气相展开揭示了模块化多基化合成酶中基底结合相关的稳定性转移
Chunyi Zhao1, Nicholas B Borotto1, Jennifer Schmidt2
1Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States.
Journal of the American Society for Mass Spectrometry
|July 25, 2024
概括
我们开发了新的碰撞诱导展开 (CIU) 方法来研究大型蛋白质复合体,如多基酸合成酶 (PKSs). 这种技术揭示了从小分子结合到庞大的酶组合中的微妙稳定性变化.
科学领域:
- 生物化学和结构生物学
- 质谱学和生物物理技术
背景情况:
- 原生质谱 (MS),离子流动性 (IM) 和碰撞诱导展开 (CIU) 是研究蛋白质-小分子相互作用的既定方法.
- 在将这些技术应用于大型多域蛋白质复合体,如多基酸合成酶 (PKSs) 的应用方面仍然存在挑战,这些蛋白质复合酶对天然产品生物合成和制药至关重要.
- 现有的方法难以检测大量蛋白质组合中的微妙结合事件,因为它们的复杂性和大小.
研究的目的:
- 开发先进的CIU方法来分析大型的多蛋白质复合体.
- 评估CIU在大规模蛋白质组合中检测轻微结合事件的定量能力.
- 为了研究大约280kDa的多基酸合成酶 (PKS) 模态复合物的稳定性.
主要方法:
- 开发和应用新的碰撞诱导展开 (CIU) 方法.
- 测试大型多域蛋白质复合物的气相稳定性,特别是PKS二极体.
- 在完整的蛋白质组合中,与基质结合相关的稳定性转移的定量分析.
主要成果:
- 成功调整CIU方法,从大型多蛋白质复合体中提取有意义的数据.
- 证明了定量CIU在基质结合时检测微妙稳定性变化的能力.
- 观察到与基质结合相对应的稳定性转移,占PKS二极体复合体总质量的0.1%以下.
结论:
- 开发的CIU方法显著推进了对大型蛋白质复合物的研究.
- 定量CIU能够检测大量蛋白质组合中的低丰度结合事件.
- 这项工作扩大了基于MS的技术的应用到PKS等复杂的生物系统.
更多相关视频
07:22How to Stabilize Protein: Stability Screens for Thermal Shift Assays and Nano Differential Scanning Fluorimetry in the Virus-X Project
Published on: February 11, 2019
28.2K
11:27X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050
Published on: May 13, 2020
4.0K
相关概念视频
03:00Molecular Chaperones and Protein Folding
17.9K
The native conformation of a protein is formed by interactions between the side chains of its constituent amino acids. When the amino acids cannot form these interactions, the protein cannot fold by itself and needs chaperones. Notably, chaperones do not relay any additional information required for the folding of polypeptides; the native conformation of a protein is determined solely by its amino acid sequence. Chaperones catalyze protein folding without being a part of the folded protein.
The...
The...
17.9K
01:22Protein Folding
117.8K
Overview
117.8K
00:49Ligand Binding and Linkage
4.8K
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...
4.8K
01:58Cooperative Allosteric Transitions
7.9K
Cooperative allosteric transitions can occur in multimeric proteins, where each subunit of the protein has its own ligand-binding site. When a ligand binds to any of these subunits, it triggers a conformational change that affects the binding sites in the other subunits; this can change the affinity of the other sites for their respective ligands. The ability of the protein to change the shape of its binding site is attributed to the presence of a mix of flexible and stable segments in the...
7.9K
02:30Stability of Substituted Cyclohexanes
12.6K
This lesson discusses the stability of substituted cyclohexanes with a focus on energies of various conformers and the effect of 1,3-diaxial interactions.
The two chair conformations of cyclohexanes undergo rapid interconversion at room temperature. Both forms have identical energies and stabilities, each comprising equal amounts of the equilibrium mixture. Replacing a hydrogen atom with a functional group makes the two conformations energetically non-equivalent.
For example, in...
The two chair conformations of cyclohexanes undergo rapid interconversion at room temperature. Both forms have identical energies and stabilities, each comprising equal amounts of the equilibrium mixture. Replacing a hydrogen atom with a functional group makes the two conformations energetically non-equivalent.
For example, in...
12.6K
01:19Allosteric Proteins-ATCase
5.7K
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...
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...
5.7K