Core D5: Phage display synthetic toxin pipeline

膜蛋白结构动力学（MPSD）联盟旨在通过连接结构，动力学和功能来实现对膜蛋白操作的机械理解。膜蛋白改变它们的构象来运作。我们的目的是研究与功能相关的不同构象状态，并绘制连接操作和静息构象的途径。 天然存在的肽毒素已成为许多膜蛋白研究的一个组成部分。该核心采用了一种新的高通量方法，该方法利用了天然肽毒素支架的结构稳健性和噬菌体展示技术的力量。其目的是产生以位点和/或状态依赖性方式以高亲和力和选择性结合特异性膜受体的新型合成毒素。该方法扩展了使用高亲和力的已验证策略 肽配体研究膜蛋白超出了少数天然毒素已被分离。 从蜘蛛、蝎子、蜗牛和蛇中分离出的肽毒素是有效的分析工具，可以促进对体外和体内通道、泵、转运蛋白和激素受体的理解，揭示这些膜蛋白在生理学中的作用及其作用机制[1]。在野生环境中，毒素作用于捕食者;它们是有效的（pM-nM亲和力），对广谱的膜靶点广泛有效。许多毒素将靶受体锁定在独特的功能状态。自然 毒素很小（~ 10 - 80个氨基酸），并且构建在弹性结构支架上，所述支架耐受广泛的残基多样性以产生具有显著不同性质的产物。 在大多数情况下，使用细菌或通过从头化学方法的肽毒素的实验室合成已被证明是直接的。与天然产物的分离相比，这些策略提高了产率，并且显著地允许掺入有用的修饰，例如残基改变，以提高靶特异性或亲和力，改变对受体功能的影响，或连接货物以递送至特定的细胞和分子位置[2，3]。 天然毒素及其合成变体已用于鉴定不同组织和亚细胞区域中的膜受体亚型[4];区分生理学和疾病中的作用[5];描绘分子机制[6];免疫纯化靶受体[7];通过阻断离子通道治疗疼痛[8];并且，在此具有独特的相关性，使用生物物理[9]、光学[3]和计算方法[10]将受体结构定义为构象状态的函数。 尽管从生物化学和遗传学研究中推断出的天然肽“毒素组”的预测多样性是巨大的（> 1100万），但对于已经分离和研究的数百种毒素中的大多数，没有确定特定的靶标。那些与已知受体结合的毒素通常具有低亲和力或与相关靶标交叉反应。这种状况很容易理解：无论是它们在野外的目的还是对靶受体的非定向搜索，都不利于分离特异性、高亲和力的毒素。在这里，这些问题是避免克隆毒素的基础上，他们的功能属性。

The Membrane Protein Structural Dynamics (MPSD) Consortium seeks to achieve mechanistic understanding of membrane protein operation by linking structure, dynamics and function. Membrane proteins change their conformation to operate. Our purpose is to study different conformational states associated with function and to map the pathway that links operating and resting conformations. Naturally-occurring peptide toxins have become an integral part of research on many membrane proteins. This core employs a new, high-throughput methodology that exploits the structural robustness of natural peptide toxin scaffolds and the power of phage display technology. The purpose is to produce novel synthetic toxins that bind to specific membrane receptors in site and/or state-dependent manner with high affinity and selectivity. This method extends the proven strategy of using high-affinity peptide ligands to study membrane proteins beyond a handful of natural toxins that have been isolated. Peptide toxins isolated from spiders, scorpions, snails and snakes have been potent analytic tools to advance understanding of channels, pumps, transporters, and hormone receptors in vitro and in vivo revealing the roles of these membrane proteins in physiology and their mechanisms of action [1]. In the wild, toxins act to immobilize prey; they are potent (pM-nM affinity) and broadly effective on a wide spectrum of membrane targets. Many toxins lock target receptors in unique functional states. Natural toxins are small (-10-80 amino acids) and are constructed on resilient structural scaffolds that tolerate wide residue diversity to yield products with markedly different properties. Laboratory synthesis of peptide toxins using bacteria or by de novo chemical methods has proven straightfoHA/ard in most cases. These strategies improve yield compared to isolation of natural products and, significantly, allow incorporation of useful modifications such as residue alterations to improve target specificity or affinity, to alter impact on receptor function, or to attach cargo for delivery to specific cellular and molecular locations [2, 3]. Natural toxins and their synthetic variants have been used to identify membrane receptor subtypes in different tissue and subcellular locales [4]; distinguish roles in physiology and disease [5]; delineate molecular mechanisms [6]; immunopurify target receptors [7]; to treat pain via blockade of ion channels [8]; and, of unique relevance here, to define receptor structure as a function of conformational state using biophysical [9], optical [3] and computational methods [10]. Even though the predicted diversity of the natural peptide "toxome" extrapolated from biochemical and genetic studies is vast (> 11 million), specific targets are not identified for most of the hundreds of toxins that have been isolated and studied. Those toxins that bind to known receptors are often of low affinity or cross-react with related targets. This state-of-affairs is easily understood: neither their purpose in the wild nor non-directed searches for target receptors favor isolation of specific, high-affinity toxins. Here, these problems are avoided by cloning toxins based on their functional attributes.