Assignment of Side-Chain ~(13)C Resonances in Perdeuterated Proteins

摘要

For moderately sized ~(13)C/~(15)N-labeled proteins, side-chain ~(13)C resonance assignments are obtained from either the HCCH-TOCSY or the HC(CC)(CO)NH experiment. The HCCH-TOCSY has been the experiment of choice because of its extremely high sensitivity and high level of correlative redundancy. The increased number of correlation peaks generated by this experiment, however, may severely aggravate the problem of spectral overlap in larger proteins such as human carbonic anhydrase Ⅱ (HCA Ⅱ, 29 kDa, 259-residue monomer) and, in the absence of prior side-chain ~(13)C chemical-shift assignments, may limit the utility of this experiment. Compared to ~1H_N resonances in β-sheet proteins and ~(15)N resonances in general, the dispersion of particular aliphatic ~1H_C and ~(13)C resonances is usually poorer. The HC(CC)(CO)NH experiment would therefore seem more appropriate for the assignment of side-chain ~(13)C resonances in larger proteins. Rapid ~(13)C and ~1H_N T_2 relaxation, however, may render this experiment too insensitive for large proteins. In this regard, the process of sequential backbone assignments in large proteins has benefited greatly from high levels of ~2H incorporation (～85%) at aliphatic sites. We have therefore adapted the HC(CC)(CO)NH experiment to perdeuterated proteins by starting the sequence of magnetization transfer steps on ~(13)C~6 instead of ~1H_C. In this communication, we present the results from the C(CC)(CO)-NH experiment on perdeuterated HCA Ⅱ (~2H-HCA Ⅱ) and estimate the theoretical increase in sensitivity over the HC(CC)-(CO)NH experiment on protonated HCA Ⅱ (~1H-HCA Ⅱ).