萝卜磷脂氢谷胱甘肽过氧化物酶(RsPHGPx)是一个定位于线粒体的蛋白质.为了阐明该蛋白质线粒体定位信号的准确切割位点,采用了免疫亲和层析方法纯化天然的RsPHGPx.用重组RsPHGPx 蛋白免疫兔子获得了抗RsPHGPx 的多克隆抗血清,以重组RsPH ...萝卜磷脂氢谷胱甘肽过氧化物酶(RsPHGPx)是一个定位于线粒体的蛋白质.为了阐明该蛋白质线粒体定位信号的准确切割位点,采用了免疫亲和层析方法纯化天然的RsPHGPx.用重组RsPHGPx 蛋白免疫兔子获得了抗RsPHGPx 的多克隆抗血清,以重组RsPH G Px 蛋白为配体,采用亲和层析技术对抗血清进行了纯化,得到了单特异性的抗RsPHGPx 的抗体.将纯化好的抗体偶联到一个N-羟基琥珀酰亚胺(NHS)预先激活的琼脂糖柱子上,装配成一个以单特异性的抗RsPHGPx 抗体为配体的免疫亲和层析柱.经过对纯化条件的摸索和优化,形成了一个简单、特异的一步法纯化方案.按照该方案,从萝卜幼苗线粒体总蛋白质提取物中纯化到一个分子质量与预期值相一致的特异蛋白质.免疫印迹分析表明,该蛋白质被抗RsPH G Px 的抗血清特异识别.酶活性分析表明,该蛋白质具有显著的PH G Px 活性.这些结果表明,纯化到的特异蛋白质是萝卜的RsPH G Px天然蛋白.这是首个关于定位于植物细胞器的PH G Px 蛋白纯化的报道.这一结果为准确测定RsPH G Px 信号肽的切割位点奠定了基础,并将有助于对植物PH G Px 的亚细胞定位机制及其生理功能的深入研究.展开更多
Protein ubiquitination is an important post-translational modification(PTM) in eukaryotic organisms that regulates a variety of cellular processes, such as protein degradation, signal transduction, apoptosis, and DNA ...Protein ubiquitination is an important post-translational modification(PTM) in eukaryotic organisms that regulates a variety of cellular processes, such as protein degradation, signal transduction, apoptosis, and DNA damage tolerance. To decipher mechanistically the diverse biological functions of ubiquitination, homogeneous ubiquitinated proteins are greatly needed.Although direct isolation from cell source and in vitro enzymatic methods can be used to produce such proteins, these methods often suffer from problems of low yield or heterogeneous products. Comparably, total chemical and semisynthetic approaches offer good alternatives to produce the ubiquitinated proteins with high purity and selectivity. This review summarizes the recent developments of protein ubiquitination strategies and the use of the synthesized proteins to help garner structural and functional insight into the inner workings of the ubiquitin system.展开更多
Available online Immunoglobulins G(IgGs)are Y-shaped globular proteins,however,their high flexibility and heterogeneity pose great challenges to their structure and conformation determinations.Geometric structure of I...Available online Immunoglobulins G(IgGs)are Y-shaped globular proteins,however,their high flexibility and heterogeneity pose great challenges to their structure and conformation determinations.Geometric structure of IgG closely correlates to its biofunctions,such as the antibody escape of human immunodeficiency virus(HIV)could attribute to the distance mismatch between the ends of two Fab arms(antigen-binding sites)and envelope glycoprotein spikes on virion surface.Herein,we report the first use of mobility capillary electrophoresis(MCE)and native mass spectrometry(nMS)to resolve the internal geometric structure and conformation of an IgG(trastuzumab)in solution phase.After proteolysis,the ellipsoid dimensions of IgG and its subunits were measured by MCE-nMS experiments.IgG was then reconstructed,in which the sizes and relative positions of these three subunits in three-dimensional space were characterized.It was found that the two Fab arms have an angle of~102.1°and a distance of~11.0 nm between the two antigen-binding sites under native condition,and the Fc arm was tilted~16.0°towards one of the Fab arms.Fc was not on the plane of Fab-Fab,but has an angle of no larger than 103.1°.Under acidic environment(pH 3.0),each subunit of the IgG would unfold into larger dimensions,and the angles between these subunits also change.With great potential for tumor imaging and therapy,the structure of F(ab')_(2)fragments was also measured and validated by molecular dynamic simulation.It was found that the electrostatic force among these three subunits and steric hindrance stemming from Fc help maintaining the angle between two Fab arms.展开更多
Snake toxin Calciseptine as a natural antagonist of L-type calcium channel has potential drug values, but its structural information remains unknown. Here, we report the total chemical synthesis of Calciseptine by usi...Snake toxin Calciseptine as a natural antagonist of L-type calcium channel has potential drug values, but its structural information remains unknown. Here, we report the total chemical synthesis of Calciseptine by using hydrazide based native chemical ligation. The crystal structure of Calciseptine was determined by racemic protein crystallography technique. Compared to the structure of its homologous family protein, we found that Calciseptine is adopting a typical three-finger structure.展开更多
Solid phase peptide synthesis(SPPS)based on Fmoc chemistry has become a commonly used technique in peptide chemistry,as it can be easily conducted using automated machine,and not requiring highly toxic HF in compariso...Solid phase peptide synthesis(SPPS)based on Fmoc chemistry has become a commonly used technique in peptide chemistry,as it can be easily conducted using automated machine,and not requiring highly toxic HF in comparison to Boc-SPPS.With the fast development in the emerging field of protein chemical synthesis,many efforts have been endeavored aiming to find more efficient methods for preparing peptide fragments required in ligation reactions.This review briefly summarizes recent advances in the engineering and modification of Fmoc-SPPS-derived peptides,which can be used as the N-terminal fragments in a native chemical ligation(NCL)or NCL-type ligation reactions.展开更多
Proteins might misfold during translation and folding or even once they are in their native states, due to stochastic fluctuations, destabilizing mutations or cellular stress. Aberrant protein species are usually dete...Proteins might misfold during translation and folding or even once they are in their native states, due to stochastic fluctuations, destabilizing mutations or cellular stress. Aberrant protein species are usually detected and either refolded or cleared by the protein quality control machinery (Ciechanover and Kwon, 2015). When misfolded protein conformers cannot be degraded, they tend to self-assemble to form aggregates, a characteristic of many neurodegenerative diseases.展开更多
文摘萝卜磷脂氢谷胱甘肽过氧化物酶(RsPHGPx)是一个定位于线粒体的蛋白质.为了阐明该蛋白质线粒体定位信号的准确切割位点,采用了免疫亲和层析方法纯化天然的RsPHGPx.用重组RsPHGPx 蛋白免疫兔子获得了抗RsPHGPx 的多克隆抗血清,以重组RsPH G Px 蛋白为配体,采用亲和层析技术对抗血清进行了纯化,得到了单特异性的抗RsPHGPx 的抗体.将纯化好的抗体偶联到一个N-羟基琥珀酰亚胺(NHS)预先激活的琼脂糖柱子上,装配成一个以单特异性的抗RsPHGPx 抗体为配体的免疫亲和层析柱.经过对纯化条件的摸索和优化,形成了一个简单、特异的一步法纯化方案.按照该方案,从萝卜幼苗线粒体总蛋白质提取物中纯化到一个分子质量与预期值相一致的特异蛋白质.免疫印迹分析表明,该蛋白质被抗RsPH G Px 的抗血清特异识别.酶活性分析表明,该蛋白质具有显著的PH G Px 活性.这些结果表明,纯化到的特异蛋白质是萝卜的RsPH G Px天然蛋白.这是首个关于定位于植物细胞器的PH G Px 蛋白纯化的报道.这一结果为准确测定RsPH G Px 信号肽的切割位点奠定了基础,并将有助于对植物PH G Px 的亚细胞定位机制及其生理功能的深入研究.
基金supported by Exploit Technologies Pte Ltd of Agency for Science,Technology and Research(A*Star)of Singapore(ETPL-QP-19-06)
文摘Protein ubiquitination is an important post-translational modification(PTM) in eukaryotic organisms that regulates a variety of cellular processes, such as protein degradation, signal transduction, apoptosis, and DNA damage tolerance. To decipher mechanistically the diverse biological functions of ubiquitination, homogeneous ubiquitinated proteins are greatly needed.Although direct isolation from cell source and in vitro enzymatic methods can be used to produce such proteins, these methods often suffer from problems of low yield or heterogeneous products. Comparably, total chemical and semisynthetic approaches offer good alternatives to produce the ubiquitinated proteins with high purity and selectivity. This review summarizes the recent developments of protein ubiquitination strategies and the use of the synthesized proteins to help garner structural and functional insight into the inner workings of the ubiquitin system.
基金supported by Ministry of Science and Technology of the People’s Republic of China instrumentation program(No.2020YFF01014502)NNSFC(No.21827810)Beijing Institute of Technology(No.2021CX006)。
文摘Available online Immunoglobulins G(IgGs)are Y-shaped globular proteins,however,their high flexibility and heterogeneity pose great challenges to their structure and conformation determinations.Geometric structure of IgG closely correlates to its biofunctions,such as the antibody escape of human immunodeficiency virus(HIV)could attribute to the distance mismatch between the ends of two Fab arms(antigen-binding sites)and envelope glycoprotein spikes on virion surface.Herein,we report the first use of mobility capillary electrophoresis(MCE)and native mass spectrometry(nMS)to resolve the internal geometric structure and conformation of an IgG(trastuzumab)in solution phase.After proteolysis,the ellipsoid dimensions of IgG and its subunits were measured by MCE-nMS experiments.IgG was then reconstructed,in which the sizes and relative positions of these three subunits in three-dimensional space were characterized.It was found that the two Fab arms have an angle of~102.1°and a distance of~11.0 nm between the two antigen-binding sites under native condition,and the Fc arm was tilted~16.0°towards one of the Fab arms.Fc was not on the plane of Fab-Fab,but has an angle of no larger than 103.1°.Under acidic environment(pH 3.0),each subunit of the IgG would unfold into larger dimensions,and the angles between these subunits also change.With great potential for tumor imaging and therapy,the structure of F(ab')_(2)fragments was also measured and validated by molecular dynamic simulation.It was found that the electrostatic force among these three subunits and steric hindrance stemming from Fc help maintaining the angle between two Fab arms.
基金supported by the National Natural Science Foundation of China (21572043, 21473176)the Ministry of Science and Technology (2016YFA0400900, 2015CB910103)the Fundamental Research Funds for the Central Universities (PA2017GDQT0021)
文摘Snake toxin Calciseptine as a natural antagonist of L-type calcium channel has potential drug values, but its structural information remains unknown. Here, we report the total chemical synthesis of Calciseptine by using hydrazide based native chemical ligation. The crystal structure of Calciseptine was determined by racemic protein crystallography technique. Compared to the structure of its homologous family protein, we found that Calciseptine is adopting a typical three-finger structure.
基金supported by the Peking University Health Science Center(BMU20130354)State Key Laboratory of Natural and Biomimetic Drugs,the National Recruitment Program of Global Youth Experts(1000 Plan)the National Natural Science Foundation of China (21502005)
文摘Solid phase peptide synthesis(SPPS)based on Fmoc chemistry has become a commonly used technique in peptide chemistry,as it can be easily conducted using automated machine,and not requiring highly toxic HF in comparison to Boc-SPPS.With the fast development in the emerging field of protein chemical synthesis,many efforts have been endeavored aiming to find more efficient methods for preparing peptide fragments required in ligation reactions.This review briefly summarizes recent advances in the engineering and modification of Fmoc-SPPS-derived peptides,which can be used as the N-terminal fragments in a native chemical ligation(NCL)or NCL-type ligation reactions.
文摘Proteins might misfold during translation and folding or even once they are in their native states, due to stochastic fluctuations, destabilizing mutations or cellular stress. Aberrant protein species are usually detected and either refolded or cleared by the protein quality control machinery (Ciechanover and Kwon, 2015). When misfolded protein conformers cannot be degraded, they tend to self-assemble to form aggregates, a characteristic of many neurodegenerative diseases.
