Polverino de Laureto P, Vinante D, Scaramella E, Frare E, Fontana A
CRIBI Biotechnology Centre, University of Padua, Italy.
Eur J Biochem. 2001 Aug;268(15):4324-33. doi: 10.1046/j.1432-1327.2001.02352.x.
Bovine alpha-lactalbumin (alpha-LA) is an alpha/beta protein which adopts partly folded states when dissolved at low pH (A-state), by removal of the protein-bound calcium at neutral pH and low salt concentration (apo-state), as well as in aqueous trifluoroethanol. Previous spectroscopic studies have indicated that the A-state of alpha-LA at pH 2.0, considered a prototype molten globule, has a native-like fold in which the helical core is mostly retained, while the beta subdomain is less structured. Here, we investigate the conformational features of three derivatives of alpha-LA characterized by a single peptide bond fission or a deletion of 12 or 19/22 amino-acid residues of the beta subdomain of the native protein (approximately from residue 34 to 57). These alpha-LA derivatives were obtained by limited proteolysis of the protein in its partly folded state(s). A nicked alpha-LA species consisting of fragments 1-,3-40 and 41-123 (nicked-LA) was prepared by thermolytic digestion of the 123-residue chain of alpha-LA in 50% (v/v) aqueous trifluoroethanol. Two truncated or gapped protein species given by fragments 1-40 and 53-123 (desbeta1-LA) or fragments 1-34 and 54-,57-123 (desbeta2-LA) were obtained by digestion of alpha-LA with pepsin in acid or with proteinase K at neutral pH in its apo-state, respectively. The two protein fragments of nicked or gapped alpha-LA are covalently linked by the four disulfide bridges of the native protein. CD measurements revealed that, in aqueous solution at neutral pH and in the presence of calcium, the three protein species maintain the helical secondary structure of intact alpha-LA, while the tertiary structure is strongly affected by the proteolytic cleavages of the chain. Temperature effects of CD signals in the far- and near-UV region reveal a much more labile tertiary structure in the alpha-LA derivatives, while the secondary structure is mostly retained even upon heating. In acid solution at pH 2.0, the three alpha-LA variants adopt a conformational state essentially identical to the molten globule displayed by intact alpha-LA, as demonstrated by CD measurements. Moreover, they bind strongly the fluorescent dye 8-anilinonaphthalene-1-sulfonate, which is considered a diagnostic feature of the molten globule of proteins. Therefore, the beta subdomain can be removed from the alpha-LA molecule without impairing the capability of the rest of the chain to adopt a molten globule state. The results of this protein dissection study provide direct experimental evidence that in the alpha-LA molten globule only the alpha domain is structured.
牛α-乳白蛋白（α-LA）是一种α/β蛋白，当在低pH值下溶解时（A态）、在中性pH值和低盐浓度下去除与蛋白结合的钙时（脱辅基态）以及在三氟乙醇水溶液中时，它会呈现部分折叠状态。先前的光谱研究表明，pH 2.0时α-LA的A态被认为是一种典型的熔球态，具有类似天然的折叠结构，其中螺旋核心大部分得以保留，而β亚结构域的结构则较少。在此，我们研究了α-LA的三种衍生物的构象特征，这些衍生物的特点是单个肽键断裂或天然蛋白β亚结构域（大约从第34至57位残基）缺失12个或19/22个氨基酸残基。这些α-LA衍生物是通过对处于部分折叠状态的蛋白质进行有限蛋白酶解获得的。由片段1 - 3 - 40和41 - 123组成的带切口的α-LA物种（带切口-LA）是通过在50%（v/v）三氟乙醇水溶液中对α-LA的123个残基链进行热解消化制备的。由片段1 - 40和53 - 123（缺失β1-LA）或片段1 - 34和54 - 57 - 123（缺失β2-LA）组成的两种截短或有缺口的蛋白质物种分别是通过在酸性条件下用胃蛋白酶消化α-LA或在脱辅基态下在中性pH值用蛋白酶K消化α-LA获得的。带切口或有缺口的α-LA的两个蛋白质片段通过天然蛋白的四个二硫键共价连接。圆二色性（CD）测量表明，在中性pH值的水溶液中且存在钙的情况下，这三种蛋白质物种保持完整α-LA的螺旋二级结构，而三级结构受到链的蛋白酶解切割的强烈影响。远紫外和近紫外区域CD信号的温度效应表明，α-LA衍生物中的三级结构更加不稳定，而即使加热二级结构也大多得以保留。在pH 2.0的酸性溶液中，如CD测量所示，这三种α-LA变体采用的构象状态与完整α-LA呈现的熔球态基本相同。此外，它们与荧光染料8-苯胺基萘-1-磺酸盐强烈结合，这被认为是蛋白质熔球态的一个诊断特征。因此，可以从α-LA分子中去除β亚结构域而不损害链的其余部分采用熔球态的能力。这项蛋白质剖析研究的结果提供了直接的实验证据，即在α-LA熔球态中只有α结构域是有结构的。
