INFLUENCE OF ALPHA-HELICES ON THE FOLDING OF APAMIN

α-螺旋对 APAMIN 折叠的影响

• 批准号: 3466881
• 负责人: JEFFREY W NELSON
• 金额: $ 9.11万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-02-01 至 1993-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3466881
• 关键词: chemical bond; chemical structure; circular dichroism; disulfide bond; insect poison; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; protein engineering; protein folding; protein sequence

The aim of this research is to understand how the stability of an alpha-helix influences the pathway and kinetics of protein folding. Apamin, a small bee venom protein which forms an alpha-helix on the C-terminal end and contains two disulfide bonds, will be used as the model system. The mechanism for folding from the unfolded state of apamin, with reduced thiols, to the native folded state, with two disulfide bonds, will be studied by trapping the disulfide intermediates with iodoacetic acid throughout the folding process. Separation of the intermediates by HPLC will determine the rates acid throughout the folding processes. Separation of the intermediates by HPLC will determine the rates of build-up and decay of each intermediate, while enzymatic cleavage, followed by separation of the fragments, will tell which cysteines are linked by disulfide bonds. In order to study the stability of the alpha-helix in apamin, the C- terminal helical part of apamin will be synthesized by solid phase peptide methods, replacing the cysteines by alanines to avoid disulfide bond formation. The stability will be studied by using the helix-stabilizing properties of trifluoroethanol. Alpha-Helix formation will be studied by circular dichroism, to measure overall helicity, and by two-dimensional NMR, to monitor the helical transition of each amino acid. After understanding the folding of natural apamin and the stability of its helix, apamin derivatives with substitutions on the C-terminal helix will be synthesized. Comparisons of the folding kinetics and helical stability of the derivatives with natural apamin will provide information about the relationship between alpha-helix stability and folding kinetics. This research will provide new insight into how the stability of an alpha-helix influences the folding of small proteins.

这项研究的目的是了解一个人的稳定性 α-螺旋影响蛋白质折叠的途径和动力学。 阿帕明，一种形成α-螺旋的小蜜蜂毒液蛋白 将使用C-末端并包含两个二硫键 作为模型系统。从表面折叠的机制 阿帕明的未折叠状态，带有还原的硫醇，恢复到天然折叠状态 具有两个二硫键的状态将通过捕获 二硫化物中间体与碘乙酸在整个反应过程中 折叠过程。用高效液相色谱分离中间体 确定整个折叠过程中的酸度。 中间产物的分离将决定产品的产率。 每种中间体的堆积和腐烂，而酶 切割，然后分离片段，将会告诉你哪一个 半胱氨酸通过二硫键连接在一起。 为了研究阿帕明中α-螺旋的稳定性，研究了C-螺旋的稳定性。 氨基酸末端螺旋部分的固相合成 多肽法，用丙氨酸取代半胱氨酸以避免 二硫键的形成。稳定性将通过使用 三氟乙醇的螺旋稳定性能。阿尔法-螺旋 将用圆二向色性来研究地层，以测量 整体螺旋度，并通过二维核磁共振来监测 每种氨基酸的螺旋过渡。 在了解了天然阿帕明的折叠和 其螺旋结构、氨基酸及其取代物的稳定性 将合成C-末端螺旋。折页的比较 天然衍生物的动力学和螺旋稳定性 阿帕明将提供有关两者之间关系的信息 α-螺旋稳定性和折叠动力学。这项研究将 为阿尔法螺旋的稳定性提供了新的见解 影响小蛋白质的折叠。