SYNTHETIC STUDIES OF PROTEIN STABILITY AND FOLDING

这项研究计划应用最先进的温和化学方法来 多肽合成制备牛胰酶类似物 抑制剂(BPTI)，然后通过一系列生物物理研究 技巧，以便学习结构、动力学和折叠。BPTI 是一个小的58个残基的蛋白质，有三个二硫键的阵列。 广泛的初步结果证明了合成和 BPTI与自然序列和两个类似物的特征 用成对的α-氨基-正丁酸取代成对的半胱氨酸残基 酸(ABU)等位线。已经表明，尽管BPTI与所有六个 由ABU(ALL-ABU-BPTI)取代的半胱氨酸表现为真正的随机 半胱氨酸14和38之间含有一个完整二硫键的BPTI线圈 ([Cys14，38]-BPTI)部分折叠，具有天然结构 疏水核心和分子其余部分的无序结构。 [Cys14，38]-BPTI代表了一种蛋白质的独特例子，这种蛋白质既形成了 一个“熔化的球体”和一个“A状态”；这些物种具有非常 类似于在前10到20毫秒内形成的瞬变中间体 蛋白质折叠过程。这项研究的具体目的有两个， 寻求合成和表征部分折叠的蛋白质， 反之，合成并表征环状或圆形 可能具有增强稳定性的变异体。拟议的BPTI 类似物可以在单个分子中包含编码 折叠的开始和最终的第三折叠，例如通过 用连接N-和C-的肽键环化All-Abu-BPTI Termini[这些在天然BPTI中是近端]，通过使用寡甘醇(或 相关的)间隔物，以连接对应于慢- 交换核心，或通过取代[Cys14，38]核心中的关键残基- BPTI。BPTI的表观“环状”和二硫键的作用 控制蛋白质稳定性和假定折叠的桥梁 途径，将通过母体分子和类似物的合成进行测试 具有一个或多个二硫化物的环状置换和环状BPTI‘S 用ABU残基和/或侧链内酰胺、硫醚取代的桥 (Carba)，或Dicarba桥。要问的问题包括：如何 疏水核心中氨基酸的性质会影响其形成吗？多么 次结构构件的顺序是否会影响 蛋白质的稳定性、结构和活性？这是怎么回事 半胱氨酸残基的顺序影响半胱氨酸的获取和稳定性 最终构象是什么？满足以下条件所需的最小共价约束是什么 实现类似原住民的构象和活动？额外的共价能吗 限制导致更稳定的蛋白质？这些研究代表了一种新的 BPTI折叠的方法，并有望带来重要的见解 这导致了“蛋白质折叠问题”。这样做的另一个目标是 工作是开发一种治疗上稳定的通用方法 有希望的小蛋白可以防止变性和蛋白分解，并用于 降低它们的免疫原性。这笔赠款的进展可能会影响到 治疗肺气肿和关节炎的药物的开发， 在其他疾病中。

This research program applies state-of-the-art mild chemical methods for peptide synthesis to prepare analogues of bovine pancreatic trypsin inhibitor (BPTI), which are then studied by a series of biophysical techniques in order to learn about structure, dynamics, and folding. BPTI is a small 58-residue protein with an array of three disulfide bridges. Extensive preliminary results have demonstrated the syntheses and characterizations of BPTI with the natural sequence, and of two analogues which replace paired half-cystine residues by paired alpha-amino-n-butyric acid (Abu) isosteres. It has been shown that whereas BPTI with all six half-cystines replaced by Abu (all-Abu-BPTI) behaves as a true random coil, BPTI with one intact disulfide between half-cystines 14 and 38 ([Cys14,38]-BPTI) is partially folded, with native-like structure in the hydrophobic core and disordered structure in the rest of the molecule. [Cys14,38]-BPTI represents a unique example of a protein which forms both a "molten globule" and an "A state;" these species have structures very similar to the transient intermediate formed in the first 10 to 20 msec of the protein folding process. Specific aims of this research are two-fold, seeking to synthesize and characterize proteins that are partially folded, and conversely, to synthesize and characterize circularized or circular permuted variants that may have enhanced stability. Proposed BPTI analogues may contain in a single molecule those sequences that encode initiation of folding and the final tertiary fold, for example by circularizing all-Abu-BPTI with a peptide bond connecting the N- and C- termini [these are proximal in native BPTI], by using oligoglycyl (or related) spacers to connect BPTI sequences corresponding to the slow- exchange core, or by substituting key residues in the core of [Cys14,38]- BPTI. The apparent "circularity" of BPTI, and the role of disulfide bridges in governing the protein's stability and postulated folding pathways, will be tested by syntheses of parent molecules and analogues of circularly permuted and circular BPTI' s with one or more disulfide bridges replaced by Abu residues, and/or side-chain lactam, thioether (carba), or dicarba bridges. Questions to be asked include: How does the nature of amino acids in the hydrophobic core affect its formation? How does the order of the secondary structural elements affect the stabilities, structures, and activities of the proteins? How does the order of the cysteine residues affect the acquisitions and stabilities of final conformations? What are the minimal covalent constraints required to achieve native-like conformations and activities? Can additional covalent constraints lead to more stable proteins? These studies represent a new approach to BPTI folding, and are expected to lead to significant insights that contribute to the "protein folding problem." A further goal of this work is to develop a general methodology for stabilizing therapeutically promising small proteins against denaturation and proteolysis, and for reducing their immunogenicity. Progress from this grant could impact on the development of drugs for the treatment of emphysema and arthritis, among other diseases.