Solution 1H NMR determination of secondary structure for the three-iron form of ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus.
Solution 1H NMR determination of secondary structure for the three-iron form of ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus.
复制标题
溶液 1H NMR 测定来自超嗜热古菌激烈火球菌的三铁形式铁氧还蛋白的二级结构。
DOI：
10.1021/bi00186a035
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发表时间：
1994
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：
Teng,Q;Zhou,ZH;Smith,ET;Busse,SC;Howard,JB;Adams,MW;LaMar,GN
1994年3月15日接收的修订版Mandarin pt ®摘要：二维NMR数据已被用于进行序列特异性分配并定义来自极端嗜热古菌Pyrococcus furiosus，Pf的氧化铁氧还蛋白Fd的三铁形式的二级结构。至少一些质子的信号位于序列中66个氨基酸中的65个，尽管有顺磁性（S=1/2）基态，但并不是所有的都能归属。未分配和缺失的信号可以定性地与质子与顺磁簇的预期接近度相关。二级结构由2D核Overhauser效应的定性分析推断，其鉴定了两个反平行/3-折叠，一个三链包括Alal-Ser 5、Val 39-Glu 41和Thr 62-Ala 66，一个双链包括Glu 26-Asn 28和Lys 32-Glu 34，以及涉及Glu 43-Glu 54的螺旋。三个紧密的I型转角位于残基Asp 7-ThrlO、Pro22-Phe 25和Asp 29-Gly 31处。与脱硫弧菌（Desulfovibrio gigas）、Dg、Fd的晶体结构的比较（Kissinger等人，1991）揭示了一个非常相似的折叠拓扑结构，虽然几个二级结构元件在Pf中相对于Dg Fd延伸。因此，涉及两个末端的β-折叠被扩展以包括两个末端残基，并并入来自内部环的第三链，该第三链通过相对于Dg Fd在Pf中的几个插入而延长。Pf Fd内部的双链/3-折叠由于两条链之间更紧密的I型转弯而略微延长。在Pf中靠近C-末端的螺旋比在Dg Fd中长三个残基，并且向N-末端移动。两个非连接的Cys残基（Cys 21和Cys 48）之间的二硫键连接在Pf Fd中是保守的，但是靠近C-末端的连接在Pf Fd中的长螺旋的中间，而不是在Dg Fd中的螺旋的N-末端。β-折叠和β-螺旋的延伸使Pf Fd中的主链氢键数目相对于Dg Fd中的主链氢键数目增加约8，并且可能有助于其显著的热稳定性（其不受95 ℃下厌氧孵育24小时的影响）。对2* 中的一个样品的定性研究揭示了大约20个缓慢交换的不稳定质子，这些质子位于各种已鉴定的二级结构元件中，除了两个质子，其弛豫性质决定了它们来自其肽NH参与与簇的配位硫原子的氢键相互作用的残基。已经从富含硫化物的海洋环境中分离出微生物，其具有在接近或高于100 ℃的温度下生长旺盛的显著特性。几乎所有这些所谓的“超嗜热菌”都被归类为古细菌（以前的古细菌）（Stetter等人，1990;亚当斯等人，1992;亚当斯，1993 a），许多是严格厌氧的、减硫生物，可能与早期生命形式有关（Woese等人，1990年）。然而，我们对在这种极端条件下提供生长的潜在独特生物化学知之甚少。特别是，我们在理解在这些高温下稳定蛋白质功能性三维结构的因素方面相对简单。对极端嗜热古菌研究最多的是强烈火球菌Pf，1，其最佳生长温度为100 ℃（Fíala & Stetter，1986），并且其几种酶和蛋白质已被纯化（亚当斯，
Revised Manuscript Received March 15, 1994® abstract: Two-dimensional NMR data have been used tomake sequence-specific assignments and define the secondary structure of the three-iron formof the oxidized ferredoxin, Fd, from the hyperthermophilic archaeon Pyrococcus furiosus, Pf. Signals for at least some protons were located for 65 of the 66 amino acids in the sequence, in spite of the paramagnetic (S=1/2) ground state, but not all could be assigned. Unassigned and missing signals could be qualitatively correlated with the expected proximity of the protons to the paramagnetic cluster. The secondary structure was deduced from qualitative analysis of the 2D nuclear Overhauser effect, which identified two antiparallel/3-sheets, one triple-stranded including Alal-Ser5, Val39-Glu41, and Thr62-Ala66, and one double-stranded consisting of Glu26-Asn28 and Lys32-Glu34, as well as an-helix involving Glu43-Glu54. Three tight type I turns are located at residues Asp7-Thrl0, Pro22-Phe25, and Asp29-Gly31. Comparison with the crystal structure of Desulfovibrio gigas, Dg, Fd (Kissinger et al., 1991) reveals a very similar folding topology, although several secondary structural elements are extended in Pf relative to Dg Fd. Thus the/3-sheet involving the two termini is expanded to include the two terminal residues and incorporates a third strand from the internal loop that is lengthened by several insertions in Pf relative to Dg Fd. The double-stranded/3-sheet in the interior of Pf Fd is lengthened slightly due to a much tighter type I turn between the two strands. The helix near the C-terminus is three residues longer in Pf than in Dg Fd, as well as being shifted toward the N-terminus. The disulfide link between the two nonligating Cys residues (Cys21 and Cys48) is conserved in Pf Fd, but the link near the C-terminus is in the middle of the long-helix in Pf Fd, instead of at the N-terminus of the helix as in Dg Fd. The extensions of the/3-sheets and-helix increase the number of main-chain hydrogen bonds in Pf Fd by approximately 8 relative to those in Dg Fd and likely contribute to its remarkable thermostability (it is unaffected by anaerobic incubation at 95 C for 24 h). Qualitative studies of a sample in 2***** reveal some 20 slowly exchanging labile protons that are located in the various identified secondary structural elements, except for two protons whose relaxation properties dictate that theyarise from residues whose peptide NHs participate in hydrogen bonding interactionwith the coordinated sulfur atoms of the cluster.During the past decade, microorganisms have been isolated from sulfide-rich marine environments with the remarkable property of thriving at temperatures near or above 100 C. Virtually all of these so-called “hyperthermophiles” are classified as Archaea (formerly Archaebacteria)(Stetter et al., 1990; Adams et al., 1992; Adams, 1993a), and many are strictly anaerobic, sulfur-reducing organisms that may be related to early life forms (Woese et al., 1990). However, we know little about the potentially unique biochemistry that affords growth under such extreme conditions. In particular, we are relatively unsophisticated in understanding the factors that stabilize the functional three-dimensional structures of proteins at these elevated temperatures. The best studied of the hyperthermophilic archaea is Pyrococcus furiosus, Pf, 1 which grows optimally at 100 C (Fíala & Stetter, 1986), and several of its enzymes and proteins have been purified (Adams,