Teng Q, Zhou Z H, Smith E T, Busse S C, Howard J B, Adams M W, La Mar G N
Department of Chemistry, University of California, Davis 95616.
Biochemistry. 1994 May 24;33(20):6316-26. doi: 10.1021/bi00186a035.
Two-dimensional 1H NMR data have been used to make sequence-specific assignments and define the secondary structure of the three-iron form of 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 beta-sheets, one triple-stranded including Ala1-Ser5, Val39-Glu41, and Thr62-Ala66, and one double-stranded consisting of Glu26-Asn28 and Lys32-Glu34, as well as an alpha-helix involving Glu43-Glu54. Three tight type I turns are located at residues Asp7-Thr10, 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 beta-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 beta-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 alpha-helix in Pf Fd, instead of at the N-terminus of the helix as in Dg Fd. The extensions of the beta-sheets and alpha-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 degrees C for 24 h).(ABSTRACT TRUNCATED AT 400 WORDS)
二维¹H NMR数据已被用于对嗜热古菌激烈火球菌（Pf）的氧化型铁氧化还原蛋白（Fd）的三铁形式进行序列特异性归属，并确定其二级结构。尽管处于顺磁（S = 1/2）基态，但序列中66个氨基酸中的65个至少有一些质子的信号已被定位，但并非所有信号都能被归属。未归属和缺失的信号可以与质子和顺磁簇的预期接近程度进行定性关联。二级结构是通过对二维核Overhauser效应的定性分析推导出来的，该效应确定了两个反平行的β-折叠片层，一个三链的包括Ala1-Ser5、Val39-Glu41和Thr62-Ala66，一个双链的由Glu26-Asn28和Lys32-Glu34组成，以及一个涉及Glu43-Glu54的α-螺旋。三个紧密的I型转角位于Asp7-Thr10、Pro22-Phe25和Asp29-Gly31残基处。与巨大脱硫弧菌（Dg）Fd的晶体结构（Kissinger等人，1991年）比较发现，尽管Pf Fd中的几个二级结构元件相对于Dg Fd有所延伸，但折叠拓扑非常相似。因此，涉及两个末端的β-折叠片层扩展到包括两个末端残基，并纳入了来自内部环的第三条链，该链在Pf中相对于Dg Fd通过几个插入而延长。Pf Fd内部的双链β-折叠片层由于两条链之间更紧密的I型转角而略有延长。Pf中靠近C末端的螺旋比Dg Fd中的长三个残基，并且向N末端移动。两个非配位半胱氨酸残基（Cys21和Cys48）之间的二硫键在Pf Fd中保守，但靠近C末端的键在Pf Fd的长α-螺旋中间，而不是像在Dg Fd中那样在螺旋的N末端。相对于Dg Fd，Pf Fd中β-折叠片层和α-螺旋的延伸使主链氢键数量增加了约8个，这可能有助于其显著的热稳定性（在95℃厌氧孵育24小时不受影响）。（摘要截断于400字）
