Protein structure determination by NMR has predominantly relied on simulated annealing-based Schisandrin B Schisandrin B conformational search for a converged fold using primarily distance constraints including constraints derived from nuclear Overhauser effects (NOEs) paramagnetic relaxation enhancement (PRE) and cysteine crosslinkings. including the reported NMR and crystal structures. This result highlights the fundamental limitation of global fold determination for homo-oligomeric proteins using ambiguous distance constraints and provides a systematic solution for exhaustive enumeration of all satisfying solutions. [5 6 7 8 For dimers separating intra- vs inter-subunit NOEs using X-filtered NOESY [9] is sufficient to resolve subunit ambiguity. For trimers and higher-order oligomers even after a distance restraint has been classified as inter-subunit it still has at least two possible assignments and is still ambiguous. ADRs consider degenerate atom pairs by using an average function derived from a mean field approximation. Although it has been demonstrated that genuine interactions can be extracted from ADRs these methods are prone to becoming trapped in local minima since they rely heavily on Schisandrin B the initial fold to remove assignment ambiguity. The energy landscapes for homo-oligomers contain a large number of minima with similarly low energy so when simulated annealing methods using ADRs become trapped in Schisandrin B local minima these methods can fail to report satisfying folds from other minima. This situation is further exacerbated in the case of homo-oligomeric membrane proteins for which dense restraint collection is often impractical [10 11 12 13 8 In the case of Diacylglycerol Kinase from (henceforth simply DAGK) a membrane-associated homo-trimer two different structures have been published. The solution NMR structure [14] of DAGK determined using ambiguously-assigned distance restraints possesses a domain-swapped subunit interface while the crystal structure [15] has a subunit with a more compact conformation and without domain-swapping. Here we show that the difference between the two structures is due to the local minimum limitations of current methodology for NMR structure determination. We demonstrate that this limitation can be mitigated by searching over topologically distinct folds using a systematic approach called (Figure 1 middle). From these schematic representations of the folds it is easy to visualize the domain-swapped configuration Rabbit Polyclonal to Cytochrome P450 4F8. of the NMR structure relative to the compact subunits of the crystal structure. Figure 1 Fold schematics clearly show helical packing for the NMR (top) and crystal (bottom) structures of DAGK. In the fold schematic the helices are shown as colored discs (the amphiphilic surface helix SH is not shown) the loop regions are shown as black … Of the deposited restraints collected for DAGK in solution there are no inter-subunit NOEs nor long range (> 4) NOEs within the same subunit. Hence the NOEs hydrogen bond restraints dihedral angle restraints and RDCs primarily constrain secondary structures within each subunit. The helices SH H1 H2 and H3 are well-restrained individually but the inter-helical linkers are relatively unrestrained giving little long-range information to pack the quaternary structure. The helical packing of DAGK and hence the overall fold is largely defined by the inter-subunit restraints: cysteine cross-linking via disulfide bonds and restraints from paramagnetic relaxation enhancement (PRE). Since the PREs are plagued by intra/inter ambiguity [7] as well as subunit ambiguity we focused on the effect of cysteine crosslinking restraints (which are only complicated by subunit ambiguity) to predict satisfying folds. The absence of a possible intra-subunit assignment makes the disulfide bond restraints much simpler to interpret so our computational approach will initially focus solely on these restraints. Therefore our goal will to be to find all possible topologically distinct folds that satisfy the disulfide bond restraints. The PRE restraints will be used later as a filter to eliminate the erroneous predictions. Fold-operator theory finds alternative folds allowed by restraints Since the restraint supplied by the disulfide bonds can be ambiguous and rather loose (dCα(also to the crystal fold where may be the move operator and may be the swap operator. These providers could be applied in virtually any purchase and the full total result may be the same. Consequently and type the basis of the finite Abelian band of purchase 36. The numerical framework of the group can be talked about in the Supplementary Info (SI) Section 1. Shape 3.