Identification and characterization of a novel periplasmic polygalacturonic acid binding protein from Yersinia enterolitica

A number of bacteria in the family Enterobacteriaceae harbor the genes comprising well-developed pectinolytic pathways (e.g. Erwinia sp.) or abridged versions of this pathway (e.g. Yersinia sp.). One of the most enigmatic components present in some of these pathways is a small gene that encodes a predicted secreted protein of approximately 160 amino acid residues with unknown function. This protein shows distant amino acid sequence similarity over its entire length to galactose-specific family 32 carbohydrate-binding modules (CBMs). Here we demonstrate the ability of the Yersinia enterocolitica example, here called YeCBM32, to bind polygalacturonic acid containing components of pectin. This binding is selective for highly polymerized galacturonic acid and shows a complex mode of polysaccharide recognition. The high resolution X-ray crystal structure (1.35 A) shows YeCBM32s overall structural similarity to galactose specific CBMs and conserved binding site location but reveals a substantially different binding site topology, which likely reflects its unique polymeric and acidic ligand. The results suggest the possibility of a unique role for YeCBM32 in polygalacturonic acid transport.     

Screening combinatorial libraries of molecularly imprinted polymer films casted on membranes in single-use membrane modules

A new and fast technique for screening combinatorial libraries of molecularly imprinted polymers is presented. The procedure is based on commercially available membrane modules which are rinsed with pre-polymerization imprinting mixtures. After the in situ polymerization and generation of MIP films on the PTFE membranes within the modules, the membranes are evaluated in terms of affinity towards the target molecule (template) R-(-)-phenylbutyric acid. Therefore, after template extraction from the freshly produced membranes a solution of this target molecule is flushed through the module. By analyzing the remaining analyte concentration in the permeate, the amount of analyte adsorbed on the membrane can be calculated and related to specific interactions with the molecular imprints. By this means, optimized recipes in terms of cross-linker to template ratios could be obtained in combination with the optimal porogen, when screening p-divinylbenzene or ethylene glycol dimethacrylate as cross-linker and porogens like acetonitrile, dimethylsulfoxide and methanol.     

Binding sub-site dissection of a carbohydrate-binding module reveals the contribution of entropy to oligosaccharide recognition at "non-primary" binding subsites

The optimal ligands for many carbohydrate-binding proteins are often oligosaccharides comprising two, three, or more monosaccharide units. The binding affinity for these sugars is increased incrementally by contributions from binding subsites on the protein that accommodate the individual monosaccharide residues of the oligosaccharide. Here, we use CsCBM6-1, a xylan-specific type B carbohydrate-binding module (CBM) from Clostridium stercorarium falling into amino acid sequence family CBM6, as a model system to investigate the structural and thermodynamic contributions of binding subsites in this protein to carbohydrate recognition. The three-dimensional structures of uncomplexed CsCBM6-1 (at 1.8 A resolution) and bound to the oligosaccharides xylobiose, xylotriose, and xylotetraose (at 1.70 A, 1.89 A, and 1.69 A resolution, respectively) revealed the sequential occupation of four subsites within the binding site in the order of subsites 2, 3, 4 then 1. Overall, binding to all of the xylooligosaccharides tested was enthalpically favourable and entropically unfavourable, like most protein-carbohydrate interactions, with the primary subsites 2 and 3 providing the bulk of the free energy and enthalpy of binding. In contrast, the contributions to the changes in entropy of the non-primary subsites 1 and 4 to xylotriose and xylotetraose binding, respectively, were positive. This observation is remarkable, in that it shows that the 10-20-fold improvement in association constants for oligosaccharides longer than a disaccharide is facilitated by favourable entropic contributions from the non-primary binding subsites.     

生物信息学方法预测蛋白质相互作用网络中的功能模块

蛋白质相互作用是大多数生命过程的基础。随着高通量实验技术和计算机预测方法的发展,在各种生物中已获得了数目十分庞大的蛋白质相互作用数据,如何从中提取出具有生物学意义的数据是一项艰巨的挑战。从蛋白质相互作用数据出发获得相互作用网络进而预测出其中的功能模块,对于蛋白质功能预测、揭示各种生化反应过程的分子机理都有着极大的帮助。我们分类概括了用生物信息学预测蛋白质相互作用功能模块的方法,以及对这些方法的评价,并介绍了蛋白质相互作用网络比较的一些方法。     

天童常绿阔叶树种栲树生殖个体大小及其生殖构件特征

对浙江天童木荷-栲树林内的常绿阔叶树种栲树(Castanopsis fargesii Franch.)的生殖个体大小、生殖构件的分布及其动态变化特征进行了研究。结果表明, 该地区栲树生殖个体的胸径在17~50 cm 间, 平均胸径为31. 2±8.0 cm, 平均年龄约36.3±6.6 年;林缘附近的生殖个体小于木荷-林内。相对稳定的群落和比较丰富的土壤养分条件有利于生殖枝数量和花序数量的增多。栲树生殖个体的数量在两年中变化较大, 部分栲树个体可以在连续年份中生殖。从枝系水平分析:在持续生殖的栲树个体上, 生殖枝数量有明显变化, 并非所有的生殖枝在两年中都可开花或结果, 保持连续生殖的枝系约占48.2%。栲树果序枝数量在连续年份有明显差异(p < 0.01), 而且果序枝上的幼蕾数、果实数量及结实率等都有明显差异(p < 0.05)。