Zakharov S D, Lindeberg M, Griko Y, Salamon Z, Tollin G, Prendergast F G, Cramer W A
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4282-7. doi: 10.1073/pnas.95.8.4282.
Atomic level structures have been determined for the soluble forms of several colicins and toxins, but the structural changes that occur after membrane binding have not been well characterized. Changes occurring in the transition from the soluble to membrane-bound state of the C-terminal 190-residue channel polypeptide of colicin E1 (P190) bound to anionic membranes are described. In the membrane-bound state, the alpha-helical content increases from 60-64% to 80-90%, with a concomitant increase in the average length of the helical segments from 12 to 16 or 17 residues, close to the length required to span the membrane bilayer in the open channel state. The average distance between helical segments is increased and interhelix interactions are weakened, as shown by a major loss of tertiary structure interactions, decreased efficiency of fluorescence resonance energy transfer from an energy donor on helix V of P190 to an acceptor on helix IX, and decreased resonance energy transfer at higher temperatures, not observed in soluble P190, implying freedom of motion of helical segments. Weaker interactions are also shown by a calorimetric thermal transition of low cooperativity, and the extended nature of the helical array is shown by a 3- to 4-fold increase in the average area subtended per molecule to 4,200 A2 on the membrane surface. The latter, with analysis of the heat capacity changes, implies the absence of a developed hydrophobic core in the membrane-bound P190. The membrane interfacial layer thus serves to promote formation of a highly helical extended two-dimensional flexible net. The properties of the membrane-bound state of the colicin channel domain (i.e., hydrophobic anchor, lengthened and loosely coupled alpha-helices, and close association with the membrane interfacial layer) are plausible structural features for the state that is a prerequisite for voltage gating, formation of transmembrane helices, and channel opening.
已经确定了几种大肠杆菌素和毒素的可溶形式的原子水平结构，但膜结合后发生的结构变化尚未得到很好的表征。本文描述了与阴离子膜结合的大肠杆菌素E1（P190）的C端190个残基通道多肽从可溶状态转变为膜结合状态时发生的变化。在膜结合状态下，α-螺旋含量从60%-64%增加到80%-90%，同时螺旋段的平均长度从12个残基增加到16或17个残基，接近开放通道状态下跨越膜双层所需的长度。螺旋段之间的平均距离增加，螺旋间相互作用减弱，这表现为三级结构相互作用的大量丧失、从P190螺旋V上的能量供体到螺旋IX上的受体的荧光共振能量转移效率降低，以及在较高温度下共振能量转移降低，而在可溶的P190中未观察到这种情况，这意味着螺旋段的运动自由度。低协同性的量热热转变也表明相互作用较弱，而螺旋阵列的延伸性质表现为每个分子在膜表面所占据的平均面积增加了3至4倍，达到4200 Ų。后者结合热容量变化分析表明，膜结合的P190中不存在发达的疏水核心。因此，膜界面层有助于促进形成高度螺旋化的延伸二维柔性网络。大肠杆菌素通道结构域的膜结合状态的特性（即疏水锚定、延长且松散耦合的α-螺旋以及与膜界面层的紧密结合）是电压门控、跨膜螺旋形成和通道开放的前提状态的合理结构特征。
