Sari N, Holden M J, Mayhew M P, Vilker V L, Coxon B
Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA.
Biochemistry. 1999 Aug 3;38(31):9862-71. doi: 10.1021/bi9906423.
The backbone dynamics of uniformly 15N-labeled reduced and oxidized putidaredoxin (Pdx) have been studied by 2D 15N NMR relaxation measurements. 15N T1 and T2 values and 1H-15N NOEs have been measured for the diamagnetic region of the protein. These data were analyzed by using a model-free dynamics formalism to determine the generalized order parameters (S2), the effective correlation time for internal motions (tau e), and the 15N exchange broadening contributions (Rex) for each residue, as well as the overall correlation time (tau(m)). Order parameters for the reduced Pdx are generally higher than for the oxidized Pdx, and there is increased mobility on the microsecond to millisecond time scale for the oxidized Pdx, in comparison with the reduced Pdx. These results clearly indicate that the oxidized protein exhibits higher mobility than the reduced one, which is in agreement with the recently published redox-dependent dynamics studied by amide proton exchange. In addition, we observed very high T1/T2 ratios for residues 33 and 34, giving rise to a large Rex contribution. Residue 34 is believed to be involved in the binding of Pdx to cytochrome P450cam (CYP101). The differences in the backbone dynamics are discussed in relation to the oxidation states of Pdx, and their impact on electron transfer. The entropy change occurring on oxidation of reduced Pdx has been calculated from the order parameters of the two forms.
通过二维(^{15}N)核磁共振弛豫测量研究了均匀(^{15}N)标记的还原型和氧化型恶臭假单胞菌铁氧还蛋白（Pdx）的主链动力学。已测量了该蛋白质抗磁区域的(^{15}N)纵向弛豫时间（(T_1)）、横向弛豫时间（(T_2)）值以及(^{1}H - ^{15}N)核Overhauser效应（NOE）。利用无模型动力学形式对这些数据进行分析，以确定每个残基的广义序参数（(S_2)）、内部运动的有效相关时间（(\tau_e)）以及(^{15}N)交换加宽贡献（(R_{ex})），以及整体相关时间（(\tau_m)）。还原型Pdx的序参数通常高于氧化型Pdx，并且与还原型Pdx相比，氧化型Pdx在微秒至毫秒时间尺度上的流动性增加。这些结果清楚地表明，氧化型蛋白质比还原型蛋白质表现出更高的流动性，这与最近发表的通过酰胺质子交换研究的氧化还原依赖性动力学一致。此外，我们观察到残基33和34的(T_1/T_2)比值非常高，导致较大的(R_{ex})贡献。残基34被认为参与了Pdx与细胞色素P450cam（CYP101）的结合。讨论了主链动力学的差异与Pdx氧化态的关系及其对电子转移的影响。根据两种形式的序参数计算了还原型Pdx氧化时发生的熵变。
