在上分子系统中Ile,Leu和Val甲基相关性的分配:对阿斯巴酸转碳胺酶的应用
Algirdas Velyvis1, Howard K Schachman, Lewis E Kay
1Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8.
Journal of the American Chemical Society
|October 29, 2009
概括
研究人员开发了分配甲基组的大型蛋白质复合物的方法,如阿斯巴酸转糖酶 (ATCase). 结合技术分配了86%的甲基,揭示了ATP结合时的结构变化.
科学领域:
- 结构生物学 结构生物学
- 生物化学 生物化学
- 生物物理学的生物物理.
背景情况:
- 使用Methyl-TROSY (横向放松优化光谱) 在大型蛋白质复合体中分配甲基是具有挑战性的.
- 了解超分子蛋白质复合体的结构和动态对于破译生物功能至关重要.
研究的目的:
- 介绍和比较大型蛋白质复合物的甲基组分配在甲基-TROSY光谱中的互补方法.
- 将这些方法应用于阿斯巴酸转糖酶 (ATCase) 酶,并研究其对核酸结合的结构反应.
主要方法:
- 从较小的碎片转移任务,使用"分裂与征服"的方法.
- 通过交换光谱学进行分配转移.
- 使用假接触转移与偏磁探测器生成任务.
主要成果:
- "分裂与征服"方法在ATCase.中分配了大约60%的甲基.
- 将所有方法结合起来,为86%的甲基组实现了赋值,提供了广泛的结构和动态探测.
- 对ATP定位后化学转移变化的分析显示,调节链的N端尾部存在显著的扰动.
结论:
- 结合相辅相成的方法可显著增强大型蛋白质复合体中的甲基组分配.
- 这项研究提供了ATCase在核酸结合时N端调节链中的结构性扰动的第一个证据.
- 这些发现为ATCase的全调节机制提供了洞察力.
相关概念视频
Allosteric Proteins-ATCase
Binding sites linkages can regulate a protein's function. For example, enzyme activity is often regulated through a feedback mechanism where the end product of the biochemical process serves as an inhibitor.
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis pathway,...
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis pathway,...
Ligand Binding and Linkage
Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked. In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence the...
¹³C NMR: Distortionless Enhancement by Polarization Transfer (DEPT)
When proton-coupled carbon-13 spectra are simplified by a broadband proton decoupling technique, structural information about the coupled protons is lost. Distortionless enhancement by polarization transfer (DEPT) is a technique that provides information on the number of hydrogens attached to each carbon in a molecule. While the DEPT experiment utilizes complex pulse sequences, the pulse delay and flip angle are specifically manipulated. The resulting signals have different phases depending on...
Mass Spectrometry of Amines
In mass spectroscopy, amines undergo fragmentation to give parent ions with odd molecule weights. This observed mass spectrum follows the nitrogen rule; a molecule with an odd number of nitrogen atoms produces a molecular ion with an odd molecular weight. Amines undergo fragmentation through α cleavage, producing nitrogen-containing cations—iminium ions—and alkyl radicals. Mass spectra of aromatic and cyclic aliphatic amines exhibit strong molecular ion peaks, but acyclic aliphatic amines show...
¹H NMR: Complex Splitting
A proton M that is coupled to a proton X results in doublet signals for M. However, NMR-active nuclei can be simultaneously coupled to more than one nonequivalent nucleus. When M is coupled to a second proton A, such as in styrene oxide, each peak in the doublet is split into another doublet.
Splitting diagrams or splitting tree diagrams are routinely used to depict such complex couplings. While drawing splitting diagrams, the splitting with the larger coupling constant is usually applied first.
Splitting diagrams or splitting tree diagrams are routinely used to depict such complex couplings. While drawing splitting diagrams, the splitting with the larger coupling constant is usually applied first.
tRNA Activation
Aminoacyl-tRNA synthetases are present in both eukaryotes and bacteria. Though eukaryotes have 20 different aminoacyl-tRNA synthetases to couple to 20 amino acids, many bacteria do not have genes for all of these aminoacyl-tRNA synthetases. Despite this, they still use all 20 amino acids to synthesize their proteins. For instance, some bacteria do not have the gene encoding the enzyme that couples glutamine with its partner tRNA. In these organisms, one enzyme adds glutamic acid to all of the...


