NMR structural and dynamic characterization of the acid-unfolded state of apomyoglobin provides insights into the early events in protein folding.
NMR structural and dynamic characterization of the acid-unfolded state of apomyoglobin provides insights into the early events in protein folding.
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DOI：
10.1021/bi002776i
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发表时间：
2001-02
期刊：
影响因子：
2.9
通讯作者：
Jian Yao;John Chung;D. Eliezer;P. Wright;H. Dyson
中科院分区：
文献类型：
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作者：
Jian Yao;John Chung;D. Eliezer;P. Wright;H. Dyson
阿肌红蛋白在 pH 2.3 的低盐条件下形成变性状态。该状态的构象倾向和多肽主链动力学已通过 NMR 进行了表征。使用针对低蛋白质浓度 (0.2 mM) 和差的化学位移分散度定制的三重共振分配策略，获得了几乎完整的主链和一些侧链共振分配。通过检查 (13)C(α)、(13)CO 和 (1)H(α) 化学位移与随机卷曲值、标量 (3)J(HN,H)(α) 耦合常数和 (1)H-(1)H NOE 的偏差，对残余二级结构的数量和位置进行了估计。如果使用适当的随机卷曲化学位移参考，化学位移构成二级结构偏好的高度可靠的指标，但在酸解折叠的脱肌红蛋白的情况下，(3)J(HN,H)(α)耦合常数对于二级结构形成的诊断效果不佳。在与折叠蛋白的 A 和 H 螺旋相对应的区域中形成大量螺旋结构，与未折叠状态处于动态平衡。此外，化学位移与随机卷曲值的偏差表明存在包围 D 螺旋并延伸到 E 螺旋第一圈的螺旋结构。使用 (15)N 弛豫数据的约简谱密度函数分析研究了酸展开的脱辅基肌红蛋白的多肽主链动力学。谱密度 J(omega(N)) 对皮秒到纳秒时间尺度上主干波动的变化特别敏感。跨越 E 螺旋 C 端一半、EF 转角和 F 螺旋的多肽中心区域表现为自由飞行的随机卷曲链，但 J(omega(N)) 有证据表明 A 和 H 螺旋区域在皮秒到纳秒时间尺度上的运动受到限制，其中倾向于在酸解折叠状态下填充螺旋二级结构。由于局部疏水簇的形成，B 和 G 螺旋的部分主链波动也受到限制。主干柔性受限的区域通常与大的埋藏表面积相关。对于位于折叠蛋白的 A 和 G 螺旋中的一些残基的 NH 共振，观察到 J(0) 显着增加，并且与微秒到毫秒时间尺度的波动相关，这种波动可能是由多肽链的这些遥远区域之间的瞬时接触引起的。我们的结果表明，在 pH 2.3 下形成的脱辅基肌红蛋白的平衡未折叠状态是预计在蛋白质折叠的最早阶段发生的事件的极好模型，提供了对自发经历局部疏水性塌陷和采样天然二级结构的多肽区域的深入了解。
Apomyoglobin forms a denatured state under low-salt conditions at pH 2.3. The conformational propensities and polypeptide backbone dynamics of this state have been characterized by NMR. Nearly complete backbone and some side chain resonance assignments have been obtained, using a triple-resonance assignment strategy tailored to low protein concentration (0.2 mM) and poor chemical shift dispersion. An estimate of the population and location of residual secondary structure has been made by examining deviations of (13)C(alpha), (13)CO, and (1)H(alpha) chemical shifts from random coil values, scalar (3)J(HN,H)(alpha) coupling constants and (1)H-(1)H NOEs. Chemical shifts constitute a highly reliable indicator of secondary structural preferences, provided the appropriate random coil chemical shift references are used, but in the case of acid-unfolded apomyoglobin, (3)J(HN,H)(alpha) coupling constants are poor diagnostics of secondary structure formation. Substantial populations of helical structure, in dynamic equilibrium with unfolded states, are formed in regions corresponding to the A and H helices of the folded protein. In addition, the deviation of the chemical shifts from random coil values indicates the presence of helical structure encompassing the D helix and extending into the first turn of the E helix. The polypeptide backbone dynamics of acid-unfolded apomyoglobin have been investigated using reduced spectral density function analysis of (15)N relaxation data. The spectral density J(omega(N)) is particularly sensitive to variations in backbone fluctuations on the picosecond to nanosecond time scale. The central region of the polypeptide spanning the C-terminal half of the E helix, the EF turn, and the F helix behaves as a free-flight random coil chain, but there is evidence from J(omega(N)) of restricted motions on the picosecond to nanosecond time scale in the A and H helix regions where there is a propensity to populate helical secondary structure in the acid-unfolded state. Backbone fluctuations are also restricted in parts of the B and G helices due to formation of local hydrophobic clusters. Regions of restricted backbone flexibility are generally associated with large buried surface area. A significant increase in J(0) is observed for the NH resonances of some residues located in the A and G helices of the folded protein and is associated with fluctuations on a microsecond to millisecond time scale that probably arise from transient contacts between these distant regions of the polypeptide chain. Our results indicate that the equilibrium unfolded state of apomyoglobin formed at pH 2.3 is an excellent model for the events that are expected to occur in the earliest stages of protein folding, providing insights into the regions of the polypeptide that spontaneously undergo local hydrophobic collapse and sample nativelike secondary structure.