Wang Y X, Jacob J, Cordier F, Wingfield P, Stahl S J, Lee-Huang S, Torchia D, Grzesiek S, Bax A
Molecular Structural Biology Unit, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892-0520, USA.
J Biomol NMR. 1999 Jun;14(2):181-4. doi: 10.1023/a:1008346517302.
A method is described which permits detection of 3hJNC' scalar couplings across hydrogen bonds in larger, perdeuterated proteins. The experiment is demonstrated for the uniformly 2H/13C/15N-enriched 30 kDa ribosome inactivating protein MAP30. The 3hJNC' interactions are smaller than 1 Hz, but their detection in an HNCO experiment is made possible through the use of constructive interference between the 15N chemical shift anisotropy and 1H-15N dipole-dipole relaxation mechanisms in a manner similar to that of recently proposed TROSY schemes. Sensitivity of the HNCO experiment depends strongly on the 15N transverse relaxation rate of the downfield 15N multiplet component and on the amide proton T1. In perdeuterated MAP30 at 40 degrees C, the average TROSY T2 was 169 ms at 750 MHz 1H frequency, and a wide range of longitudinal relaxation rates was observed for the amide protons.
本文描述了一种方法，该方法可用于检测较大的全氘代蛋白质中通过氢键的3hJNC'标量耦合。该实验在均匀富集2H/13C/15N的30 kDa核糖体失活蛋白MAP30上得到了验证。3hJNC'相互作用小于1 Hz，但通过利用15N化学位移各向异性与1H-15N偶极-偶极弛豫机制之间的相长干涉，类似于最近提出的TROSY方案，在HNCO实验中可以检测到它们。HNCO实验的灵敏度强烈依赖于低场15N多重峰组分的15N横向弛豫率以及酰胺质子的T1。在40℃的全氘代MAP30中，在750 MHz 1H频率下平均TROSY T2为169 ms，并且观察到酰胺质子的纵向弛豫率范围很广。
