The assignment of the aliphatic 13C resonances of trimeric Bacillus Subtilis chorismate mutase, a protein with a molecular mass of 44kDa, consisting of three 127-residue monomers is presented by use of two-dimensional (2D) 13C-start and 13C-observe NMR experiments. These experiments start with 13C excitation and end with 13C observation while relying on the long transverse relaxation times of 13C spins in uniformly deuterated and 13C,15N-labeled large proteins. Gains in sensitivity are achieved by the use of a paramagnetic relaxation enhancement agent to reduce 13C T 1 relaxation times with little effect on 13C T 2 relaxation times. Such 2D 13C-only NMR experiments circumvent problems associated with the application of conventional experiments for side-chain assignment to proteins of larger sizes, for instance, the absence or low concentration of the side-chain 1H spins, the transfer of the side-chain spin polarization to the 1HN spins for signal acquisition, or the necessity of a quantitative reprotonation of the methyl moieties in the otherwise fully deuterated side-chains. We demonstrate that having obtained a nearly complete assignment of the side-chain aliphatic 13C resonances, the side-chain 1H chemical shifts can be assigned in a semiautomatic fashion using 3D 15N-resolved and 13C-resolved NOESY experiments measured with a randomly partially protonated protein sample. We also discuss perspectives for structure determination of larger proteins by using novel strategies which are based on the 1H,1H NOEs in combination with multiple residual dipolar couplings between adjacent 13C spins determined with 2D 13C-only experiment
