Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Campus-Vienna-Biocenter 5, 1030, Vienna, Austria.
J Biomol NMR. 2010 Mar;46(3):205-16. doi: 10.1007/s10858-009-9394-z. Epub 2009 Dec 22.
Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR spectroscopy has emerged as a powerful tool for quantifying the kinetics and thermodynamics of millisecond exchange processes between a major, populated ground state and one or more minor, low populated and often invisible 'excited' conformers. Analysis of CPMG data-sets also provides the magnitudes of the chemical shift difference(s) between exchanging states (|Deltavarpi|), that inform on the structural properties of the excited state(s). The sign of Deltavarpi is, however, not available from CPMG data. Here we present one-dimensional NMR experiments for measuring the signs of (1)H(N) and (13)C(alpha) Deltavarpi values using weak off-resonance R (1rho ) relaxation measurements, extending the spin-lock approach beyond previous applications focusing on the signs of (15)N and (1)H(alpha) shift differences. The accuracy of the method is established by using an exchanging system where the invisible, excited state can be converted to the visible, ground state by altering conditions so that the signs of Deltavarpi values obtained from the spin-lock approach can be validated with those measured directly. Further, the spin-lock experiments are compared with the established H(S/M)QC approach for measuring the signs of chemical shift differences. For the Abp1p and Fyn SH3 domains considered here it is found that while H(S/M)QC measurements provide signs for more residues than the spin-lock data, the two different methodologies are complementary, so that combining both approaches frequently produces signs for more residues than when the H(S/M)QC method is used alone.
Carr-Purcell-Meiboom-Gill (CPMG) 弛豫弥散 NMR 光谱学已成为一种强大的工具，可用于量化毫秒交换过程中主要、丰富的基态与一个或多个次要、低丰度且通常不可见的“激发”构象之间的动力学和热力学。CPMG 数据的分析还提供了交换状态之间的化学位移差(|Deltavarpi|)的大小，这些信息反映了激发态的结构特性。然而，CPMG 数据无法提供 Deltavarpi 的符号。在这里，我们提出了一维 NMR 实验，用于使用弱非共振 R(1rho)弛豫测量来测量(1)H(N)和(13)C(alpha)Deltavarpi 值的符号，将自旋锁定方法扩展到以前仅关注(15)N 和(1)H(alpha) 位移差异符号的应用之外。该方法的准确性通过使用交换系统来建立，在该系统中，可以通过改变条件将不可见的激发态转换为可见的基态，从而可以通过自旋锁定方法获得的 Deltavarpi 值的符号与直接测量的值进行验证。此外，还比较了自旋锁定实验与用于测量化学位移差异符号的 H(S/M)QC 方法。对于这里考虑的 Abp1p 和 Fyn SH3 结构域，发现虽然 H(S/M)QC 测量提供了比自旋锁定数据更多残基的符号，但这两种不同的方法是互补的，因此将这两种方法结合起来通常比单独使用 H(S/M)QC 方法产生更多残基的符号。
