The small‐angle and wide‐angle X‐ray scattering set‐up at beamline BL9 of DELTA

The multi‐purpose experimental endstation of beamline BL9 at the Dortmund Electron Accelerator (DELTA) is dedicated to diffraction experiments in grazing‐incidence geometry, reflectivity and powder diffraction measurements. Moreover, fluorescence analysis and inelastic X‐ray scattering experiments can be performed. Recently, a new set‐up for small‐angle and wide‐angle X‐ray scattering utilizing detection by means of an image‐plate scanner was installed and is described in detail here. First small‐angle X‐ray scattering experiments on aqueous solutions of lysozyme with different cosolvents and of staphylococcal nuclease are discussed. The application of the set‐up for texture analysis is emphasized and a study of the crystallographic texture of natural bio‐nanocomposites, using lobster and crab cuticles as model materials, is presented.     

银纳米颗粒对溶菌酶纤维化影响的浓度效应

纳米颗粒对蛋白质淀粉纤维化过程的影响机制对扩大其在生物学诊断和纳米药物应用中起到至关重要的作用. 在本研究中,通过拉曼光谱结合原子力显微镜和硫磺素T荧光光谱实验研究不同浓度银纳米颗粒条件下的溶菌酶淀粉纤维化过程. 利用四个具有代表性的拉曼光谱指标在分子水平上监测蛋白质的三级结构和二级结构的转化过程,如：Trp费米共振双峰(1340 cm^-1和1360 cm^{-1-1-1相似文献}   

Structural Compactness in Hen Egg White Lysozyme Induced by Bisphenol S: A Spectroscopic and Molecular Dynamics Simulation Approach

The endocrine disrupting compound Bisphenol and its analogues are widely used in food packaging products and can cause serious health hazards. The protein, Lysozyme (Lyz), showing anti-microbial properties, is used as a “natural” food and dairy preservative. Herein, we explored the interaction between Lyz and Bisphenol S (BPS) by multi-spectroscopic and theoretical approaches. Lyz interacts with BPS through static quenching, where hydrophobic force governed the underlying interaction. Molecular docking results reveal that tryptophan plays a vital role in binding, corroborated well with near UV-CD studies. A decrease in the radius of gyration (from 1.43 nm to 1.35 nm) of Lyz substantiates the compactness of the protein conformation owing to such an interaction. This structural alteration experienced by Lyz may alter its functional properties as a food preservative. Consequently, this can degrade the quality of the food products and thereby lead to severe health issues.     

Changes in the characteristics of acid‐treated clay after the inclusion of proteins

The aim of this work is to study the difference in the characteristics of commercial clay after the inclusion of two proteins. Bovine serum albumin and egg white lysozyme were immobilized on the structure of commercial montmorillonite, which was previously treated with a hydrochloric acid solution. Studies were carried out at two different concentrations, fixing the pH to ensure the charge of biomolecules and clay surface. Acid‐treated clay containing proteins was characterized by X‐ray diffraction, Fourier transformed infrared spectroscopy, thermogravimetric analysis, and nitrogen adsorption at 77 K. The powders showed similar thermal behavior after the inclusion of proteins but with variations in the amount of mass loss in each sample. Moreover, changes in the surface characteristics of the final solid were observed, depending on both the concentration and the nature of the incorporated protein. The differences observed in the acid‐treated clay characteristics after the inclusion of each type of protein are discussed. The characterization of materials after the inclusion of protein molecules can be useful to understand the adsorption mechanism of biomolecules on solid surfaces. Copyright © 2014 John Wiley & Sons, Ltd.     

阴离子表面活性剂与溶菌酶相互作用的相行为

研究了pH值为7时蛋白质溶菌酶与六种阴离子表面活性剂CnH2n 1SO4Na /CnH2n 1SO3Na (n＝8、10、12)混合体系的相行为.用紫外光谱方法考察了表面活性剂分子结构(头基、链长)、盐(缓冲溶液)对混合体系相行为的影响.结果表明,同浓度缓冲溶液中和在碳链相同时,CnH2n 1SO4Na比CnH2n 1SO3Na更易与溶菌酶结合;在头基相同时,表面活性剂与溶菌酶的结合强度随表面活性剂的链长增加而增加.盐浓度为0～50 mmol/L时,混合体系中的表面活性剂更容易形成胶团;盐浓度增大到100 mmol/L时,混合体系中的表面活性剂更易与蛋白质结合.     

A g-Type Lysozyme from Deep-Sea Hydrothermal Vent Shrimp Kills Selectively Gram-Negative Bacteria

Lysozyme is a key effector molecule of the innate immune system in both vertebrate and invertebrate. It is classified into six types, one of which is the goose-type (g-type). To date, no study on g-type lysozyme in crustacean has been documented. Here, we report the identification and characterization of a g-type lysozyme (named LysG1) from the shrimp inhabiting a deep-sea hydrothermal vent in Manus Basin. LysG1 possesses conserved structural features of g-type lysozymes. The recombinant LysG1 (rLysG1) exhibited no muramidase activity and killed selectively Gram-negative bacteria in a manner that depended on temperature, pH, and metal ions. rLysG1 bound target bacteria via interaction with bacterial cell wall components, notably lipopolysaccharide (LPS), and induced cellular membrane permeabilization, which eventually caused cell lysis. The endotoxin-binding capacity enabled rLysG1 to alleviate the inflammatory response induced by LPS. Mutation analysis showed that the bacterial binding and killing activities of rLysG1 required the integrity of the conserved α3 and 4 helixes of the protein. Together, these results provide the first insight into the activity and working mechanism of g-type lysozyme in crustacean and deep-sea organisms.     

Unraveling the Compositional and Molecular Features Involved in Lysozyme-Benzothiazole Derivative Interactions

In this work we present a computational analysis together with experimental studies, focusing on the interaction between a benzothiazole (BTS) and lysozyme. Results obtained from isothermal titration calorimetry, UV-vis, and fluorescence were contrasted and complemented with molecular docking and machine learning techniques. The free energy values obtained both experimentally and theoretically showed excellent similarity. Calorimetry, UV-vis, and 3D/2D-lig-plot analysis revealed that the most relevant interactions between BTS and lysozyme are based on a predominance of aromatic, hydrophobic Van der Waals interactions, mainly aromatic edge-to-face (T-shaped) π-π stacking interactions between the benzene ring belonging to the 2-(methylthio)-benzothiazole moiety of BTS and the aromatic amino acid residue TRP108 of the lysozyme receptor. Next, conventional hydrogen bonding interactions contribute to the stability of the BTS-lysozyme coupling complex. In addition, mechanistic approaches performed using elastic network models revealed that the BTS ligand theoretically induces propagation of allosteric signals, suggesting non-physiological conformational flexing in large blocks of lysozyme affecting α-helices. Likewise, the BTS ligand interacts directly with allosteric residues, inducing perturbations in the conformational dynamics expressed as a moderate conformational softening in the α-helices H1, H2, and their corresponding β-loop in the lysozyme receptor, in contrast to the unbound state of lysozyme.     

基于异硫氰酸荧光素标记溶菌酶和荷正电纳米金构建荧光共振能量转移平台检测广谱细菌

通过异硫氰酸荧光素标记的溶菌酶(FITC-Lys)和聚乙烯亚胺修饰的荷正电纳米金粒子(PEI-AuNPs)与细菌结合,构建了一个基于荧光共振能量转移(FRET)的平台用于广谱细菌检测.待检样本中存在细菌时,荷正电的FITC-Lys(能量供体)通过与细胞壁肽聚糖的识别作用和静电作用与细菌结合,荷正电的PEI-AuNPs(...     

蛋清溶菌酶基因的密码子优化及其在毕赤酵母中的分泌表达

作为一种天然的抑菌物质,溶菌酶对抗生素耐药菌有较强的杀伤作用,具有无耐药性、无残留等特点,被认为在诸如动物饲料、药品等领域可有效代替抗生素,改善滥用抗生素所引起的一系列问题,市场前景巨大。通过微生物发酵大规模生产重组溶菌酶符合市场发展趋势,由于目前生产的菌株表达量偏低,限制了溶菌酶的应用和产业化发展。为提高蛋清溶菌酶的发酵表达量,根据毕赤酵母密码子的偏爱性,对其编码基因进行密码子优化,并构建了真核分泌表达载体pPIC9K-coEWL,经遗传霉素G418抗性筛选后,获得了一株酵母转化子(G-p-coEWL),其对G418的抗性浓度高达15 mg·mL^-1。在28℃、pH 6.0、转速240 r·min^-1和甲醇浓度1%的诱导条件下,酵母重组子G-p-coEWL经摇瓶培养72 h,实现了蛋清溶菌酶的分泌表达。在发酵上清液中,总蛋白浓度为 607 mg·L^-1,酶活达到677 U·mL^-1。此外,本实验还比较了葡聚糖凝胶柱Sephadex G-50和强酸性阳离子交换柱SP-Sepharose FF两种方法对目的蛋白的分离效果,得到 Sephadex G-50的分离效果更好,并在14.4 kDa处得到单一的溶菌酶条带。     

溶液热历史对蛋白质晶体生长影响的内在原因研究

从溶液中聚集体的角度研究了溶液的热历史改变生长出的蛋白质晶体的数目和尺寸的内在原因. 将在281 和309 K下保存1 d的两组溶菌酶溶液按不同比例混合, 加入沉淀剂生长晶体. 随着高温溶液的比例增加, 生长出的晶体数目减少, 同时溶液中生长基元的尺寸增大. 在5周内, 采用动态光散射对281, 293和309 K三种温度下保存的溶菌酶溶液中聚集体的变化情况进行监测, 发现溶液中均存在大小不同的两部分聚集体, 称之为小聚集体与多聚体. 前者的尺寸基本不随保存时间而变化, 而后者尺寸随保存时间增加而减小, 减小的速度与保存温度有关. 多聚体的尺寸经过5周后和小聚集体基本相同. 研究结果表明, 处于无序聚集阶段的溶液的均一化程度和成核阶段生长基元的尺寸受到了溶液热历史的影响, 并最终对晶体的数目产生影响.