very good agreement with X-ray crystal structure for the center part of this dodecamer and, as expected, lack several of the unusual features observed for the terminal few basepairs, which have been attributed to Mg 2+ coordination and intermolecular contacts.

Subsequently measured H3'i-1-31Pi dipolar couplings measured for the dodecamer were found to be in better agreement with the NMR structure than with X-ray structures, despite the fact that none of the dipolar couplings used in the NMR structure determination contained direct information on the phosphodiester linkage. Other independent parameters for validating this result will also be discussed.

In favorable cases, when a sufficiently large number of dipolar couplings can be measured, structures can be calculated exclusively from dipolar couplings. However, in practice the dipolar coupling NMR approach appears of most immediate value as a complement to either conventional NMR or X-ray crystallography. Dipolar couplings are particularly suited for studying "pliable" systems, whose structure is difficult to study by conventional NMR, and for which the conformation in the crystalline state is subject to crystal packing and co-crystallization agents. Dipolar couplings can identify precisely the location and nature of differences in structure that may occur in solution relative to the crystalline state, particularly in multi-domain systems.

Zhengrong Wu,(1) Masatsune Kainosho,(2) Nico Tjandra,(3) and Ad Bax(1)

Laboratory of Chemical Physics(1), NIDDK, NIH, Bethesda, Maryland, USA; Tokyo Metropolitan University(2), 1-1 Minami-ohsawa, Hachioji, Tokyo 192-0397, Japan; Laboratory of Biophysical Chemistry(3), NHLBI, NIH, Bethesda, Maryland 20892.
Email: bax@nih.gov