A novel strategy for the assignment of side-chain resonances in completely deuterated large proteins using 13C spectroscopy.
The assignment of the aliphatic (13)C resonances of trimeric Bacillus Subtilis chorismate mutase, a protein with a molecular mass of 44 kDa, consisting of three 127-residue monomers is presented by use of two-dimensional (2D) (13)C-start and (13)C-observe NMR experiments. These experiments start with (13)C excitation and end with (13)C observation while relying on the long transverse relaxation times of (13)C spins in uniformly deuterated and (13)C,(15)N-labeled large proteins. Gains in sensitivity are achieved by the use of a paramagnetic relaxation enhancement agent to reduce (13)C T(1) relaxation times with little effect on (13)C T(2) relaxation times. Such 2D (13)C-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 (1)H spins, the transfer of the side-chain spin polarization to the (1)H(N) 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 (13)C resonances, the side-chain (1)H chemical shifts can be assigned in a semiautomatic fashion using 3D (15)N-resolved and (13)C-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 (1)H,(1)H NOEs in combination with multiple residual dipolar couplings between adjacent (13)C spins determined with 2D (13)C-only experiments.
Eletsky
Alexander
A
Laboratory of Physical Chemistry, Swiss Federal Institute of Technology, ETH-Hönggerberg, CH-8093 Zürich, Switzerland.
Moreira
Osvaldo
O
Kovacs
Helena
H
Pervushin
Konstantin
K
D000595:Amino Acid Sequence; D024322:Amino Acids, Aromatic; D001412:Bacillus subtilis / Q000201:enzymology; D002247:Carbon Isotopes; D002826:Chorismate Mutase / Q000737:chemistry; D003903:Deuterium; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*
D024322:Amino Acids, Aromatic; D002247:Carbon Isotopes; D011506:Proteins; D003903:Deuterium; D002826:Chorismate Mutase