Homocysteine Thiolactone Modification of Ribonuclease A: Thermodynamics and Kinetics.

IF 3.2 4区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Proteins-Structure Function and Bioinformatics Pub Date : 2025-04-04 DOI:10.1002/prot.26824

Kabira Sabnam, Swagata Dasgupta

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Abstract

Homocysteine thiolactone is a metabolite associated with various diseases at elevated levels in humans. Lysine residues in proteins are modified through N-homocysteinylation and homocysteinylated proteins are prone to form dimers and oligomers through disulfide cross-linkages. This study investigates the effects of N-homocysteinylation on Ribonuclease A (RNase A). The formation of dimers and higher oligomers in RNase A have been confirmed by SDS-PAGE and MALDI-ToF. Agarose-gel assays revealed an altered ribonucleolytic activity due to Lys modification. Fluorescence spectroscopy indicates local changes in the Tyr microenvironment. CD melting studies reveal that β-sheet formation is slightly enhanced with a reduction in the α-helical content in case of modified RNase A. However, the similar melting temperature of both native and modified RNase A indicates overall structural integrity with local changes in secondary structural components. ITC and UV-visible kinetics show reduced ribonucleolytic activity in homocysteinylated RNase A compared to the unmodified enzyme. These findings provide insights into the structural and functional consequences of RNase A homocysteinylation, contributing to our understanding of hyperhomocysteinemia-related pathologies.

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同半胱氨酸硫内酯修饰核糖核酸酶 A：热力学和动力学。

同型半胱氨酸硫内酯是一种与人类多种疾病相关的代谢物。蛋白质中的赖氨酸残基通过n -同型半胱氨酸化修饰，同型半胱氨酸化的蛋白质容易通过二硫交键形成二聚体和低聚物。本研究探讨了n -同型半胱氨酸化对核糖核酸酶A （RNase A）的影响。通过SDS-PAGE和MALDI-ToF证实了RNase A中二聚体和高低聚物的形成。琼脂糖凝胶分析显示，由于赖氨酸修饰，核糖核溶解活性发生了改变。荧光光谱显示Tyr微环境的局部变化。CD熔化研究表明，改性RNase a的α-螺旋含量降低，β-薄片的形成略有增强。然而，天然RNase a和改性RNase a的熔化温度相似，表明其整体结构完整，但二级结构组分发生了局部变化。ITC和紫外可见动力学表明同型半胱氨酸化RNase A的核糖核分解活性比未修饰的酶降低。这些发现为RNase A同型半胱氨酸化的结构和功能后果提供了见解，有助于我们理解高同型半胱氨酸血症相关病理。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Proteins-Structure Function and Bioinformatics 生物-生化与分子生物学

CiteScore

5.90

自引率

3.40%

发文量

172

审稿时长

3 months

期刊介绍： PROTEINS : Structure, Function, and Bioinformatics publishes original reports of significant experimental and analytic research in all areas of protein research: structure, function, computation, genetics, and design. The journal encourages reports that present new experimental or computational approaches for interpreting and understanding data from biophysical chemistry, structural studies of proteins and macromolecular assemblies, alterations of protein structure and function engineered through techniques of molecular biology and genetics, functional analyses under physiologic conditions, as well as the interactions of proteins with receptors, nucleic acids, or other specific ligands or substrates. Research in protein and peptide biochemistry directed toward synthesizing or characterizing molecules that simulate aspects of the activity of proteins, or that act as inhibitors of protein function, is also within the scope of PROTEINS. In addition to full-length reports, short communications (usually not more than 4 printed pages) and prediction reports are welcome. Reviews are typically by invitation; authors are encouraged to submit proposed topics for consideration.