Department of Chemistry and ‡Department of Molecular Biophysics and Biochemistry, Yale University , New Haven, Connecticut 06520, United States.
Biochemistry. 2013 Sep 17;52(37):6548-58. doi: 10.1021/bi400973e. Epub 2013 Aug 30.
To date, little work has been conducted on the relationship between solute and buffer molecules and conformational exchange motion in enzymes. This study uses solution NMR to examine the effects of phosphate, sulfate, and acetate in comparison to MES- and HEPES-buffered references on the chemical shift perturbation and millisecond, chemical, or conformational exchange motions in the enzyme ribonuclease A (RNase A), triosephosphate isomerase (TIM) and HisF. The results indicate that addition of these solutes has a small effect on (1)H and (15)N chemical shifts for RNase A and TIM but a significant effect for HisF. For RNase A and TIM, Carr-Purcell-Meiboom-Gill relaxation dispersion experiments, however, show significant solute-dependent changes in conformational exchange motions. Some residues show loss of millisecond motions relative to the reference sample upon addition of solute, whereas others experience an enhancement. Comparison of exchange parameters obtained from fits of dispersion data indicates changes in either or both equilibrium populations and chemical shifts between conformations. Furthermore, the exchange kinetics are altered in many cases. The results demonstrate that common solute molecules can alter observed enzyme millisecond motions and play a more active role than what is routinely believed.
迄今为止，人们对溶质和缓冲分子与酶中构象交换运动之间的关系研究甚少。本研究使用溶液 NMR 来检测磷酸盐、硫酸盐和醋酸盐与 MES 和 HEPES 缓冲参考物相比，对核糖核酸酶 A（RNase A）、磷酸丙糖异构酶（TIM）和 HisF 中化学位移扰动和毫秒、化学或构象交换运动的影响。结果表明，这些溶质的添加对 RNase A 和 TIM 的（1）H 和（15）N 化学位移只有很小的影响，但对 HisF 的影响很大。然而，对于 RNase A 和 TIM，Carr-Purcell-Meiboom-Gill 弛豫分散实验显示出构象交换运动中显著的溶质依赖性变化。一些残基在添加溶质后相对于参考样品失去了毫秒运动，而其他残基则增强了运动。从分散数据拟合得到的交换参数的比较表明，平衡态种群和构象之间的化学位移都发生了变化。此外，在许多情况下，交换动力学也发生了改变。结果表明，常见的溶质分子可以改变观察到的酶毫秒运动，并发挥比通常认为的更积极的作用。
