Takei J, Reményi A, Clarke A R, Dempsey C E
Department of Biochemistry and Molecular Recognition Centre, School of Medical Sciences, University of Bristol, U.K.
Biochemistry. 1998 Apr 21;37(16):5699-708. doi: 10.1021/bi9729007.
Two cysteine substitutions of bee venom melittin have been synthesized to investigate the effects of disulfide cross-linking on the self-association properties of the peptide in solution. K23C melittin (mltK23C) was designed to link nonpolar surfaces of the amphipathic melittin helix on the basis of the close juxtaposition of pairs of K23 side chains in the crystal of the native melittin tetramer. K23Q/Q25C melittin (mltQ25C) was designed to link the polar surfaces of the peptide such that self-association in membrane bound states might be stabilized. The mltK23C disulfide dimer, (mltK23C)2, is highly structured at low pH under conditions where native melittin, and the mltK23C monomer, are unstructured. High-resolution NMR, circular dichroism, and fluorescence spectroscopy established that (mltK23C)2 is a helical monomer (pseudodimer) with stable helical segments between residues 2-13 and 15-25. Although the symmetrical nature of the pseudodimer prevented high-resolution structure determination, analysis of calculated hydrogen bond lengths, chemical shifts, near-UV circular dichroism, and urea denaturation demonstrated similarities with alpha-helical coiled coils and with the structure of native melittin in methanol. Stopped flow fluorescence showed that (mltK23C)2 underwent pH- and divalent anion-linked dimerization to a melittin-like pseudotetramer, indicating that a pair of disulfide bonds could be accommodated in a structure similar to the native melittin crystal structure. Despite incorporation of two disulfide bonds into the melittin tetramer, the folding free energy (DeltaGw) of [(mltK23C)2]2 was similar to that for the native melittin tetramer under the condition used. Incorporation of a disulfide bond on the polar helix face in melittin did not stabilize helical structure in the absence of self-association. Instead, this molecule underwent pH- and divalent anion-linked self-association to an ill-defined aggregate which precipitated.
已合成蜂毒溶血素的两个半胱氨酸取代物，以研究二硫键交联对该肽在溶液中自缔合特性的影响。基于天然溶血素四聚体晶体中K23侧链对的紧密并列，设计了K23C溶血素（mltK23C）以连接两亲性溶血素螺旋的非极性表面。设计K23Q/Q25C溶血素（mltQ25C）以连接肽的极性表面，从而可能稳定膜结合状态下的自缔合。mltK23C二硫键二聚体(mltK23C)2在低pH下具有高度结构化，而此时天然溶血素和mltK23C单体是无结构的。高分辨率核磁共振、圆二色性和荧光光谱表明(mltK23C)2是一种螺旋单体（假二聚体），在残基2 - 13和15 - 25之间具有稳定的螺旋段。尽管假二聚体的对称性质妨碍了高分辨率结构的确定，但对计算出的氢键长度、化学位移、近紫外圆二色性和尿素变性的分析表明，它与α - 螺旋卷曲螺旋以及甲醇中天然溶血素的结构相似。停流荧光显示(mltK23C)2经历了pH和二价阴离子连接的二聚化，形成类似溶血素的假四聚体，表明一对二硫键可以容纳在类似于天然溶血素晶体结构的结构中。尽管在溶血素四聚体中引入了两个二硫键，但在所用条件下，[(mltK23C)2]2的折叠自由能(ΔGw)与天然溶血素四聚体的相似。在没有自缔合的情况下，在溶血素的极性螺旋面上引入二硫键并不能稳定螺旋结构。相反，该分子经历了pH和二价阴离子连接的自缔合，形成不明确的聚集体并沉淀。
