Core D5: Phage display synthetic toxin pipeline

膜蛋白质结构动力学(MPSD)联合会试图通过将结构、动力学和功能联系起来来实现对膜蛋白质运行的机械理解。膜蛋白改变其构象以进行操作。我们的目的是研究与功能相关的不同构象状态，并绘制连接工作构象和静止构象的途径。 自然产生的多肽毒素已成为许多膜蛋白研究的重要组成部分。该核心采用了一种新的高通量方法，利用了天然多肽毒素支架的结构健壮性和噬菌体展示技术的力量。其目的是产生新的合成毒素，以高亲和力和选择性与特定的膜受体结合。此方法扩展了已证实的使用高亲和力的策略 除了已分离的几种天然毒素外，还可用于研究膜蛋白的多肽配体。 从蜘蛛、蝎子、蜗牛和蛇中分离出的多肽毒素已经成为一种强有力的分析工具，可以在体外和体内促进对通道、泵、转运体和激素受体的了解，揭示这些膜蛋白在生理上的作用及其作用机制[1]。在野外，毒素起到固定猎物的作用；它们是有效的(PM-NM亲和力)，并对广泛的膜目标广泛有效。许多毒素将靶标受体锁定在独特的功能状态。天然 毒素很小(-10-80个氨基酸)，构建在弹性结构支架上，可以容忍广泛的残基多样性，从而产生具有明显不同性质的产品。 在大多数情况下，使用细菌或通过从头化学方法在实验室合成多肽毒素已被证明是直接的。与分离天然产物相比，这些策略提高了产量，并且显著地允许加入有用的修饰，例如残基改变，以提高靶标特异性或亲和力，改变对受体功能的影响，或将货物附着到特定的细胞和分子位置[2，3]。 天然毒素及其合成变异体已被用来鉴定不同组织和亚细胞部位的膜受体亚型[4]；区分生理和疾病中的角色[5]；描绘分子机制[6]；免疫纯化靶受体[7]；通过阻断离子通道来治疗疼痛[8]；以及，在这里具有独特相关性的是，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。 尽管从生化和遗传学研究推断出的天然多肽“毒素”的预测多样性是巨大的(1100万英镑)，但在已分离和研究的数百种毒素中，大多数都没有确定特定的靶标。那些与已知受体结合的毒素通常是低亲和力的，或者与相关靶点发生交叉反应。这种情况很容易理解：无论是它们在野外的目的，还是对目标受体的非定向搜索，都不利于分离特定的高亲和力毒素。在这里，根据毒素的功能属性克隆毒素可以避免这些问题。

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.