1H NMR investigation of the paramagnetic cluster environment in Pyrococcus furiosus three-iron ferredoxin: sequence-specific assignment of ligated cysteines independent of tertiary structure.
1H NMR investigation of the paramagnetic cluster environment in Pyrococcus furiosus three-iron ferredoxin: sequence-specific assignment of ligated cysteines independent of tertiary structure.
复制标题
强烈火球菌三铁铁氧还蛋白顺磁簇环境的 1H NMR 研究：连接半胱氨酸的序列特异性分配，与三级结构无关。
DOI：
10.1021/bi00002a027
复制
发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
LaMar,GN
中科院分区：
文献类型：
--
作者：
Gorst,CM;Yeh,YH;Teng,Q;Calzolai,L;Zhou,ZH;Adams,MW;LaMar,GN
1994年11月4日收到的修订稿件摘要：分析用于检测S=V2附近的铁还蛋白的顺磁松弛质子的一维和二维核磁共振数据，以对超细移位连接的半胱氨酸信号进行特殊排序，确定这些半胱氨酸所驻留的二级结构元件的性质，并定义该簇与远离该簇的先前描述的二级结构的其余部分的三级接触[Teng，Q.，周，Z.-H.，Busse，S.C.，Howard，J.B.，Adams，M.Ww，&La Mar，[Teng，Q.，周，Z.-H.，Busse，S.C.，Howard，J.B.，Adams，M.Ww，&La Mar，G.N.(1994)生物化学33,6316-6326]。对六个结构表征的立方铁氧还蛋白(FD)簇中连接半胱氨酸的簇合物的几何结构的检查揭示了与簇铁的距离模式(S)，这表明每个半胱氨酸都将显示一个主干质子，这将允许检测到相邻残基的主干的偶极连接性。预计簇共识结合序列中的第一个和最后一个半胱氨酸将显示弱松弛的肽NH和强松弛的CAH信号，而该序列中的两个中心半胱氨酸将显示强松弛的肽NH和弱松弛的CaH峰。在3Fe P.Furiosus Fd中，清楚地观察到三个连接的半胱氨酸的这些偶极接触，提供了第一个序列特异性的连接半胱氨酸的指定，这不需要明确地知道蛋白质的三级结构。该方法对立方铁氧还蛋白有非常广泛的应用。稳态NOES和短混合时间NOESY实验的组合证明了Cys17通过Leu20是在一个短螺旋上，而Cys56可能启动了I型转变，正如在Desulfovibriogas的3Fe FD的晶体结构中观察到的那样[Kisinger，C.R.，Sieker，L.C.，Adman，E.T.，&Jensen，L.H.(1991)J.Mol。比奥尔。219,693-715]。观察到的分辨或部分分辨信号的驰豫速率与它们与团簇中各种铁的接近程度有关，如对D.gias Fd中的同源残基所确定的那样，提供了关于团簇三级接触的额外定性信息。
Revised Manuscript Received November 4, 1994® abstract: One-and two-dimensional* H NMR data tailored to detect paramagnetically relaxed protons near the S= V2, three-iron—sulfur cluster of the ferredoxin from the hyperthermophile Pyrococcus furiosus are analyzed to sequence specifically assign the hyperfine shifted ligated cysteine signals, to determine the nature of the secondary structural elements on which these cysteines reside, and to define the tertiary contacts of the cluster with the remainder of the previously characterizedsecondary structure remote from the cluster [Teng, Q., Zhou, Z.-H., Busse, S. C., Howard, J. B., Adams, M. WW, & La Mar, G. N.(1994) Biochemistry 33, 6316—6326]. Inspection of the geometry of the clusterligating cysteines in the sixstructurally characterizedcubane ferredoxin (Fd) clustersreveals a pattern of distances from the cluster iron (s) that indicate that each Cys will exhibit one backbone proton that will allow the detection of dipolar connectivities to the backbone of adjacent residues. It is expected that the first and last of the Cys in the cluster consensus binding sequence will exhibitweakly relaxed peptide NH and strongly relaxed CaH signals, while the two central Cys in that sequence will exhibitstrongly relaxed peptide NH but weakly relaxed CaHpeaks. These dipolar contacts are clearly observed for the three ligated Cys in 3Fe P. furiosus Fd, providing the first sequence specific assignment of ligated cysteines which do not explicitly require knowledge of the tertiary structure of the protein. This approach is proposed to have very general application to cubane ferredoxins. A combination of steady-state NOEs and short mixing time NOESY experiments demonstrate that Cys17 is on a short helix through Leu20 and that Cys56 likely initiates a type I turn, as observed in the crystal structure of the 3Fe Fd for Desulfovibriogigas [Kissinger, C. R., Sieker, L. C., Adman, E. T., & Jensen, L. H.(1991) J. Mol. Biol. 219, 693—715]. The observed relaxation rates of resolved or partially resolved signals are shown to correlate with their proximity to the various iron in the cluster, as determined for the homologous residues in D. gigas Fd, providing additional qualitative information on tertiary contacts of the cluster.