Guan Lan, Murphy Franklin D, Kaback H Ronald
Howard Hughes Medical Institute, Department of Physiology, Molecular Biology Institute, University of California, Los Angeles, CA 90095-1662, USA.
Proc Natl Acad Sci U S A. 2002 Mar 19;99(6):3475-80. doi: 10.1073/pnas.052703699.
Intermolecular thiol cross-linking was used to determine surface-exposed positions in 250 lactose permease mutants containing single-Cys replacements in each transmembrane helix. Significant cross-linking of monomers to produce homodimers is observed in nine mutants with a 5-A-long cross-linking agent containing bis-methane thiosulfonate reactive groups [position 78 (helix III); positions 185, 186, and 187 (helix VI); positions 263, 275, and 278 (helix VIII); and positions 308 (helix IX) and 398 (helix XII)]. The results are consistent with a current helix-packing model of the permease. Seven of the nine mutants that exhibit intermolecular cross-linking are located at or near the cytoplasmic ends of transmembrane helices; two are near periplasmic ends. The results suggest that only those Cys replacements accessible from the aqueous phase and not from the hydrophobic core of the membrane are susceptible to cross-linking because of the much higher reactivity of the thiolate anion relative to the thiol. Single-Cys mutants at positions 278 (helix VIII) and 398 (helix XII), which are located in opposite sides of the 12-helix bundle, exhibit similar rates of cross-linking with sigmoid kinetics. Furthermore, cross-linking is markedly decreased at 0 degrees C, suggesting that lateral diffusion of the permease within the plane of the membrane is important for intermolecular cross-linking. The findings confirm previous observations indicating that intermolecular cross-linking is a stochastic process resulting from random collisions and support a number of other lines of evidence that lactose permease is a monomer.
分子间硫醇交联用于确定250个乳糖通透酶突变体中每个跨膜螺旋含单个半胱氨酸取代的表面暴露位置。使用含有双甲烷硫代磺酸盐反应基团的5埃长交联剂，在9个突变体中观察到单体显著交联形成同型二聚体[位置78（螺旋III）；位置185、186和187（螺旋VI）；位置263、275和278（螺旋VIII）；以及位置308（螺旋IX）和398（螺旋XII）]。结果与通透酶当前的螺旋堆积模型一致。表现出分子间交联的9个突变体中有7个位于跨膜螺旋的胞质端或其附近；2个靠近周质端。结果表明，由于硫醇盐阴离子的反应性远高于硫醇，只有那些可从水相而非膜的疏水核心接近的半胱氨酸取代才易于交联。位于12螺旋束相对两侧的位置278（螺旋VIII）和398（螺旋XII）处的单半胱氨酸突变体表现出相似的交联速率，动力学呈S形。此外，在0℃时交联明显减少，这表明通透酶在膜平面内的侧向扩散对分子间交联很重要。这些发现证实了先前的观察结果，即分子间交联是由随机碰撞导致的随机过程，并支持了许多其他证据表明乳糖通透酶是单体。
