1H-NMR assignments and conformational studies of melanin concentrating hormone in water using two-dimensional NMR.
1H-NMR assignments and conformational studies of melanin concentrating hormone in water using two-dimensional NMR.
复制标题
使用二维 NMR 对水中黑色素浓缩激素进行 1H-NMR 分配和构象研究。
DOI：
10.1002/bip.360301314
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发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Hruby,VJ
中科院分区：
文献类型：
--
作者：
Matsunaga,TO;Gehrig,CA;Hruby,VJ
材料和方法SMCCH 是作为三氟乙酸（TFA）盐合成和分离的？通过使水溶液通过以 C1 形式生成的二乙氨基乙基 (DEAE) Sephadex A-25 阴离子交换树脂，将 TFA 交换为氯离子。将产物冻干，然后在90％HzO/10％H 2 O中重构至7mM(Aldrich，低顺磁性，TI＝48.1s)。用 1 N HCl 将 pH 调节至 4.6。所有 nmr 测量均在 Bruker AM-500 光谱仪上以 500.13 MHz 进行。所有二维光谱均使用时间比例相位增量 (TPPI) 方法以纯吸收模式记录。二维相敏 NOESY 光谱用脉冲序列记录到-9O0-t1-9Oo-~,-9O0-t2。”确定了 6.5 ps 的 90”脉冲。在单个 tl 值 (3 ps) 内，混合时间 T 的变化范围为 50 到 500 ms。所有 NOES 均在 200 ms 的混合时间下测定和定量。混合时间的 10% 变化用于抑制位移差异大于 0.1 ppm 的自旋之间的零量子相干性。使用脉冲序列 to-90"-t,-SL,-(MLEV17)-SL,-t2 记录 2D TOCSY，其中 SL 是 2.5 ms 微调脉冲，沿 x 轴定向，以散焦不平行于 x 轴的磁化强度。代替微调脉冲，在混合时间之前和之后还使用 2 个滤波器进行 TOCSY 实验。13 在 TOCSY 实验中，脉冲序列以相反模式运行，以此外，将 90' 脉冲的持续时间调整为 20 到 28 ps 之间，以允许 30 到 100 ms 的持续时间，以解决在 -90-tl-900-A-900-tz 脉冲序列中使用的 DQF COZY，其中 tl、t2 和 A 分别是已校正的演化、检测和固定延迟周期。通过在频域中应用线性频率相关相位校正，获得了光谱宽度为 6024 Hz 的 NOESY 和 DQF COSY 光谱，并以 8032 Hz 的光谱宽度记录了 TOCSY 实验，以观察远程连通性（即，通常为 512）。
MATERIALS AND METHODSMCH was synthesized and isolated as the trifluoroacetate (TFA) salt? The TFA was exchanged for chloride by passing an aqueous solution thru a diethylaminoethyl (DEAE) Sephadex A-25 anion exchange resin generated in the C1-form. The product was lyophilized, then reconstituted to 7 m M in 90% HzO/lO%'H20 (Aldrich, low paramagnetic, TI= 48.1 s). The pH was adjusted to 4.6 with 1 N HCI.All nmr measurements were conducted at 500.13 MHz on a Bruker AM-500 spectrometer. All 2D spectra were recorded in the pure absorption mode using the time proportional phase incremention (TPPI) method."." Twodimensional phase-sensitive NOESY spectra were recorded with the pulse sequence to-9O0-t1-9Oo-~,-9O0-t2." A 90" pulse of 6.5 ps was determined. Within a single tl value (3 ps) mixing times T, varied from 50 to 500 ms. All NOES were determined and quantitated at a mixing time of 200 ms. A 10% variation in the mixing time was used to suppress zero-quantum coherence between spins with shift differences greater than 0.1 ppm. The 2D TOCSY were recorded with the pulse sequence to-90"-t,-SL,-(MLEV17)-SL,-t2, where SL, is a 2.5 ms trim pulse, directed along the x axis, to defocus magnetization not parallel to the x axis. In lieu of trim pulses, TOCSY experiments were also performed with a 2 filter before and after the mixing time. 13 In TOCSY experiments, the pulse sequence was run in the inverse mode to adjust the 90'pulse to between 20 and 28 ps in duration. In addition, the number of MLEV17 phase cycles was adjusted to allow between 30 and 100 ms duration for resolution of remote connectivities. DQF COSY used at,,-90-tl-900-A-900-tz pulse sequence, where tl, t2, and A are the evolution, detection, and fixed delay periods, respectively. Baseline distortions were corrected by applying linear frequencydependent phase corrections in the frequency domain. 14 The NOESY and DQF COSY spectra were acquired with a spectral width of 6024 Hz. TOCSY experiments were recorded with a spectral width of 8032 Hz to observe remote connectivities (ie, all Arg and Val). Usually 512