The Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan, Israel.
Biophys J. 2011 Jun 22;100(12):2991-9. doi: 10.1016/j.bpj.2011.03.065.
The effect of an inert small molecule osmolyte, trimethyl amine N-oxide (TMAO), upon the conformational equilibria of Escherichia coli adenylate kinase was studied using time-resolved FRET. The relative populations of open and closed clefts between the LID and the CORE domains were measured as functions of the concentrations of the substrate ATP over the concentration range 0-18 mM and TMAO over the concentration range 0-4 M. A model was constructed according to which the enzyme exists in equilibrium among four conformational states, corresponding to combinations of open and closed conformations of the LID-CORE and AMP-CORE clefts. ATP is assumed to bind only to those conformations with the closed LID-CORE cleft, and TMAO is assumed to be differentially excluded as a hard spherical particle from each of the four conformations in accordance with calculations based upon x-ray crystallographic structures. This model was found to describe quantitatively the dependence of the fraction of the closed LID-CORE cleft upon the concentrations of both ATP and TMAO over the entire range of concentrations with just five undetermined parameters.
使用时间分辨荧光共振能量转移（FRET）技术研究了惰性小分子渗透剂三甲基胺 N-氧化物（TMAO）对大肠杆菌腺苷酸激酶构象平衡的影响。作为底物 ATP 浓度在 0-18 mM 范围内和 TMAO 浓度在 0-4 M 范围内的函数，测量了 LID 和 CORE 结构域之间的裂隙的开和闭的相对群体。根据该模型，酶存在于四个构象状态之间的平衡中，对应于 LID-CORE 和 AMP-CORE 裂隙的开和闭构象的组合。假设 ATP 仅结合于具有闭合的 LID-CORE 裂隙的那些构象，并且 TMAO 被假定根据基于 X 射线晶体结构的计算从四个构象中的每一个以硬球颗粒的形式被有差异地排除。发现该模型可以定量描述在整个浓度范围内，封闭的 LID-CORE 裂隙的分数对 ATP 和 TMAO 的浓度的依赖性，只需五个未确定的参数。
