INTERMEDIATES AND TRANSBILAYER PEPTIDES

描述：膜融合是一种重要的和普遍存在的现象， 生物学 然而，生物膜融合的机制仍然是生物膜融合的主要机制之一。 膜生物学的核心奥秘 “梗”论被广泛引用 作为生物膜融合的基础，但没有直接证据表明， 即使是纯脂质膜融合也是通过这种机制发生的。 有 有证据表明，脂质双层融合和双层/反相 过渡通过密切相关的机制进行，涉及相同的 中间结构。 卡夫瑞博士会用这些信件来测试 茎理论的两个方面作为生物膜融合的基础， 测试关于融合诱导的跨膜结构域的作用的假设 proteins. 首先，他将使用独特的实验系统， 用时间分辨x射线测定熔合中间结构 衍射法 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， 在经历双层/倒置六方相的系统中的情况 过渡 在这项研究中，卡夫瑞博士将建立条件， 增加这些数组的大小和顺序，并将尝试确定 使用时间分辨X射线衍射直接得到中间结构。 这将是聚变机制的第一次直接演示。 第二，茎理论预测，膜融合和细胞分裂的速率， 大体积脂质/水体系中的双层/倒置立方相变是 由相同中间结构的衰变速率控制。 如果 茎理论是正确的，降低双层断裂张力应 两种速度并行。 已显示跨膜肽 在低浓度脂质下基本上降低了这种断裂张力 膜。 因此，卡夫瑞博士和他的同事们会在 仔细表征的跨双层肽脂质系统，测量 它们对膜张力的影响，并测量两者的速率。 单层脂质体的相转变和膜融合速率 含有这些肽。 后一项实验还将测试 这一假说认为，融合催化蛋白的跨膜结构域在 病毒对融合活动很重要，因为它们可以破坏 同样的融合中间体。 在流感病毒血凝素（HA）中， 跨膜结构域对于融合活性是至关重要的： 该结构域的脂质锚破坏HA融合活性，但脂质 混合活性不起作用。 这种活动与卡夫瑞博士 假说.

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.