Jordan J B, Shobana S, Andersen O S, Hinton J F
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, USA.
Biochemistry. 2006 Nov 28;45(47):14012-20. doi: 10.1021/bi061560z.
Tryptophan residues often are found at the lipid-aqueous interface region of membrane-spanning proteins, including ion channels, where they are thought to be important determinants of protein structure and function. To better understand how Trp residues modulate the function of membrane-spanning channels, we have examined the effects of Trp replacements on the structure and function of gramicidin A channels. Analogues of gramicidin A in which the Trp residues at positions 9, 11, 13, and 15 were sequentially replaced with Gly were synthesized, and the three-dimensional structure of each analogue was determined using a combination of two-dimensional NMR techniques and distance geometry-simulated annealing structure calculations. Though Trp --> Gly substitutions destabilize the beta6.3-helical gA channel structure, it is possible to determine the structure of analogues with Trp --> Gly substitutions at positions 11, 13, and 15, but not for the analogue with the Trp --> Gly substitution at position 9. The Gly11-, Gly13-, and Gly15-gA analogues form channels that adopt a backbone fold identical to that of native gramicidin A, with only small changes in the side chain conformations of the unsubstituted residues. Single-channel current measurements show that the channel function and lifetime of the analogues are significantly affected by the Trp --> Gly replacements. The conductance variations appear to be caused by sequential removal of the Trp dipoles, which alter the ion-dipole interactions that modulate ion movement. The lifetime variations did not appear to follow a clear pattern.
色氨酸残基常常出现在跨膜蛋白的脂-水界面区域，包括离子通道，人们认为它们是蛋白质结构和功能的重要决定因素。为了更好地理解色氨酸残基如何调节跨膜通道的功能，我们研究了色氨酸替换对短杆菌肽A通道结构和功能的影响。合成了短杆菌肽A的类似物，其中9、11、13和15位的色氨酸残基依次被甘氨酸取代，并结合二维核磁共振技术和距离几何-模拟退火结构计算确定了每个类似物的三维结构。尽管色氨酸替换为甘氨酸会使β6.3-螺旋短杆菌肽A通道结构不稳定，但仍有可能确定11、13和15位色氨酸被甘氨酸取代的类似物的结构，而9位色氨酸被甘氨酸取代的类似物则无法确定其结构。甘氨酸11、甘氨酸13和甘氨酸15的短杆菌肽A类似物形成的通道，其主链折叠与天然短杆菌肽A相同，只是未被取代残基的侧链构象有微小变化。单通道电流测量表明，色氨酸替换为甘氨酸对类似物的通道功能和寿命有显著影响。电导变化似乎是由色氨酸偶极子的依次去除引起的，这改变了调节离子移动的离子-偶极相互作用。寿命变化似乎没有明显的规律。
