Measurement of C-13(alpha)-C-13(beta) dipolar couplings in N-15,C-13,H-2-labeled proteins: Application to domain orientation in maltose binding protein

TROSY-based HN(CO)CA 2D and 3D pulse schemes are presented for measurement of C-13(alpha)-C-13(beta) dipolar couplings in high molecular weight N-15, C-13, H-2-labeled proteins. In one approach, C-13(alpha)-C-13(beta) dipolar couplings are obtained directly from the time modulation of cross-peak int ensities in a set of 2D N-15-(HN)-H-1 correlated spectra recorded in both t he presence and absence of aligning media. In a second approach 3D data set s are recorded with C-13(alpha)-C-13(beta) couplings encoded in a frequency dimension. The utility of the experiments is demonstrated with an applicat ion to an N-15, C-13, H-2-labeled sample of the ligand-free form of maltose binding protein. A comparison of experimental dipolar couplings with those predicted from the X-ray structure of the apo form of this two-domain prot ein established that ene relative orientation of the domains in solution an d in the crystal state are very Similar. This is in contrast to the situati on for maltose binding protein in complex with beta -cyclodextrin where the solution structure can be generated from the crystal state via a 11 degree s domain closure.