Chemical shift perturbation (CSP, chemical shift mapping or complexation-induced changes in chemical shift, CIS) follows changes in the chemical shifts of a protein when a ligand is added, and uses these to determine the location of the binding site, the affinity of the ligand, and/or possibly the structure of the complex. A key factor in determining the appearance of spectra during a titration is the exchange rate between free and bound, or more specifically the off-rate koff. When koff is greater than the chemical shift difference between free and bound, which typically equates to an affinity Kd weaker than about 3μM, then exchange is fast on the chemical shift timescale. Under these circumstances, the observed shift is the population-weighted average of free and bound, which allows Kd to be determined from measurement of peak positions, provided the measurements are made appropriately. (1)H shifts are influenced to a large extent by through-space interactions, whereas (13)Cα and (13)Cβ shifts are influenced more by through-bond effects. (15)N and (13)C' shifts are influenced both by through-bond and by through-space (hydrogen bonding) interactions. For determining the location of a bound ligand on the basis of shift change, the most appropriate method is therefore usually to measure (15)N HSQC spectra, calculate the geometrical distance moved by the peak, weighting (15)N shifts by a factor of about 0.14 compared to (1)H shifts, and select those residues for which the weighted shift change is larger than the standard deviation of the shift for all residues. Other methods are discussed, in particular the measurement of (13)CH3 signals. Slow to intermediate exchange rates lead to line broadening, and make Kd values very difficult to obtain. There is no good way to distinguish changes in chemical shift due to direct binding of the ligand from changes in chemical shift due to allosteric change. Ligand binding at multiple sites can often be characterised, by simultaneous fitting of many measured shift changes, or more simply by adding substoichiometric amounts of ligand. The chemical shift changes can be used as restraints for docking ligand onto protein. By use of quantitative calculations of ligand-induced chemical shift changes, it is becoming possible to determine not just the position but also the orientation of ligands.

中文翻译：

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使用化学位移扰动来表征配体结合

化学位移扰动（CSP，化学位移图谱或复合诱导的化学位移变化，CIS）跟随添加配体时蛋白质化学位移的变化，并使用这些来确定结合位点的位置，配体，和/或可能是复合物的结构。在滴定期间确定光谱外观的一个关键因素是游离和结合之间的交换率，或者更具体地说是解离率 koff。当 koff 大于游离和结合之间的化学位移差异时，这通常等同于弱于约 3μM 的亲和力 Kd，则化学位移时间尺度上的交换速度很快。在这些情况下，观察到的偏移是自由和束缚的总体加权平均值，这允许通过测量峰值位置来确定 Kd，只要测量适当。(1)H 位移在很大程度上受空间相互作用的影响，而 (13)Cα 和 (13)Cβ 位移受直通键效应的影响更大。(15)N 和 (13)C' 位移受直通键和直通空间（氢键）相互作用的影响。因此，为了根据位移变化确定结合配体的位置，最合适的方法通常是测量 (15)N HSQC 光谱，计算峰移动的几何距离，将 (15)N 位移加权系数为与 (1)H 位移相比约 0.14，并选择那些加权位移变化大于所有残基位移标准偏差的残基。讨论了其他方法，特别是 (13)CH3 信号的测量。缓慢到中间的汇率会导致线变宽，并使 Kd 值很难获得。没有很好的方法来区分由于配体直接结合引起的化学位移变化和由于变构变化引起的化学位移变化。多个位点的配体结合通常可以通过同时拟合许多测量的位移变化来表征，或者更简单地通过添加亚化学计量量的配体。化学位移变化可用作将配体对接到蛋白质上的限制。通过使用配体引起的化学位移变化的定量计算，不仅可以确定配体的位置，还可以确定配体的方向。没有很好的方法来区分由于配体直接结合引起的化学位移变化和由于变构变化引起的化学位移变化。多个位点的配体结合通常可以通过同时拟合许多测量的位移变化来表征，或者更简单地通过添加亚化学计量量的配体。化学位移变化可用作将配体对接到蛋白质上的限制。通过使用配体引起的化学位移变化的定量计算，不仅可以确定配体的位置，还可以确定配体的方向。没有很好的方法来区分由于配体直接结合引起的化学位移变化和由于变构变化引起的化学位移变化。多个位点的配体结合通常可以通过同时拟合许多测量的位移变化来表征，或者更简单地通过添加亚化学计量量的配体。化学位移变化可用作将配体对接到蛋白质上的限制。通过使用配体诱导的化学位移变化的定量计算，不仅可以确定配体的位置，还可以确定配体的方向。化学位移变化可用作将配体对接到蛋白质上的限制。通过使用配体引起的化学位移变化的定量计算，不仅可以确定配体的位置，还可以确定配体的方向。化学位移变化可用作将配体对接到蛋白质上的限制。通过使用配体引起的化学位移变化的定量计算，不仅可以确定配体的位置，还可以确定配体的方向。