Ishima Rieko, Baber James, Louis John M, Torchia Dennis A
Molecular Structural Biology Unit, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892-4307, USA.
J Biomol NMR. 2004 Jun;29(2):187-98. doi: 10.1023/B:JNMR.0000019249.50306.5d.
A constant-time, Carr-Purcell-Meiboom-Gill (CPMG) transverse relaxation, R(2), dispersion experiment for carbonyl carbons was designed and executed to detect micros-ms time-scale dynamics of protein backbone carbonyl sites. Because of the large (ca. 55 Hz) C(alpha)-C' J-coupling, the carbonyl signal intensity is strongly modulated as the spacing between CPMG pulses is varied, in uniformly (13)C enriched proteins, unless care is taken to minimize the perturbation of the C(alpha) magnetization by the CPMG pulses. CPMG pulse trains consisting of either a band-selective pulse, such as RE-BURP, or rectangular (with an excitation null in the C(alpha) region of the spectrum) pulses were employed in order to minimize C' signal modulation by C(alpha)-C' J-coupling. The performance of these types of CPMG refocusing pulses was assessed by computer simulation, and by comparing dispersion profiles measured for (1) uniformly [(13)C,(15)N, (2)H] ((2)H at non-labile hydrogen sites) labeled, and (2) uniformly (15)N/selectively-(13)C' labeled samples of HIV-1 protease bound to a potent inhibitor, DMP323. In addition, because the uniformly (13)C/(15)N/(2)H labeled sample was well suited to measure (15)N and (1)H R(2) dispersion as well as (13)C' dispersion, conformational exchange in the inter subunit beta-sheet hydrogen-bond network of the inhibitor-bound protease was elucidated using relaxation dispersion data of all three types of nuclei.
设计并实施了一种用于羰基碳的恒时 Carr-Purcell-Meiboom-Gill（CPMG）横向弛豫 R(2) 色散实验，以检测蛋白质主链羰基位点的微秒至毫秒时间尺度动力学。由于较大的（约 55 Hz）Cα-C' J 耦合，在均匀（13）C 富集的蛋白质中，当 CPMG 脉冲之间的间距变化时，羰基信号强度会受到强烈调制，除非采取措施尽量减少 CPMG 脉冲对 Cα 磁化的扰动。为了尽量减少 Cα-C' J 耦合对 C' 信号的调制，采用了由带选择性脉冲（如 RE-BURP）或矩形脉冲（在光谱的 Cα 区域有激发零点）组成的 CPMG 脉冲序列。通过计算机模拟以及比较针对（1）均匀标记的[(13)C,(15)N, (2)H]（在非不稳定氢位点的（2）H）和（2）均匀（15）N/选择性（13）C' 标记的与强效抑制剂 DMP323 结合的 HIV-1 蛋白酶样品测量的色散曲线，评估了这些类型的 CPMG 重聚焦脉冲的性能。此外，由于均匀（13）C/（15）N/（2）H 标记的样品非常适合测量（15）N 和（1）H R(2) 色散以及（13）C' 色散，因此利用所有三种类型原子核的弛豫色散数据阐明了抑制剂结合蛋白酶的亚基间β-折叠氢键网络中的构象交换。
