A new strategy for backbone resonance assignment in large proteins using a MQ-HACACO experiment

A new strategy of backbone resonance assignment is proposed based on a combination of the most sensitive TROSY-type triple resonance experiments such as TROSY-HNCA and TROSY-HNCO with a new 3D multiple-quantum HACACO experiment. The favourable relaxation properties of the multiple-quantum coherences and signal detection using the C-13' antiphase coherences optimize the performance of the proposed experiment for application to larger proteins. In addition to the H-1(N), N-15, C-13(alpha) and C-13' chemical shifts the 3D multiple-quantum HACACO experiment provides assignment for the H-1(alpha) resonances in constrast to previously proposed experiments for large proteins. The strategy is demonstrated with the 44 kDa uniformly N-15, C-13-labeled and fractionally 35% deuterated trimeric B. subtilis Chorismate Mutase measured at 20 degreesC and 9 degreesC. Measurements at the lower temperature indicate that the new strategy can be applied to even larger proteins with molecular weights up to 80 kDa.