NON-CHANNEL FORMS OF GRAMICIDIN IN LIPID MEMBRANE BY DQC ESR

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 在先前使用自旋标记短杆菌肽A（GAsl）进行的通道形成的工作中，我们发现非通道形式在错配脂质（DPPC，DSPC）的凝胶相中占优势。我们现在已经解决了非通道形式的分配，并研究了短杆菌肽/脂质系统中的复合物平衡，该系统包括通道（即头对头二聚体[HHD]），非通道二聚体和单体。为了研究非通道形式的自旋标记的GA，其可以聚集，我们标记了同一短杆菌肽分子上的两个不同位点，而不是测量附接在两个二聚体形成分子上的相同位置处的自旋标记之间的距离。自旋标记之间的分子内距离可以用作特定构象的指纹。不同短杆菌肽分子上的自旋标记之间的相互作用通过用未标记的短杆菌肽基本上稀释双自旋标记的短杆菌肽来消除。合成并应用了几种新的自旋标记短杆菌肽化合物。在辛醇中，短杆菌肽主要以双螺旋（DH）左手反平行二聚体的形式存在，我们确定了这种结构的自旋间距。具有N-和C-末端标记的GAD和在N-末端单标记的GALN基本上削弱了它们形成HHD的倾向。在大多数脂质中，我们观察到两种不同构象的GAD。主馏分中的自旋间距离（20.0 <$）与单体状态一致。双螺旋（31.6 <$，与辛醇完全相同）是一种次要组分，其在饱和脂质系列中的比例按以下顺序变化：DLPC<DMPC<DSPC<DPPC。我们还表明，短杆菌肽单体和DH存在于所有研究的脂质相，只有在聚集体。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In previous work on channel formation using spin labeled Gramicidin A (GAsl) we found that the non-channel form prevails in the gel phase of mismatching lipids (DPPC, DSPC). We have now addressed the assignment of non-channel forms and studied the complex equilibrium in the gramicidin/lipid system, which includes channels (i.e. head-to-head dimers [HHD]), non-channel dimers, and monomers. To study non-channel forms of spin labeled GA, which can aggregate, we labeled two different sites on the same gramicidin molecule, instead of measuring the distance between spin labels attached at the same position on both dimer-forming molecules. This intramolecular distance between the spin labels could be used as a fingerprint of the particular conformation. Interactions between spin labels on different gramicidin molecules were eliminated by substantially diluting the double spin-labeled gramicidin by unlabeled gramicidin. Several new spin labeled gramicidin compounds were synthesized and used. In octanol, gramicidin exists mainly as a double helical (DH) left-handed antiparallel dimer, and we determined interspin distances for this structure. GAD with both N- and C-termini labeled and GALN, single-labeled at the N-terminus, substantially impair their propensity to HHD formation. In most lipids we observed two different conformations of GAD. The interspin distance in the main fraction (20.0¿) is consistent with the monomeric state. Double helices (31.6¿, exactly as in octanol) are a minor component with the fraction changing in the saturated lipid series in the following order: DLPC<DMPC<DSPC<DPPC. We also showed by DQC that both gramicidin monomers and DH exist in all studied lipid phases only in aggregates.