Redox-dependent 1H NMR spectral features and tertiary structural constraints on the C-terminal region of putidaredoxin.
Redox-dependent 1H NMR spectral features and tertiary structural constraints on the C-terminal region of putidaredoxin.
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Putidaredoxin C 末端区域的氧化还原依赖性 1H NMR 光谱特征和三级结构限制。
DOI：
10.1021/bi00187a007
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Patera,A
中科院分区：
文献类型：
--
作者：
Pochapsky,TC;Ratnaswamy,G;Patera,A
材料和方法从细菌培养物中获得氧化的Pdx，并如前所述进行纯化（Pochapsky和Ye，1991）。纯化后，将缓冲液更换为氩气吹扫的H2O和D2 O的90%/10%混合物，其用pH 7.4的0.05 M Tris-t3缓冲。在装有预平衡的P-2尺寸排阻凝胶（BioRad）的离心强制柱上完成缓冲液交换。然后将样品（通常为4 mM）置于隔片密封的5-mm NMR管中，并用氩气吹扫该管。通过将适量的新鲜连二亚硫酸钠置于小瓶中，然后将小瓶隔片密封并用氩气吹扫，制备0.1M连二亚硫酸钠溶液。然后通过注射器将与用于NMR样品制备的相同的适当量的氩气吹扫的缓冲液引入小瓶中，并将所得溶液用氩气吹扫几分钟。使用微升注射器用连二亚硫酸盐溶液滴定NMR管中的样品。接近-0.8 ppm的宽峰（对应于三个质子）的积分是完全还原的有用NMR标记（图1）。本文所述的所有NMR光谱变化都是可逆的，并且精确地对应于还原，如通过化学计量法测定的（Cushman等人，1967年）。在以500.13 MHz操作的Bruker AMX-500光谱仪（Brandeis University）或以600.14 MHz操作的Bruker AMX-600光谱仪（Bruker Institute，Billerica，MA）上进行NMR实验。在AMX-500光谱仪上使用8064 Hz的1H谱宽进行实验，而在AMX-600光谱仪上使用10 000 Hz的谱宽。在所有情况下通过预饱和获得水抑制。在co2维度中，通常使用2048个复杂点的数字分辨率。NOESY、2 Q和HOHAHA实验总共使用400 ti增量，而使用500 tj增量
MATERIALS AND METHODSOxidized Pdx was obtained from bacterial cultures and purified as described previously (Pochapsky & Ye, 1991). After purification, buffer was exchanged for an argon-purged 90%/10% mixture of H20 and D20, which was buffered with 0.05 M Tris-t^ at pH 7.4. Buffer exchange was accomplished on a centrifugally forced column packed with preequilibrated P-2 size exclusion gel (BioRad). The sample (typically 4 mM) was then placed in a septum-sealed 5-mm NMR tube and the tube purged with argon.A 0.1 M sodium dithionite solution was prepared by placing the appropriate amount of fresh sodium dithionite in a vial that was then septum-sealed and purged with argon. The appropriate amount of argon-purged buffer identical to that used for NMR sample preparation was then introduced by syringe into the vial and the resulting solution purged with argon for several minutes. A microliter syringe was used to titrate the sample in the NMR tube with the dithionite solution. The integration of a broad peak near-0.8 ppm, corresponding to three protons, is a useful NMR marker for complete reduction (Figure 1). All NMR spectral changes described herein are reversible and correspond precisely with reduction, as determined spectrophotometrically (Cushman et al., 1967). NMR experiments were performed on either a Bruker AMX-500 spectrometer operating at 500.13 MHz (Brandeis University) or a Bruker AMX-600 spectrometer operating at 600.14 MHz (Bruker Institute, Billerica, MA). A ‘H spectral width of 8064 Hz was used for experiments on the AMX-500 spectrometer, while a spectral width of 10 000 Hz was used on the AMX-600 spectrometer. Water suppression was obtained in all cases by presaturation. A digital resolution of 2048 complex points was routinely used in the co2 dimension. A total of 400 ti increments was used for NOESY, 2Q, and HOHAHA experiments, while 500 tj increments were used