INTERMEDIATES AND TRANSBILAYER PEPTIDES

描述：膜融合是一种关键且普遍存在的现象 生物学。然而，生物膜融合的机制仍然是 膜生物学的中心谜团。“茎”理论被广泛引用。 作为生物膜融合的基础，目前还没有直接的证据 即使是纯脂膜融合也是通过这种机制发生的。的确有 有证据表明，脂质双层融合和双层/倒相 过渡通过密切相关的机制进行，涉及相同的 中间结构。卡弗里博士将利用这封信来测试 作为生物膜融合基础的秸秆理论的两个方面，以及 验证关于融合诱导跨膜结构域作用的假说 蛋白质。首先，他将使用一种独特的实验系统来尝试直接 用时间分辨x射线测定聚变中间体结构 绕射。最近的时间分辨冷冻电子显微镜结果表明 在特定的条件下，熔合中间体的瞬时有序阵列存在 经历双层/倒置六角相的系统的情况 过渡。在这项研究中，卡弗里博士将建立 增加这些阵列的大小和顺序，并将尝试确定 直接用时间分辨x射线衍射法测定中间体结构。 这将是聚变机制的首次直接证明。 其次，茎理论预测了膜融合和膜融合的速度 体脂/水体系的双层/倒立方相变 由相同中间结构的衰减率控制。如果 柄理论是正确的，降低双层断裂张力应 同时提高这两个比率。跨膜脑多肽已被证明可以 在低浓度的脂类中显著降低这种破裂张力 膜。因此，卡弗里博士和同事们将添加 仔细鉴定跨双层多肽到脂质系统，测量 它们对膜张力的影响，并测量两者的速率 单层脂质体的相变与膜融合速率 含有这些多肽。后一项实验还将测试 假设融合催化蛋白的跨膜结构域在 病毒对核聚变活性非常重要，因为它们会破坏核聚变的稳定性 同样的聚变迭代。流感病毒血凝素(HA)中， 跨膜结构域对融合活性至关重要：取代 该结构域的脂质锚定破坏HA融合活性，但脂质 混合活动不起作用。这种活动与卡弗里博士的 假设。

DESCRIPTION: Membrane fusion is a critical and ubiquitous phenomenon in biology. However, the mechanism of biomembrane fusion remains one of the central mysteries of membrane biology. The "stalk" theory is widely cited as the basis for biological membrane fusion, yet there is no direct evidence that even pure lipid membrane fusion occurs via this mechanism. There is evidence that both lipid bilayer fusion and bilayer/inverted phase transitions proceed via closely-related mechanisms, involving the same intermediate structures. Dr. Caffrey will use this correspondence to test two aspects of the stalk theory as a basis for biomembrane fusion, and also test a hypothesis about the role of transmembrane domains of fusion-inducing proteins. First, he will use a unique experimental system to attempt direct determination of fusion intermediate structure, using time-resolved x-ray diffraction. Recent time-resolved cryoelectron microscopy results show that transient, ordered arrays of fusion intermediates exist under special circumstances in systems undergoing the bilayer/inverted hexagonal phase transition. In this study Dr. Caffrey will establish conditions that increase the size of and order in these arrays, and will attempt to determin directly the intermediate structure using time-resolved x-ray diffraction. This would be the first direct demonistration of a fusion mechanism. Second, the stalk theory predicts that the membrane fusion and the rate of the bilayer/inverted cubic phase transition in bulk lipid/water systems are controlled by the rate of decay of the same intermediate structure. If the stalk theory is correct, reducing the bilayer rupture tension should increase both rates in parallel. Transmembrabe peptides have been shown to substantially reduce this rupture tension at low concentrations in lipid membranes. Therefore, Dr. Caffrey and collegues will add traces of carefully characterized transbilayer peptides to lipid systems, measure their effect on the membrane tensions, and measure the rates of both the phase transition and the rate of membrane fusion in unilmaellar liposomes containing these peptides. The latter experiment will also test the hypothesis that the transmembrane domains of fusion-catalyzing proteins in viruses are imoportant to fusion activity because they serve to destabilize the same sort of fusion itermediate. In influenza virus hemagglutinin (HA), the transmembrane domain is crtical for fusion activity: substituting a lipid anchor for this domaim destroys HA fusion activity, but the lipid mixing activity inact. Such activity is consistent with Dr. Caffrey's hypothesis.