Interaction between two discontiguous chain segments from the beta-sheet of Escherichia coli thioredoxin suggests an initiation site for folding.
Interaction between two discontiguous chain segments from the beta-sheet of Escherichia coli thioredoxin suggests an initiation site for folding.
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大肠杆菌硫氧还蛋白β-折叠的两个不连续链段之间的相互作用表明了折叠的起始位点。
DOI：
10.1021/bi000761e
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发表时间：
2000
期刊：
影响因子：
2.9
通讯作者：
Georgescu,RE
中科院分区：
文献类型：
--
作者：
Tasayco,ML;Fuchs,J;Yang,XM;Dyalram,D;Georgescu,RE
比较折叠和折叠/结合过程的方法非常适合缩小驱动蛋白质折叠的序列区域。我们将氧化的大肠杆菌硫氧还蛋白（Trx）的小的单一α/β结构域分解成三个互补片段（N，残基1 - 37; M，残基38 - 73;和C，残基74 - 108），以通过远紫外CD和NMR光谱研究它们的分离和重组。孤立的碎片显示出最小的椭圆度约。197 nm的远紫外CD光谱中没有浓度依赖性，Hα的化学位移接近无规卷曲值，并且在三维NMR光谱中没有中程和长程NOE连接性。这些片段表现为无序单体。只有含有N-和C-片段的二元或三元混合物的远紫外CD光谱与其各自光谱的总和不同，这表明这些片段的折叠和/或结合。事实上，在加入未标记的互补片段后，Trx β折叠的疏水性β 2和β 4区域的交叉峰分别从分离的标记N和C片段的1H − 15 N HSQC光谱中消失。消失的交叉峰表明β 2和β 4区之间的相互作用，它们在较低温度下的再现表明主要由疏水力保持的异聚体的解折叠和/或解离。我们的研究结果表明，Trx的折叠是通过拉链连接两个不连续的疏水链段（β 2和β4）开始的，这两个链段对应于天然β折叠的相邻链。
The approach of comparing folding and folding/binding processes is exquisitely poised to narrow down the regions of the sequence that drive protein folding. We have dissected the small single α/β domain of oxidizedEscherichia colithioredoxin (Trx) into three complementary fragments (N, residues 1−37; M, residues 38−73; and C, residues 74−108) to study them in isolation and upon recombination by far-UV CD and NMR spectroscopy. The isolated fragments show a minimum of ellipticity of ca. 197 nm in their far-UV CD spectra without concentration dependence, chemical shifts of Hαthat are close to the random coil values, and no medium- and long-range NOE connectivities in their three-dimensional NMR spectra. These fragments behave as disordered monomers. Only the far-UV CD spectra of binary or ternary mixtures that contain N- and C-fragments are different from the sum of their individual spectra, which is indicative of folding and/or binding of these fragments. Indeed, the cross-peaks corresponding to the rather hydrophobic β2and β4regions of the β-sheet of Trx disappear from the1H−15N HSQC spectra of isolated labeled N- and C-fragments, respectively, upon addition of the unlabeled complementary fragments. The disappearing cross-peaks indicate interactions between the β2and β4regions, and their reappearance at lower temperatures indicates unfolding and/or dissociation of heteromers that are predominantly held by hydrophobic forces. Our results argue that the folding of Trx begins by zippering two discontiguous and rather hydrophobic chain segments (β2and β4) corresponding to neighboring strands of the native β-sheet.