Janssen O E, Chen B, Büttner C, Refetoff S, Scriba P C
Department of Medicine, Klinikum Innenstadt, Ludwig-Maximilians-University, Munich, Federal Republic of Germany.
J Biol Chem. 1995 Nov 24;270(47):28234-8. doi: 10.1074/jbc.270.47.28234.
Thyroxine-binding globulin (TBG) is the main transport protein for thyroxine (T4) in blood. It shares considerable sequence homology with alpha 1-antitrypsin (AT) and other members of the serine proteinase inhibitor (serpin) superfamily of proteins. The crystallographic structure of AT has been determined and was found to represent the archetype of the serpins. This model has been used for structure-function correlations of TBG. Sequence analysis of the heat-resistant variant TBG-Chicago (TBG-CH) revealed a substitution of the normal tyrosine 309 with phenylalanine. For further analysis, vectors containing the coding regions of normal TBG (TBG-N) and TBG-CH were constructed, transcribed in vitro, and expressed in Xenopus oocytes. Both TBGs were secreted into the culture medium and could not be distinguished by gel electrophoresis. Scatchard analysis of T4 binding to TBG-N and -CH revealed no significant differences in binding affinity. The rate of heat denaturation of TBGs was determined by measurement of residual T4 binding capacity after incubation at 60 degrees C for various periods of time. The half-life values of denaturation of TBG-N and -CH were 7 and 132 min, respectively. The tyrosine 309 to phenylalanine substitution of TBG-CH involves a highly conserved phenylalanine residue of the serpins. The respective phenylalanine 312 of AT ties the alpha-helix hI1 to the molecule, thus stabilizing the tertiary structure. A substitution with tyrosine would disrupt this interaction. Accordingly, stabilization of the TBG molecule by replacement of tyrosine with phenylalanine in position 309 causes the increased heat stability of TBG-CH.
甲状腺素结合球蛋白（TBG）是血液中甲状腺素（T4）的主要转运蛋白。它与α1-抗胰蛋白酶（AT）以及丝氨酸蛋白酶抑制剂（serpin）超家族的其他蛋白质成员具有相当大的序列同源性。AT的晶体结构已被确定，并且被发现代表了丝氨酸蛋白酶抑制剂的原型。该模型已被用于TBG的结构-功能相关性研究。对耐热变体TBG-芝加哥（TBG-CH）的序列分析显示，正常的酪氨酸309被苯丙氨酸取代。为了进一步分析，构建了包含正常TBG（TBG-N）和TBG-CH编码区的载体，进行体外转录，并在非洲爪蟾卵母细胞中表达。两种TBG都分泌到培养基中，并且通过凝胶电泳无法区分。对T4与TBG-N和-CH结合的Scatchard分析显示结合亲和力没有显著差异。通过测量在60℃孵育不同时间后的残余T4结合能力来确定TBG的热变性速率。TBG-N和-CH的变性半衰期值分别为7分钟和132分钟。TBG-CH中酪氨酸309到苯丙氨酸的取代涉及丝氨酸蛋白酶抑制剂的一个高度保守的苯丙氨酸残基。AT相应的苯丙氨酸312将α-螺旋hI1与分子相连，从而稳定三级结构。用酪氨酸取代会破坏这种相互作用。因此，通过在309位用苯丙氨酸取代酪氨酸来稳定TBG分子，导致了TBG-CH热稳定性的增加。
