DARL1 & YARF1 & HMNT

DARL1

• 批准号: 6281299
• 负责人: DAGMAR RINGE
• 金额: $ 1.21万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-15 至 1999-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6281299
• 关键词: biological products; biomedical resource; proteins; radiography; structural biology

This project is focussed on the elucidation of structures of proteins in the secretory pathway. The key protein in vesicle formation is the ADP ribosylation factor that sequesters the proteins required for vesicle formation to the membrane. The protein behaves like an on/off switch where the GDP form is soluble in the cytosol and the GTP form is membrane associated. One of the elements that is essential for membrane association is a myristyl group at the N-terminus of the protein, without which membrane association does not occur. So far, the structure of the human ARF1 has been determined, and structures of yeast ARF1 and ARF2 are underway. In addition to the ARF's, cells also contain ARF-related proteins, called ARL's, that seem to have a function similar to that of ARF, but not identical. We have crystallized ARL1 from drosophila in hopes of obtaining a structure that might indicate what the differences between ARL and ARF are, and how they relate to cellular function. These crystals are too small for in-house data collection. During the trip to CHESS (8.-11. April 1998) a number of ARL crystals were screened, however, none of them diffracted to high enough resolution (better than 8A) for useful data collection. The enzyme responsible for myristylation at the N-terminus of the ARF's and ARL's is N-myristyl transferase. The structure of this enzyme from yeast has been determined, but is sufficiently different from the human enzyme by sequence comparison that we are determining the structure of the human enzyme. The protein crystallizes slowly and produces very small crystals that do not diffract well enough for in-house data collection. A 85% complete data set (to 3A resolution) of the hNMT was obtained at CHESS (8.-11. April 1998) under cryo conditions. We are in the process of using the yeast NMT coordinates to these data in molecular replacement attempts. So far, we have obtained two promising solutions that have to be analyzed further before we have a definitive answer.

该项目的重点是阐明 分泌途径中的蛋白质。 囊泡中的关键蛋白 形成是隔离蛋白质的 ADP 核糖基化因子 膜上形成囊泡所需的。 该蛋白质的行为 就像一个开关，GDP 形式可溶于细胞质中，并且 GTP 形式是膜相关的。 其中的元素之一是 膜结合所必需的是肉豆蔻基 蛋白质的 N 末端，没有它，膜缔合就不会发生 发生。 至此，人类ARF1的结构已经确定， 酵母ARF1和ARF2的结构正在进行中。 此外 除了 ARF 之外，细胞还含有 ARF 相关蛋白，称为 ARL， 看起来和ARF有类似的功能，但又不完全相同。 我们 从果蝇中结晶了 ARL1，希望获得 可能表明 ARL 和 ARF 之间差异的结构 以及它们与细胞功能的关系。 这些水晶也 适合内部数据收集。 在国际象棋之旅中 (8.-11. 1998年4月）筛选了许多ARL晶体，但是没有一个 它们衍射到足够高的分辨率（优于 8A）以用于有用的 数据收集。 负责肉豆蔻酰化的酶 ARF 和 ARL 的 N 末端是 N-肉豆蔻基转移酶。 这 酵母中这种酶的结构已被确定，但 通过序列比较，与人类酶有很大不同 我们正在确定人类酶的结构。 这 蛋白质缓慢结晶并产生非常小的晶体 对于内部数据收集而言，衍射效果不够好。 完成85% hNMT 的数据集（3A 分辨率）是在 CHESS (8.-11. 1998 年 4 月）在低温条件下。 我们正在使用 酵母 NMT 在分子替换尝试中与这些数据相协调。 到目前为止，我们已经获得了两个有希望的解决方案 在我们得到明确答案之前进行进一步分析。