Novel heteronuclear methods of assignment transfer from a diamagnetic to a paramagnetic protein: application to rat cytochrome b5.
Novel heteronuclear methods of assignment transfer from a diamagnetic to a paramagnetic protein: application to rat cytochrome b5.
复制标题
从抗磁性蛋白到顺磁性蛋白的分配转移的新型异核方法：在大鼠细胞色素 b5 上的应用。
DOI：
10.1021/bi00083a037
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Waskell,L
中科院分区：
文献类型：
--
作者：
Guiles,RD;Basus,VJ;Sarma,S;Malpure,S;Fox,KM;Kuntz,ID;Waskell,L
1993年5月27日收到的修订版Mandarin pt摘要：已经获得了大鼠铁细胞色素65的两种平衡形式的骨架和侧链共振的15 N和共振分配，使用来自抗磁性蛋白的已知分配的新的异源分配转移方法的组合[Guiles，RD，Basus，V. J.，孔茨岛D、& Waskell，L. A.（1992）Biochemistry 31，11365-11375]和依赖于磁化率张量的分量的取向的准确确定的计算方法。酰胺质子共振归属的转移利用了酰胺15 N共振对伪接触效应的明显不敏感性，这在15 N-H杂原子相关光谱的叠加中是明显的。酰胺-质子共振分配暂时转移到顺磁形式的蛋白质的已知的抗磁性分配使用常规的分配策略，采用600 MHz的COSY，HOHAHA和NOESY光谱的氧化蛋白质确认。正如在大鼠亚铁细胞色素<$5中观察到的那样，由于血红素的两种不同取向，超过40%的所有残留物表现出NMR可检测的异质性。这两种形式的完整分配，使准确确定的磁化率张量的两种构象的血红素的方向。这两种形式的磁化率张量的z分量的方向是不可区分的，而面内分量似乎相差约6。面内敏感性成分的取向差异无疑主要是由于血红素的相对轴向旋转在5和10之间，这由在亚铁细胞色素光谱中观察到的与蛋白质的NOESY接触指示[Guiles，R. D、Basus，V.J.，Kuntz，ID，& Waskell，LA（1992）Biochemistry 31，11365-11375; Pochapsky，TC，Sligar，S. G.，McLachlan，S. J.，& LaMar，G. N.等人（1990）J. Am. 112，5258-5263]，相对较少的金属蛋白质的质子化学位移已经被广泛地分配在多于一种的氧化态中。细胞色素c（Wand等人，1989; Feng等人，1989年; Gao等人，1991）、牛细胞色素5（Guiles等人，1990; Veitch等人，1990）和硫氧还蛋白（Dyson等人，1988年）是这种系统的几个显著的例子。这种系统的数量相对较少是由于以下原因造成的困难：（1）顺磁系统中的有效弛豫产生宽谱线，因此，可能严重地使重叠问题复杂化，以及（2）接触或伪接触（偶极）效应，其经常导致谐振位置的大的偏移，导致作为传统的双极性的基础的典型连接性图案的混乱，维度分配策略（Wüthrich，1986）。这项工作的重点是非各向同性的顺磁系统与relativelyshort弛豫时间，如血红素蛋白质，这两个困难都遇到。
Revised Manuscript Received May 27, 1993 abstract: 15N and resonance assignments for backbone and side-chain resonances of both equilibrium forms of rat ferricytochrome 65 have been obtained, using a combination of novel heteronuclear assignment transfer methods from the knownassignments of the diamagnetic protein [Guiles, RD, Basus, V. J., Kuntz, I. D., & Waskell, L. A.(1992) Biochemistry 31, 11365-11375] and computational methods which depend on an accurate determination of the orientation of the components of the susceptibility tensor. The transfer of amide proton resonance assignments takes advantage of the apparent insensitivity of amide 15N resonances to pseudocontact effects, evident in overlays of 15N-'H heteronuclear correlation spectra. Amide-proton resonance assignments tentatively transferred from the known diamagnetic assignments to the paramagnetic form of the protein were confirmed using conventional assignment strategies employing 600-MHz COSY, HOHAHA, and NOESY spectra of the oxidized protein. As was observed in rat ferrocytochrome¿> 5, more than 40% of all residues exhibited NMR detectable heterogeneity due to the two different orientations of the heme. Complete assignment of both forms enabled accurate determination of the orientation of the susceptibility tensor for both conformations of the heme. The orientation of the z-component of the susceptibility tensors for thetwo forms are indistinguishable, while the in-plane components appear to differ by about 6. Differences in the orientation of the in-planesusceptibility components are undoubtedly due dominantly to the relative axialrotation of theheme of between 5 and 10 indicated by the NOESY contacts to the protein observed in the spectra of the ferrocytochrome [Guiles, R. D., Basus, V. J., Kuntz, ID, & Waskell, LA (1992) Biochemistry 31, 11365-11375; Pochapsky, TC, Sligar, S. G., McLachlan, S. J., & LaMar, G. N.(1990) J. Am. Chem. Soc. 112, 5258-5263], Proton chemical shifts of relatively few metalloproteins have been extensively assigned in more than one oxidation state. Cytochrome c (Wand et al., 1989; Feng et al., 1989; Gao et al., 1991), bovine cytochrome¿> 5 (Guiles et al., 1990; Veitch et al., 1990), and thioredoxin (Dyson et al., 1988) are a few notable examples of such systems. The relatively small number of such systems has been due to difficulties caused by (1) efficient relaxation in paramagnetic systems which yields broad lines and, as a result, can severely complicate overlap problems and (2) contact or pseudocontact (dipolar) effects that often result in large shifts in resonance positions resulting in a scrambling of typical connectivity patterns which are the basis of conventional two-dimensional assignment strategies (Wüthrich, 1986). Thiswork focuses on nonisotropic paramagnetic systems with relativelyshort relaxation times such as heme proteins where both difficulties are encountered.