SYNTHETIC STUDIES OF PROTEIN STABILITY AND FOLDING

蛋白质稳定性和折叠的综合研究

• 批准号: 2190292
• 负责人: George Barany
• 金额: $ 16.34万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2190292
• 关键词: peptide chemical synthesis; protein folding; protein structure function; trypsin inhibitors

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.

该研究计划采用最先进的温和化学方法， 肽合成制备牛胰蛋白酶类似物 抑制剂（BPTI），然后通过一系列生物物理学研究 技术，以了解结构，动力学和折叠。BPTI 是一个小的58个残基的蛋白质，具有三个二硫键的阵列。 广泛的初步结果表明，合成和 BPTI与自然序列的特征，以及两个类似物 其用成对的α-氨基-N-丁酸取代成对的半胱氨酸残基 酸（Abu）等排体。已经表明，虽然BPTI与所有六个 半胱氨酸被Abu取代（全Abu-BPTI）表现为真随机 螺旋，BPTI，在半胱氨酸14和38之间具有一个完整的二硫键 （[Cys 14，38]-BPTI）部分折叠，在细胞中具有天然样结构。 疏水核心和分子其余部分的无序结构。 [Cys 14，38]-BPTI代表了一种蛋白质的独特实例， 一个“熔融球”和一个“A状态”;这些物种的结构非常 类似于在第一个10到20毫秒中形成的瞬态中间体， 蛋白质折叠过程这项研究的具体目标有两个方面， 寻求合成和表征部分折叠的蛋白质， 反过来，合成和表征循环或循环 可以具有增强的稳定性的置换变体。拟议的BPTI 类似物可以在单个分子中含有那些编码 折叠的起始和最终的三级折叠，例如通过 用连接N-和C-的肽键环化全-Abu-BPTI， 末端[这些在天然BPTI中是近端的]，通过使用寡甘氨酰（或 相关）间隔区连接对应于慢- 交换核心，或通过取代[Cys 14，38]- BPTI。BPTI的表观“环状性”和二硫化物的作用 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 被Abu残基和/或侧链内酰胺、硫醚取代的桥 （卡巴）或二甲卡巴桥。要问的问题包括： 疏水核中氨基酸的性质影响其形成？如何 二级结构元素的顺序是否影响 蛋白质的稳定性、结构和活性？如何 半胱氨酸残基的顺序影响了半胱氨酸的获得和稳定性。 最后的构造？什么是最小的共价约束所需的 实现类似于本地人的构造和活动？可以额外共价 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，并预计将导致重大见解 导致了“蛋白质折叠问题”。“这项工作的另一个目标是 我们的工作是开发一种通用的方法， 有前途的小蛋白质对变性和蛋白水解，并为 降低其免疫原性。这笔赠款的进展可能会影响 用于治疗肺气肿和关节炎的药物的开发， 以及其他疾病。