Guan L, Weinglass A B, Kaback H R
Howard Hughes Medical Institute, University of California Los Angeles, Los Angeles, CA 90095-1662, USA.
J Mol Biol. 2001 Sep 7;312(1):69-77. doi: 10.1006/jmbi.2001.4933.
Plasmids encoding "split" lactose permease constructs with discontinuities in either the periplasmic loop between helices V and VI (N(5)/C(7)) or between helices VI and VII (N(6)/C(6)) were used to localize helix VI within the tertiary structure by site-directed thiol cross-linking. A total of 57 double-Cys pairs, with one Cys residue in helix VI and another in helix V or VIII, were studied with homobifunctional cross-linking agents. Significant cross-linking is observed between the periplasmic ends of helices V (position 158 or 161) and VI (position 170) with rigid 6 or 10 A reagents. Furthermore, the Cys residue at position 170 (helix VI) also cross-links to a Cys residue at either position 264 or 265 (helix VIII) with a 21 A cross-linking agent. The data indicate that helices V, VI and VIII are in close proximity at the periplasmic face of the membrane, with helix VI significantly closer to helix V. In addition, beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside induces a significant increase in cross-linking efficiency between helices VI and VIII and between helices V and VIII, with no significant change in cross-linking between helices V and VI.
编码“分裂”乳糖通透酶构建体的质粒，其在螺旋V和VI之间的周质环（N(5)/C(7)）或螺旋VI和VII之间（N(6)/C(6)）存在间断，通过定点硫醇交联用于在三级结构中定位螺旋VI。使用同双功能交联剂研究了总共57个双半胱氨酸对，其中一个半胱氨酸残基在螺旋VI中，另一个在螺旋V或VIII中。使用刚性的6或10埃试剂时，在螺旋V（位置158或161）和VI（位置170）的周质端之间观察到显著交联。此外，位置170（螺旋VI）处的半胱氨酸残基也用21埃交联剂与位置264或265（螺旋VIII）处的半胱氨酸残基交联。数据表明，螺旋V、VI和VIII在膜的周质面紧密相邻，其中螺旋VI与螺旋V显著更接近。此外，β-D-吡喃半乳糖基1-硫代-β-D-吡喃半乳糖苷诱导螺旋VI和VIII之间以及螺旋V和VIII之间的交联效率显著增加，而螺旋V和VI之间的交联没有显著变化。
