Preparing to read the ubiquitin code: a middle‐out strategy for characterization of all lysine‐linked diubiquitins

Multiple studies demonstrate that ubiquitination of proteins codes for regulation of cell differentiation, apoptosis, endocytosis and many other cellular functions. There is great interest in and considerable effort being given to defining the relationships between the structures of polyubiquitin modifications and the fates of the modified proteins. Does each ubiquitin modification achieve a specific effect, much like phosphorylation, or is ubiquitin like glycosylation, where there is heterogeneity and redundancy in the signal? The sensitive analytical tools needed to address such questions readily are not yet mature. To lay the foundation for mass spectrometry (MS)‐based studies of the ubiquitin code, we have assembled seven isomeric diubiquitins with all‐native sequences and isopeptide linkages. Using these compounds as standards enables the development and testing of a new MS‐based strategy tailored specifically to characterize the number and sites of isopeptide linkages in polyubiquitin chains. Here, we report the use of Asp‐selective acid cleavage, separation by reverse phase high‐performance liquid chromatography and characterization by tandem MS to distinguish and characterize all seven isomeric lysine‐linked ubiquitin dimers. Copyright © 2014 John Wiley & Sons, Ltd.     

Probing Transient Conformational States of Proteins by Solid‐State R1ρ Relaxation‐Dispersion NMR Spectroscopy

The function of proteins depends on their ability to sample a variety of states differing in structure and free energy. Deciphering how the various thermally accessible conformations are connected, and understanding their structures and relative energies is crucial in rationalizing protein function. Many biomolecular reactions take place within microseconds to milliseconds, and this timescale is therefore of central functional importance. Here we show that R_1ρ relaxation dispersion experiments in magic‐angle‐spinning solid‐state NMR spectroscopy make it possible to investigate the thermodynamics and kinetics of such exchange process, and gain insight into structural features of short‐lived states.     

Fluorescently Labelled ATP Analogues for Direct Monitoring of Ubiquitin Activation

Simple and robust assays to monitor enzymatic ATP cleavage with high efficiency in real-time are scarce. To address this shortcoming, we developed fluorescently labelled adenosine tri-, tetra- and pentaphosphate analogues of ATP. The novel ATP analogues bear — in contrast to earlier reports — only a single acridone-based dye at the terminal phosphate group. The dye's fluorescence is quenched by the adenine component of the ATP analogue and is restored upon cleavage of the phosphate chain and dissociation of the dye from the adenosine moiety. Thereby the activity of ATP-cleaving enzymes can be followed in real-time. We demonstrate this proficiency for ubiquitin activation by the ubiquitin-activating enzymes UBA1 and UBA6 which represents the first step in an enzymatic cascade leading to the covalent attachment of ubiquitin to substrate proteins, a process that is highly conserved from yeast to humans. We found that the efficiency to serve as cofactor for UBA1/UBA6 very much depends on the length of the phosphate chain of the ATP analogue: triphosphates are used poorly while pentaphosphates are most efficiently processed. Notably, the novel pentaphosphate-harbouring ATP analogue supersedes the efficiency of recently reported dual-dye labelled analogues and thus, is a promising candidate for broad applications.     

Noncovalent dimerization of ubiquitin

Another kind of dynamics: Ubiquitin noncovalently dimerizes with a dissociation constant of approximately 5?mM. The two subunits adopt an array of relative orientations, utilizing an interface also for binding to other proteins (see picture). Quaternary fluctuation among members of the dimer ensemble constitutes a different kind of dynamics that complements the tertiary dynamics of each ubiquitin subunit.     

Overexpression of EAR1 and SSH4 that encode PPxY proteins in the multivesicular body provides stability to tryptophan permease Tat2, allowing yeast cells to grow under high hydrostatic pressure

Tryptophan uptake in yeast Saccharomyces cerevisiae is susceptible to high hydrostatic pressure and it limits the growth of tryptophan auxotrophic (Trp^?) strains under pressures of 15–25 MPa. The susceptibility of tryptophan uptake is accounted for by the pressure-induced degradation of tryptophan permease Tat2 occurring in a Rsp5 ubiquitin ligase-dependent manner. Ear1 and Ssh4 are multivesicular body proteins that physically interact with Rsp5. We found that overexpression of either of the EAR1 or SSH4 genes enabled the Trp^? cells to grow at 15–25 MPa. EAR1 and SSH4 appeared to provide stability to the Tat2 protein when overexpressed. The result suggests that Ear1 and Ssh4 negatively regulate Rsp5 on ubiquitination of Tat2. Currently, high hydrostatic pressure is widely used in bioscience and biotechnology for structurally perturbing macromolecules such as proteins and lipids or in food processing and sterilizing microbes. We suggest that hydrostatic pressure is an operative experimental parameter to screen yeast genes specifically for regulation of Tat2 through the function of Rsp5 ubiquitin ligase.     

NMR技术在蛋白质-蛋白质相互作用研究中的应用——泛素-蛋白水解酶体通路研究实例介绍

蛋白质-蛋白质相互作用在多种细胞内生理活动中发挥关键性作用,而蛋白质复合物结构信息的获得主要依赖于X-射线衍射技术和核磁共振技术2种主要技术手段的使用. 需要指出的是,虽然大部分蛋白质复合物的结构解析使用了X-射线衍射技术,然而在包括无法获得蛋白质复合物晶体、 蛋白质与蛋白质结合强度较弱以及蛋白质复合物系统具有复杂的动力学行为等几种情况下,核磁共振技术是可用于蛋白质复合物结构测定的唯一手段. 用于蛋白质-蛋白质相互作用研究的NMR技术主要有化学位移扰动分析、分子间NOE的检测、顺磁弛豫增强技术、残余偶极耦合检测技术等几种. 该文将结合这几种技术在泛素-蛋白水解酶体通路领域的应用实例对它们的工作原理以及可提供的信息做出总结介绍.     

双光束光解离在牛泛素蛋白离子的自顶向下质谱分析中的应用研究

自顶向下(Top-down)质谱分析方法是将完整蛋白质离子碎片化,从而在分子水平上提供更加精准、丰富的与蛋白质结构相关的生物学信息.该文首次将3μm红外激光与210 nm紫外激光共同引入到傅里叶变换离子回旋共振质谱仪(FT-ICR MS)的分析池中,获得了牛泛素蛋白离子的自顶向下质谱.通过优化两束激光被引入的时间序列,...     

铽离子-镧系金属结合标签的荧光探针的改进

镧系金属因其具有较窄的发射光谱带,较大的斯托克斯位移以及毫秒级的荧光寿命,而被广泛应用于荧光检测中,其中Tb~(3+)、Eu~(3+)最为常用.镧系金属结合标签(Lanthanide Binding Tag,LBT)可以与蛋白质融合表达,并且一般不会影响蛋白的结构和功能,这些特点使LBT被广泛应用于蛋白质结构与功能研究中.LBT能够特异性地结合镧系金属离子,并利用LBT上色氨酸的吲哚环作为"天线"吸收外部能量,再将能量传递给镧系金属离子,进而激发镧系金属离子产生荧光.该文以经典模式蛋白泛素(Ubiquitin,Ub)作为媒介,将LBT引入到Ub的碳端,采用定点突变的方法增加LBT上吲哚环的数量,观察Ub-LBT[结合铽离子(Tb~(3+))]荧光量子产率的变化.结果表明在LBT结构中增加吲哚环的数量能够提高LBT(结合Tb~(3+))的荧光量子产率.     

Acid-sensitive auxiliary assisted atypical diubiquitin synthesis exploiting thiol-ene coupling

Atypical ubiquitin (Ub) chains are generally involved in intracellular physiological processes, while the molecular mechanisms underlying their regulation remain unclear. In this work, we report an acid-sensitive auxiliary group based bifunctional handle that can prepare Lys27-, Lys29- and Lys33-diUb analogs by thiol-ene coupling (TEC) in combination with native chemical ligation (NCL). A prominent advantage of this method is the rapid and effective removal of acid-sensitive auxiliary groups after the formation of the isopeptide bond mimic. Collectively, this work illustrates the utility of the new strategy in the simple and efficient production of homogeneous atypical diUb analogs for biochemical and biophysical studies.