Alexandrescu A T, Ng Y L, Dobson C M
Oxford Centre for Molecular Sciences, University of Oxford, England.
J Mol Biol. 1994 Jan 14;235(2):587-99. doi: 10.1006/jmbi.1994.1015.
The protein alpha-lactalbumin exists in a partially folded molten globule state at pH 2.0, the A state. This state is believed to be compact, possessing a similar amount of secondary structure to the native state but having a flexible tertiary structure comprised mainly of non-specific hydrophobic clustering of residues. Addition of trifluoroethanol (TFE) to bovine, human and guinea pig alpha-lactalbumin at pH 2.0 has been found in each case to induce a conformational transition in the A state as monitored by circular dichroism, nuclear magnetic resonance chemical shifts, and 1-anilinonaphthalene-8-sulphonate binding. The mid-point of this transition is near 15% (v/v) TFE and is effectively complete by 50% (v/v) TFE at 315 K. Far ultraviolet circular dichroism ellipticities at 208 nm and 220 nm, usually taken as a measure of the degree of helical character, are substantially more negative in the TFE state than in the A state. Furthermore, backbone amide protons protected from solvent exchange in the A state are generally at least as strongly protected in the TFE state; patterns of protection appear similar in the two states and include at least part of both the B and C alpha-helices. One major difference from the A state is nevertheless evident: the ability to bind the fluorescent probe 1-anilinonaphthalene-8-sulphonate, characteristic of molten globule states, is lost in the TFE state. Like the A state, the TFE state of alpha-lactalbumin shows little chemical shift dispersion of side-chain resonances. Extensive line broadening in the nuclear magnetic resonance spectra, characteristic of slow conformational averaging in the A state, is, however, much reduced in the TFE state. The line narrowing observed in the TFE state has made it possible to obtain directly sequence-specific assignments for about 25% of the 123 residues of bovine alpha-lactalbumin in 50% (v/v) TFE. Two helices are amongst regions of structure so far identified from short-range backbone nuclear Overhauser enhancement (NOE) connectivities in two-dimensional spectra of the TFE state. One of the helices (residues 86 to 96) corresponds to the C-helix in the native structure. The other (residues 35 to 41) corresponds, however, to a region of the sequence that is not helical in the native state. The partially folded state of alpha-lactalbumin formed in TFE, therefore, supports both native and non-native secondary structure in the absence of persistent long-range tertiary structure.
蛋白质α-乳白蛋白在pH 2.0时以部分折叠的熔融球状状态存在，即A状态。据信这种状态是紧密的，具有与天然状态相似数量的二级结构，但具有主要由残基的非特异性疏水聚集组成的灵活三级结构。在pH 2.0时，向牛、人和豚鼠的α-乳白蛋白中添加三氟乙醇（TFE），在每种情况下都发现会诱导A状态的构象转变，这通过圆二色性、核磁共振化学位移和1-苯胺基萘-8-磺酸盐结合来监测。这种转变的中点接近15%（v/v）TFE，在315 K时50%（v/v）TFE时有效地完成。通常作为螺旋特征程度度量的208 nm和220 nm处的远紫外圆二色性椭圆率在TFE状态下比在A状态下更负。此外，在A状态下免受溶剂交换保护的主链酰胺质子在TFE状态下通常至少同样受到强烈保护；两种状态下的保护模式似乎相似，包括B和Cα-螺旋的至少一部分。然而，与A状态的一个主要区别是明显的：在TFE状态下失去了结合荧光探针1-苯胺基萘-8-磺酸盐的能力，这是熔融球状状态的特征。与A状态一样，α-乳白蛋白的TFE状态显示侧链共振的化学位移分散很小。然而，在A状态下特征为缓慢构象平均的核磁共振谱中的广泛线宽展宽在TFE状态下大大减少。在TFE状态下观察到的线窄化使得能够直接获得牛α-乳白蛋白123个残基中约25%在50%（v/v）TFE中的序列特异性归属。从TFE状态的二维谱中的短程主链核Overhauser增强（NOE）连接性迄今已鉴定出的结构区域中包括两个螺旋。其中一个螺旋（残基86至96）对应于天然结构中的C-螺旋。然而，另一个（残基35至41）对应于天然状态下不是螺旋的序列区域。因此，在TFE中形成的α-乳白蛋白的部分折叠状态在没有持久的长程三级结构的情况下支持天然和非天然二级结构。
