分子变异,结晶条件和空间组对联结蛋白复合体的影响:对细菌三酶的案例研究
Orly Dym1, Nidhi Aggarwal2, Yacov Ashani2
1Department of Life Sciences Core Facilities, Weizmann Institute of Science, Rehovot, Israel.
Acta crystallographica. Section D, Structural biology
|October 20, 2023
概括
研究三酶-有机酸盐复合体发现,蛋白标签,结晶条件和空间组显著影响联结体结合可视化. 仔细的实验设计对于精确的酶-连接体相互作用结构分析至关重要.
科学领域:
- 生物化学 生物化学
- 结构生物学 结构生物学
- 酶动力学 酶动力学
背景情况:
- 细菌三酶 (PTE) 是一种生物清除酶,能水解有机酸盐,包括神经毒剂和杀虫剂.
- 了解酶-连接体相互作用对于开发有效的抑制剂和排毒策略至关重要.
研究的目的:
- 研究分子结构,表达/净化连接体,结晶条件和空间组如何影响有机连接体-酶复合物的可视化.
- 通过PTE分析有机酸盐结合和水解的结构基础.
主要方法:
- 分析了12个来自定向进化的PTE结构 (apo和holo形式) 的晶体结构,分辨率高达1.38 Å.
- 在四个空间组中使用三个不同的条件进行结晶,有或没有N终端标签.
- 对PTE-有机酸盐产品复合物的表征,以确定金属离子协调和基质结合点.
主要成果:
- N-终端标签可以阻碍酶的活性部位,阻碍带结合.
- 晶体空间组显著影响结合联体的可视化.
- 结晶沉物可以干扰连接体结合,可能导致假阳性.
- 确定了有机酸盐替代物的特定结合口袋,并阐明了α-和β-Zn2+离子在稳定过渡状态中的作用.
结论:
- 诸如蛋白质标签和结晶条件等实验因素极大地影响了酶-连接体复合体的结构确定.
- 对有机酸盐与三酶结合的准确可视化需要仔细的实验设计和数据分析.
- 在活性位点中的Zn2+离子都可能在基质水解的催化机制中发挥作用.
相关概念视频
02:40Ligand Binding Sites
12.9K
Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...
12.9K
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
02:54Protein Kinases and Phosphatases
13.2K
Proteins undergo chemical modifications that trigger changes in the charge, structure, and conformation of the proteins. Phosphorylation, acetylation, glycosylation, nitrosylation, ubiquitination, lipidation, methylation, and proteolysis are various protein modifications that regulate protein activity. Such modifications are usually enzyme-driven.
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
13.2K
02:58Crystal Field Theory - Octahedral Complexes
26.6K
Crystal Field Theory
To explain the observed behavior of transition metal complexes (such as colors), a model involving electrostatic interactions between the electrons from the ligands and the electrons in the unhybridized d orbitals of the central metal atom has been developed. This electrostatic model is crystal field theory (CFT). It helps to understand, interpret, and predict the colors, magnetic behavior, and some structures of coordination compounds of transition metals.
CFT focuses on...
To explain the observed behavior of transition metal complexes (such as colors), a model involving electrostatic interactions between the electrons from the ligands and the electrons in the unhybridized d orbitals of the central metal atom has been developed. This electrostatic model is crystal field theory (CFT). It helps to understand, interpret, and predict the colors, magnetic behavior, and some structures of coordination compounds of transition metals.
CFT focuses on...
26.6K
01:19Allosteric Proteins-ATCase
5.8K
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...
5.8K