Two-dimensional NMR characterization of the deoxymyoglobin heme pocket.
Two-dimensional NMR characterization of the deoxymyoglobin heme pocket.
复制标题
脱氧肌红蛋白血红蛋白袋的二维核磁共振表征。
作者：
Busse,SC;Jue,T
材料和方法样品制备.将马心肌红蛋白（Sigma）不经进一步纯化溶解于D2 O缓冲液（25 mM磷酸盐，pH 7.4）中以制备10 mM溶液。再结晶除去任何未溶解的物质。然后将样品转移到用气密隔膜密封的5-mm NMR样品管中。用真空泵和氮气进行抽空-吹扫循环，使样品脱气并除去过量的氧气。将2 - 3当量的连二亚硫酸钠溶于0.1 mL D2 O缓冲液中，通过隔膜注入，除去剩余的氧，并将血红素从Fe（III）还原为Fe（II）。对于氧基Mb，Sephadex G-25分子大小分离步骤在D20缓冲液（1 mM EDTA和5 mM TRIS，pH 8.4）中的met Mb的初始连二亚硫酸盐还原之后。在超滤池（Amicon）中浓缩样品，产生最终的10 mM氧Mb溶液。将0.5当量的连二亚硫酸盐注入脱氧Mb样品的脱气密封管中，然后得到氧Mb和脱氧Mb的50/50混合物。尽管蛋白质制备过程使Mb从Fe（II）到Fe（III）的自氧化速率最小化，但仍有约10%的met Mb在1小时内出现，可从不同的光学和NMR信号中检测到（Antonini & Brunori，1971）。匪r用General Electric（GE）Omega 300、GE Omega 500或Bruker AMX 400光谱仪记录NMR光谱。在GE Omega 300上对脱氧肌红蛋白进行变温研究需要10 kHz的谱宽、32次扫描、用于水抑制的预饱和辐射和1 Hz指数变迹。HOD线参考25 ℃下的4.76ppm，其相对于DSS校准。在不同温度下，调整HOD参考线以反映其温度依赖性。可变温度控制单元将样品温度精确地保持并记录在±0.5 ℃范围内。在布鲁克AMX 400光谱仪上进行的2D TOCSY（Braunschweiler & Ernst，1983）实验利用先前报道的脉冲序列（Ranee，1987; Ranee & Cavanagh，1990）和TPPI（Marion & Wuthrich，1983）进行相敏检测。该光谱需要12 kHz的谱宽，256个具有IK复合点的实验，640或960次扫描，以及300-400 ms的重复时间。足够的脉冲功率允许各向同性混合（WALTZ）脉冲自旋锁定整个脱氧Mb光谱窗口。典型的90脉冲范围为10至14 µ m;自旋锁定时间在5至10 ms之间变化。2D数据集第一行的信号相位证实了自旋锁定的效率。预饱和脉冲降低了残余水共振。NOESY（Jeener等人，1979）的实验参数与TOCSY的实验参数一致。然而，NOESY混合时间在20和75 ms之间变化。正弦钟形平方函数，在每个维度上移动45，过滤采集数据。通过IK真实的点将零填充到IK的最终大小提高了数字分辨率。
MATERIALS AND METHODSSample Preparations. Horse heart myoglobin (Sigma), without further purification, was dissolved in a D2O buffer (25 mM phosphate, pH 7.4) to make a 10 mM solution. Centrifugation removed any undissolved material. The sample was then transferred to a 5-mm NMR sample tube, sealed with a gas-tight septum. An evacuation-purge cycle, with a vacuum pump and nitrogen, degassed the sample and removed the excess oxygen. Two to three equivalents of sodium dithionite, dissolved in 0.1 mL of D2O buffer and injected through the septum, removed the remaining oxygenand reduced the heme from Fe (III) to Fe (II). For oxy Mb, a Sephadex G-25 molecular size separation step followed the initial dithionite reduction of met Mb in a D20 buffer (1 mM EDTA and 5 mM TRIS, pH 8.4). Concentrating the sample in an ultrafiltration cell (Amicon) produced a final 10 mM oxy Mb solution. Injecting 0.5 equivalent of dithionite into a degassed, sealed tube of deoxy Mb sample then yielded the 50/50 mixture of oxy and deoxy Mb. Although the protein preparation procedure minimized the Mb autoxidation rate from Fe (II) to Fe (III), about 10% met Mb still appeared within 1 h, detectable from boththe distinct optical and NMR signals (Antonini & Brunori, 1971). NMR. NMR spectra were recorded with a General Electric (GE) Omega 300, a GE Omega500, or a Bruker AMX 400 spectrometer. Variable-temperature studies of deoxymyoglobin on the GE Omega 300 required a 10-kHz spectral width, 32 scans, presaturating irradiation for watersuppression, and 1-Hz exponential apodization. The HOD line was referenced to 4.76 ppm at 25 0 C, which was calibrated against DSS. At different temperatures, the HOD reference line was adjusted to reflect its temperature dependence. The variable temperature control unit maintained and recorded accurately the sample temperature within±0.5 C. 2D TOCSY (Braunschweiler & Ernst, 1983) experiments on the Bruker AMX 400 spectrometer utilized previously reported pulsesequences (Ranee, 1987; Ranee & Cavanagh, 1990) and TPPI (Marion & Wuthrich, 1983) for phasesensitive detection. The spectra required a 12-kHz spectral width, 256 experiments with IK complex points, 640 or 960 scans, and a 300-400-ms repetition time. Sufficient pulse power permitted the isotropic mixing (WALTZ) pulse to spin-lock the entire deoxy Mb spectral window. Typical 90 pulses ranged from 10 to 14 µß; spin-lock time varied between 5 and 10 ms. The signal phase of the first row of the 2D data set confirmed the efficiency of the spin lock. A presaturating pulse reduced the residual water resonance. The NOESY (Jeener et al., 1979) experimental parametersparallel the TOCSY ones. However, the NOESY mixingtimes varied between 20 and 75 ms. A sine bell squared function, shifted by 45 in each dimension, filtered the acquisition data. Zero filling to a final size of IK by IK real points improved the digital resolution.