NMR hydrogen exchange of the OB-fold protein LysN as a function of denaturant: The most conserved elements of structure are the most stable to unfolding
NMR hydrogen exchange of the OB-fold protein LysN as a function of denaturant: The most conserved elements of structure are the most stable to unfolding
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DOI：
10.1006/jmbi.1999.2813
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发表时间：
1999-06-18
影响因子：
5.6
通讯作者：
Lamour, FP
中科院分区：
文献类型：
--
作者：
Alexandrescu, AT;Jaravine, VA;Lamour, FP
LysN的结构包含OB-fold基序，该基序由结构保守的五链β -桶状结构组成，在β - 3和β - 4链之间有一个保守性较差的α -螺旋结构。两个额外的α -螺旋，独特的LysN结构，侧面的OB-fold的N端。利用核磁共振天然态氢交换测量研究了LysN的展开稳定性，并利用222 nm的椭圆率和350 nm的荧光监测了平衡展开转变。分光光度测定表明，δ G(u)(0)近似于6 kcal/ mol， m近似于3 kcal/(molM)，存在明显的两态展开跃迁。相比之下，核磁共振氢交换测量显示了δ G(u)(0)和m值的分布，这表明蛋白质可以进行亚全局展开。氢交换中最大的δ G(u)(0)值是在蛋白质的-薄片中的残基。这些值反映了蛋白质的完全展开，比圆二色性或荧光法获得的值高3至4千卡/摩尔。这种差异可能是由于在残留物水平分辨率下测量的核磁共振氢交换参数与反映α -螺旋和β -链展开跃迁的未解决的叠加的分光光度参数的比较。从蛋白质α螺旋残基子集的氢交换中得到的最大G(u)(0)值与从圆二色性或荧光中得到的G(u)(0)值一致。然而，根据氢交换数据，LysN的三个α -螺旋的平均稳定性比β -薄片低3千卡/摩尔。与氢交换证明的LysN亚全局展开一致，缺乏蛋白质第一α -螺旋的缺失突变体保留了合作折叠结构。结合以往OB-fold蛋白SN和CspA的研究结果，目前的结果表明，OB-fold基序中最保守的结构元件是最耐变性的。在这三种蛋白质中，变性稳定性与序列疏水性有关。(C) 1999学术出版社。
The structure of LysN contains an OB-fold motif composed of a structurally conserved five-stranded beta-barrel capped by a poorly conserved alpha-helix between strands beta 3 and beta 4. Two additional alpha-helices, unique to the LysN structure, flank the N terminus of the OB-fold. The stability of LysN to unfolding has been investigated with NMR native state hydrogen exchange measurements as a function of guanidinium hydrochloride concentration, and equilibrium unfolding transitions monitored by ellipticity at 222 nm and fluorescence at 350 nm. The spectrophotometric measurements suggest an apparent two-state unfolding transition with Delta G(u)(0) similar to 6 kcal/ mol and m similar to 3 kcal/(molM). By contrast, NMR hydrogen exchange measurements manifest a distribution of Delta G(u)(0) and m values which indicate that the protein can undergo subglobal unfolding. The largest Delta G(u)(0) values from hydrogen exchange are for residues in the beta-sheet of the protein. These values, which reflect complete unfolding of the protein, are between 3 and 4 kcal/mol higher than those obtained from circular dichroism or fluorescence. This discrepancy may be due to the comparison of NMR hydrogen exchange parameters measured at residue-level resolution, with spectrophotometric parameters that reflect an unresolved superposition of unfolding transitions of the alpha-helices and beta-strands. The largest Delta G(u)(0) values obtained from hydrogen exchange for the subset of residues in the alpha-helices of the protein, agree with the Delta G(u)(0) values obtained from circular dichroism or fluorescence. Based on the hydrogen exchange data, however, the three alpha-helices of LysN are on average 3 kcal/mol less stable than the beta-sheet. Consistent with the subglobal unfolding of LysN evinced by hydrogen exchange, a deletion mutant that lacks the first alpha-helix of the protein retains a cooperatively folded structure. Taken together with previous results on the OB-fold proteins SN and CspA, the present results for LysN suggest that the most conserved elements of structure in the OB-fold motif are the most resistant to denaturation. Ln all three proteins, stability to denaturation correlates with sequence hydrophobicity. (C) 1999 Academic Press.