Characterizing a monotopic membrane enzyme. Biochemical, enzymatic and crystallization studies on Aquifex aeolicus sulfide:quinone oxidoreductase

Monotopic membrane proteins are membrane proteins that interact with only one leaflet of the lipid bilayer and do not possess transmembrane spanning segments. They are endowed with important physiological functions but until now only few of them have been studied. Here we present a detailed biochemical, enzymatic and crystallographic characterization of the monotopic membrane protein sulfide:quinone oxidoreductase. Sulfide:quinone oxidoreductase is a ubiquitous enzyme involved in sulfide detoxification, in sulfide-dependent respiration and photosynthesis, and in heavy metal tolerance. It may also play a crucial role in mammals, including humans, because sulfide acts as a neurotransmitter in these organisms. We isolated and purified sulfide:quinone oxidoreductase from the native membranes of the hyperthermophilic bacterium Aquifex aeolicus. We studied the pure and solubilized enzyme by denaturing and non-denaturing polyacrylamide electrophoresis, size-exclusion chromatography, cross-linking, analytical ultracentrifugation, visible and ultraviolet spectroscopy, mass spectrometry and electron microscopy. Additionally, we report the characterization of its enzymatic activity before and after crystallization. Finally, we discuss the crystallization of sulfide:quinone oxidoreductase in respect to its membrane topology and we propose a classification of monotopic membrane protein crystal lattices. Our data support and complement an earlier description of the three-dimensional structure of A. aeolicus sulfide:quinone oxidoreductase (M. Marcia, U. Ermler, G. Peng, H. Michel, Proc Natl Acad Sci USA, 106 (2009) 9625-9630) and may serve as a reference for further studies on monotopic membrane proteins. (C) 2010 Elsevier B.V. All rights reserved.

Diversity and Distribution of Sediment NirS-Encoding Bacterial Assemblages in Response to Environmental Gradients in the Eutrophied Jiaozhou Bay, China

A gene-clone-library-based molecular approach was used to study the nirS-encoding bacteria-environment relationship in the sediments of the eutrophic Jiaozhou Bay. Diverse nirS sequences were recovered and most of them were related to the marine cluster I group, ubiquitous in estuarine, coastal, and marine environments. Some NirS sequences were unique to the Jiaozhou Bay, such as the marine subcluster VIIg sequences. Most of the Jiaozhou Bay NirS sequences had their closest matches originally detected in estuarine and marine sediments, especially from the Chesapeake Bay, indicating similarity of the denitrifying bacterial communities in similar coastal environments in spite of geographical distance. Multivariate statistical analyses indicated that the spatial distribution of the nirS-encoding bacterial assemblages is highly correlated with environmental factors, such as sediment silt content, NH4+ concentration, and OrgC/OrgN. The nirS-encoding bacterial assemblages in the most hypernutrified stations could be easily distinguished from that of the least eutrophic station. For the first time, the sedimentological condition was found to influence the structure and distribution of the sediment denitrifying bacterial community.

胸苷酸合成酶和二氢叶酸还原酶mRNA亲和肽的发现及mRNA体外展示技术的优化

胸苷酸合成酶（thymidylate synthase，简称TS）和二氢叶酸还原酶（dihydrofolate reductase, 简称DHFR）都是叶酸依赖性酶，在维持DNA合成和DNA修复上发挥关键作用，并且多年来一直是肿瘤研究和化疗的重要靶点。我们前期的研究发现，TS和DHFR在翻译水平上存在负反馈调控机制。人TS和DHFR可以与其自身的mRNA结合，从而抑制mRNA的表达，化疗药物可以与TS或者DHFR相互作用，形成的复合物不能与TS mRNA结合， 使负反馈机制丧失。因此深入研究TS和DHFR的翻译调控机理，对阐明肿瘤抗药性机制，对发现新的抗肿瘤药物和肿瘤的治疗都具有十分重要的意义。 本论文利用mRNA体外展示技术，构建多肽库（约10万亿种多肽分子），利用多种实验手段将mRNA体外展示技术进行优化，提高了多肽库融合肽的产量，提高了mRNA体外展示技术筛选的特异性。将TS mRNA分子上的顺式因子TS30 RNA固定于磁珠上，将融合肽库与顺式因子作用，经过6轮循环，由多肽库中获得了与TS mRNA高度亲和的多肽序列，体外结合实验证明亲和肽可以与TS全长mRNA结合，体外翻译实验证明多肽可以抑制TS mRNA的翻译。并且利用phage display技术由噬菌体肽库（12个氨基酸随机肽库）经过四轮筛选，分别筛选到TS和DHFR的亲和肽，凝胶阻滞实验证明它们分别能与TS和DHFR mRNA结合。 本论文利用的展示技术可以广泛应用于特异靶点的蛋白质筛选，并且本论文筛选到的TS和DHFR亲和肽可以作为TS和DHFR的抑制剂，从而为获得新型的抗肿瘤药物奠定基础。