Morozova-Roche L A, Arico-Muendel C C, Haynie D T, Emelyanenko V I, Van Dael H, Dobson C M
Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, England.
J Mol Biol. 1997 May 23;268(5):903-21. doi: 10.1006/jmbi.1997.0996.
Native state 1H NMR resonance assignments for 125 of the 129 residues of equine lysozyme have enabled measurement of the hydrogen exchange kinetics for over 60 backbone amide and three tryptophan indole hydrogen atoms in the native state. Native holo equine lysozyme hydrogen exchange protection factors are as large as 10(6), the most protected residues being located in elements of secondary structure. High exchange protection in the domain interface correlates with the binding of Ca2+ in this region. Equine lysozyme differs from most non-Ca2+ binding lysozymes in forming a highly populated partially folded state at low pH. The protein in this A-state at pH 2.0 has been found to bind 1-anilino-naphthalene-8-sulphonate with the enhancement of fluorescent intensity and blue shift in the spectral maximum characteristic of molten globules. NMR spectra indicate that the A-state is globally much less ordered than native equine lysozyme but does not contain significant regions of random coil structure. The amides most protected against hydrogen exchange in the A-state (protection factors up to 10(2) at 5 degrees C) correspond to residues of three of the four alpha-helices of the native state; the side-chains of these residues form a hydrophobic cluster that includes five aromatic residues. Circular dichroism and tryptophan fluorescence indicate that these residues are substantially more constrained than similar residues in "classical" molten globules. Taken together, the data suggest a model for the A-state of equine lysozyme in which a more ordered core is surrounded by a less ordered but still compact polypeptide chain.
马溶菌酶129个残基中的125个残基的天然状态1H NMR共振归属，使得能够测量天然状态下60多个主链酰胺和3个色氨酸吲哚氢原子的氢交换动力学。天然全马溶菌酶的氢交换保护因子高达10^6，保护作用最强的残基位于二级结构元件中。结构域界面处的高交换保护与该区域中Ca2+的结合相关。马溶菌酶与大多数非Ca2+结合的溶菌酶不同，在低pH值下会形成高度聚集的部分折叠状态。已发现pH 2.0时处于这种A状态的蛋白质能结合1-苯胺基萘-8-磺酸盐，荧光强度增强，光谱最大值出现蓝移，这是熔球态的特征。NMR光谱表明，A状态在整体上比天然马溶菌酶的有序程度低得多，但不包含显著的无规卷曲结构区域。在A状态下对氢交换保护作用最强的酰胺（5℃时保护因子高达10^2）对应于天然状态下四个α螺旋中三个的残基；这些残基的侧链形成一个疏水簇，其中包括五个芳香族残基。圆二色性和色氨酸荧光表明，这些残基比“经典”熔球态中的类似残基受到的限制要大得多。综合来看，这些数据提出了一个马溶菌酶A状态的模型，其中一个更有序的核心被一个不太有序但仍然紧密的多肽链所包围。
