Residual dipolar couplings for pairs of proximate magnetic nuclei in macromolecules can easily be measured using highresolution NMR methods when the molecules are dissolved in dilute liquid crystalline media. The resulting couplings can in principle be used to constrain the relative orientation of molecular fragments in macromolecular systems to build a complete structure. However, determination of relative fragment orientations based on a single set of residual dipolar couplings is inherently hindered by the multi-valued nature of the angular dependence of the dipolar interaction. Even with unlimited dipolar data, this gives rise to a fourfold degeneracy in fragment orientations. In this Communication, we demonstrate a procedure based on an order tensor analysis that completely removes this degeneracy by combining residual dipolar coupling measurements from two alignment media. Application is demonstrated on 15 N-1 H residual dipolar coupling data acquired on the protein zinc rubredoxin from Clostridium pasteurianum dissolved in two different bicelle media. © 2000 Academic Press Key Words: NMR; order matrix; bicelle; rubredoxin; protein fold. INTRODUCTIONThe measurement of residual dipolar couplings between protein backbone nuclei can provide an alternative to NOE information in the determination of protein folds (1)(2)(3)(4)(5)(6)(7)(8). These measurements can constrain the relative orientation of molecular fragments regardless of their separation in space, a fact of considerable value when studying loosely connected protein domains or backbone segments of proteins that are separated by unassigned side chain atoms (5,6,9). However, relative fragment orientations cannot be uniquely determined from a single set of residual dipolar coupling measurements, no matter how numerous the measurements (6, 9 -11). This limitation ultimately arises from the multi-valued character of angular dependent residual dipolar coupling function,where is the angle between the internuclear vector and the magnetic field (1, 12). For a single residual dipolar coupling measurement, it is easy to see that one can only constrain the orientation of an internuclear vector into two "cones" of orientation (10). More measurements restrict solutions to intersection of cones but a single allowed solution is never found (10).Using an order matrix analysis of multiple measurements from noncollinear vectors within a single fragment, it is easy to show that uncertainty in orientation reduces only to a fourfold degeneracy (6,9,(11)(12)(13)(14). In this analysis, residual dipolar coupling measurements are used to determine the five independent elements of a symmetric and traceless 3 ϫ 3 order matrix (11,13). These five parameters can be reformulated in terms of an alignment of axes (three angles) for a principal order frame, and values of a principal order parameter (S zz ), and an asymmetry parameter ( ϭ ͉(S xx Ϫ S yy )/S zz )͉) (11,13). Because the order parameters are themselves insensitive to axis inversion, there are four possible ways to direct a...

No abstract

The structures of apo- and holorubredoxins from Pyrococcus furiosus (PfRd) and Clostridium pasteurianum (CpRd) have been investigated and compared using residual dipolar couplings to probe the origin of thermostability. In the native, metal (Fe or Zn) containing form, both proteins can maintain native structure at very high temperatures (>70 degrees C) for extended periods of time. Significant changes in either structure or backbone dynamics between 25 and 70 degrees C are not apparent for either protein. A kinetic difference with respect to metal loss is observed as in previous studies, but the extreme stability of both proteins in the presence of metal makes thermodynamic differences difficult to monitor. In the absence of metal, however, a largely reversible thermal denaturation can be monitored, and a comparison of the two apoproteins can offer insights into the origin of stability. Below denaturation temperatures apo-PfRd is found to have a structure nearly identical to that of the native holo form, except immediately adjacent to the metal binding site. In contrast, apo-CpRd is found to have a structure distinctly different from that of its holo form at low temperatures. This structure is rapidly lost upon heating, unfolding at approximately 40 degrees C. A PfRd mutant with the hydrophobic core mutated to match that of CpRd shows no change in thermostability in the metal-free state. A metal-free chimera with residues 1-15 of CpRd and the remaining 38 residues of PfRd is severely destabilized and is unfolded at 25 degrees C. Hence, the hydrophobic core does not seem to be the key determinant of thermostability; instead, data point to the hydrogen bond network centered on the first 15 residues or the interaction of these 15 residues with other parts of the protein as a possible contributor to the thermostability.