从2009年诺贝尔化学奖看同步辐射装置和技术在生物化学研究中的重要作用

文章介绍了获得2009年诺贝尔化学奖的原核细胞核糖体结构解析的历程.在这个历时20年的探索过程中,同步辐射装置起到了重要的作用.同步辐射这种大型的科学装置为前沿的科学研究提供了不可缺少的支撑,文章通过核糖体结构解析的过程,阐述了这种支撑所具有的明显的地域特征.     

单分子荧光检测在生命科学中的应用

文章对单分子荧光检测在分子马达、离子通道、信号分子、蛋白折叠、蛋白构象变化动力学、酶活性反应、细胞过程实时观察等生命科学领域中的应用进行了介绍.这些研究结果表明,单分子荧光检测在研究生物大分子的活动规律与机制方面不但有着无法替代的优越性,而且有着广阔的发展空间.     

钨(Ⅵ)-二溴羟基苯基荧光酮配合物光谱探针测定蛋白质

研究了W(Ⅵ)- 二溴羟基苯基荧光酮(DBHPF)配合物与蛋白质的相互作用及光谱性质.提出了以W(Ⅵ)-二溴羟基苯基荧光酮配合物作为光谱探针测定微量蛋白质的方法.实验结果表明,在pH为 2.43的 Hac-NaAc缓冲介质中,在表面活性剂溴化十六烷基三甲基胺(CTMAB)存在下,DBHPF - W(Ⅵ)在室温10 min 后与蛋白质形成稳定的复合物,最大吸收波长在573 nm处.蛋白质浓度在0～5 mg·L-1 范围内符合比尔定律,复合物的表观摩尔吸光系数为ε=7.81×105 L·mol-1·cm-1.该方法具有很高的灵敏度和选择性,可直接用于人体尿液、新生牛血清中蛋白质的测定,回收率分别为103%和107%.     

动态散射光信号的高速采集的实现

根据动态光散射原理中散射光信号涨落求取纳米级颗粒粒径的方法,介绍了应用TDS5052B数字示波器构建的高速信号采集实验装置,分别阐述了应用LabVIEW虚拟仪器软件设计的基于多倍延迟时间的高速的散射光子信号的采集、甄别与计数程序.实现了单通道的32 ms内散射光子信号采集,并得到了合理的实验结果.     

An Aldehyde Responsive,Cleavable Linker for Glucose Responsive Insulins

A glucose responsive insulin (GRI) that responds to changes in blood glucose concentrations has remained an elusive goal. Here we describe the development of glucose cleavable linkers based on hydrazone and thiazolidine structures. We developed linkers with low levels of spontaneous hydrolysis but increased level of hydrolysis with rising concentrations of glucose, which demonstrated their glucose responsiveness in vitro. Lipidated hydrazones and thiazolidines were conjugated to the LysB29 side-chain of HI by pH-controlled acylations providing GRIs with glucose responsiveness confirmed in vitro for thiazolidines. Clamp studies showed increased glucose infusion at hyperglycemic conditions for one GRI indicative of a true glucose response. The glucose responsive cleavable linker in these GRIs allow changes in glucose levels to drive the release of active insulin from a circulating depot. We have demonstrated an unprecedented, chemically responsive linker concept for biopharmaceuticals.     

Combined Chemical Modification and Collision Induced Unfolding Using Native Ion Mobility-Mass Spectrometry Provides Insights into Protein Gas-Phase Structure

Native mass spectrometry is now an important tool in structural biology. Thus, the nature of higher protein structure in the vacuum of the mass spectrometer is an area of significant interest. One of the major goals in the study of gas-phase protein structure is to elucidate the stabilising role of interactions at the level of individual amino acid residues. A strategy combining protein chemical modification together with collision induced unfolding (CIU) was developed and employed to probe the structure of compact protein ions produced by native electrospray ionisation. Tractable chemical modification was used to alter the properties of amino acid residues, and ion mobility-mass spectrometry (IM-MS) utilised to monitor the extent of unfolding as a function of modification. From these data the importance of specific intramolecular interactions for the stability of compact gas-phase protein structure can be inferred. Using this approach, and aided by molecular dynamics simulations, an important stabilising interaction between K6 and H68 in the protein ubiquitin was identified, as was a contact between the N-terminus and E22 in a ubiquitin binding protein UBA2.     

Electrochemical Studies of CO2-Reducing Metalloenzymes

Only two enzymes are capable of directly reducing CO₂: CO dehydrogenase, which produces CO at a [NiFe₄S₄] active site, and formate dehydrogenase, which produces formate at a mononuclear W or Mo active site. Both metalloenzymes are very rapid, energy-efficient and specific in terms of product. They have been connected to electrodes with two different objectives. A series of studies used protein film electrochemistry to learn about different aspects of the mechanism of these enzymes (reactivity with substrates, inhibitors…). Another series focused on taking advantage of the catalytic performance of these enzymes to build biotechnological devices, from CO₂-reducing electrodes to full photochemical devices performing artificial photosynthesis. Here, we review all these works.     

Tuning the Reactivity of a Substrate for SNAP-Tag Expands Its Application for Recognition-Driven DNA-Protein Conjugation

Recognition-driven modification has been emerging as a novel approach to modifying biomolecular targets of interest site-specifically and efficiently. To this end, protein modular adaptors (MAs) are the ideal reaction model for recognition-driven modification of DNA as they consist of both a sequence-specific DNA-binding domain (DBD) and a self-ligating protein-tag. Coupling DNA recognition by DBD and the chemoselective reaction of the protein tag could provide a highly efficient sequence-specific reaction. However, combining an MA consisting of a reactive protein-tag and its substrate, for example, SNAP-tag and benzyl guanine (BG), revealed rather nonselective reaction with DNA. Therefore new substrates of SNAP-tag have been designed to realize sequence-selective rapid crosslinking reactions of MAs with SNAP-tag. The reactions of substrates with SNAP-tag were verified by kinetic analyses to enable the sequence-selective crosslinking reaction of MA. The new substrate enables the distinctive orthogonality of SNAP-tag against CLIP-tag to achieve orthogonal DNA-protein crosslinking by six unique MAs.     

Near-Ultraviolet Circular Dichroism and Two-Dimensional Spectroscopy of Polypeptides

A fully quantitative theory of the relationship between protein conformation and optical spectroscopy would facilitate deeper insights into biophysical and simulation studies of protein dynamics and folding. In contrast to intense bands in the far-ultraviolet, near-UV bands are much weaker and have been challenging to compute theoretically. We report some advances in the accuracy of calculations in the near-UV, which were realised through the consideration of the vibrational structure of the electronic transitions of aromatic side chains.     

Conformational Selection Mechanism Provides Structural Insights into the Optimization of APC-Asef Inhibitors

Metastasis is the major cause of death in colorectal cancer and it has been proven that inhibiting an interaction between adenomatous polyposis coli (APC) and Rho guanine nucleotide exchange factor 4 (Asef) efficaciously restrain metastasis. However, current inhibitors cannot achieve a satisfying effect in vivo and need to be optimized. In the present study, we applied molecular dynamics (MD) simulations and extensive analyses to apo and holo APC systems in order to reveal the inhibitor mechanism in detail and provide insights into optimization. MD simulations suggested that apo APC takes on a broad array of conformations and inhibitors stabilize conformation selectively. Representative structures in trajectories show specific APC-ligand interactions, explaining the different binding process. The stability and dynamic properties of systems elucidate the inherent factors of the conformation selection mechanism. Binding free energy analysis quantitatively confirms key interface residues and guide optimization. This study elucidates the conformation selection mechanism in APC-Asef inhibition and provides insights into peptide-based drug design.