Measurement and interpretation of 15N-1H residual dipolar couplings in larger proteins.

A decade ago, Dr. L.E. Kay and co-workers described an ingenious HNCO-based triple-resonance experiment from which several protein backbone RDCs can be measured simultaneously (Yang et al. (1999) [1]). They implemented a J-scaling technique in the (15)N dimension of the 3D experiment to obtain the NH RDCs. We have used this idea to carry out J-scaling in a 2D (15)N-(1)H-TROSY experiment and have found it to be an excellent method to obtain NH RDCs for larger proteins upto 70 kDa, far superior to commonly used HSQC in-phase/anti-phase and HSQC/TROSY comparisons. Here, this method, dubbed "RDC-TROSY" is discussed in detail and the limits of its utility are assessed by simulations. Prominent in the latter analysis is the evaluation of the effect of amide proton flips on the "RDC-TROSY" linewidths. The details of the technical and computational implementations of these methods for the determination of domain orientations in 45-60 kDa Hsp70 chaperone protein constructs are described.