Kontaxis G, Clore G M, Bax A
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892-0520, USA.
J Magn Reson. 2000 Mar;143(1):184-96. doi: 10.1006/jmre.1999.1979.
Various strategies are described and compared for measurement of one-bond J(NH) and J(NC') splittings in larger proteins. In order to evaluate the inherent resolution obtainable in the various experiments, relaxation rates of (15)N-(1)H(N) coupled and heteronuclear decoupled resonances were measured at 600- and 800-MHz field strengths for both perdeuterated and protonated proteins. A comparison of decay rates for the two (15)N-¿H(N)¿ doublet components shows average ratios of 4.8 and 3.5 at 800- and 600-MHz (1)H frequency, respectively, in the perdeuterated proteins. For the protonated proteins these ratios are 3.2 (800 MHz) and 2.4 (600 MHz). Relative to the regular HSQC experiment, the enhancement in TROSY (15)N resolution is 2.6 (perdeuterated; 800 MHz), 2.0 (perdeuterated; 600 MHz), 2.1 (protonated; 800 MHz), and 1.7 (protonated; 600 MHz). For the (1)H dimension, the upfield (1)H(N)-¿(15)N¿ component on average relaxes slower than the downfield (1)H(N)-¿(15)N¿ component by a factor of 1.8 (perdeuterated; 800 MHz) and 1.6 (perdeuterated; 600 MHz). The poor resolution for the upfield (15)N-¿(1)H¿ doublet component in slowly tumbling proteins makes it advantageous to derive the J(NH) splitting from the difference in frequency between the narrow downfield (15)N doublet component and either the (1)H-decoupled (15)N resonance or the peak position in an experiment which J-scales the frequency of the upfield doublet component but maintains some of the advantages of the TROSY experiment.
本文描述并比较了多种用于测量较大蛋白质中一键J(NH)和J(NC')分裂的策略。为了评估各种实验可获得的固有分辨率，在600兆赫和800兆赫场强下，分别对全氘代和质子化蛋白质测量了(15)N-(1)H(N)耦合和异核去耦共振的弛豫速率。对于全氘代蛋白质，在800兆赫和600兆赫(1)H频率下，两种(15)N-¿H(N)¿双峰组分的衰减速率比较显示平均比率分别为4.8和3.5。对于质子化蛋白质，这些比率分别为3.2(800兆赫)和2.4(600兆赫)。相对于常规HSQC实验，TROSY(15)N分辨率的提高分别为2.6(全氘代；800兆赫)、2.0(全氘代；600兆赫)、2.1(质子化；800兆赫)和1.7(质子化；600兆赫)。对于(1)H维度，在全氘代蛋白质中，上场(1)H(N)-¿(15)N¿组分的平均弛豫速度比下场(1)H(N)-¿(15)N¿组分慢1.8倍(800兆赫)和1.6倍(600兆赫)。在慢翻滚蛋白质中，上场(15)N-¿(1)H¿双峰组分的分辨率较差，因此从窄下场(15)N双峰组分与(1)H去耦(15)N共振之间的频率差或在对上场双峰组分频率进行J标度但保留TROSY实验一些优点的实验中的峰位置推导J(NH)分裂是有利的。
