Conformations of the signal recognition particle protein Ffh from Escherichia coli as determined by FRET

The signal recognition particle (SRP) initiates the co-translational targeting of proteins to the plasma membrane in bacteria by binding to the N-terminal signal sequence emerging from the translating ribosome. SRP in Escherichia coli is composed of one protein, Ffh, and 4.5S RNA. In the present work, we probe the structure of Ffh alone and in the complex with 4.5S RNA by measuring distances between different positions within Ffh and between Ffh and 4.5S RNA by fluorescence resonance energy transfer (FRET). According to the FRET distances, NG and M domains in free Ffh are in close contact, as in the A/A arrangement in the crystal structure of Ffh from Thermus aquaticus, in agreement with the formation of a crosslink between cysteine residues at two critical positions in the G and M domains. Upon Ffh binding to 4.5S RNA or a 61 nucleotide fragment comprising internal loops A-C, the G and M domains move apart to assume a more open conformation, as indicated by changes of FRET distances. The movement is smaller when Ffh binds to a 49 nucleotide fragment of 4.5S RNA comprising only internal loops A and B, i.e. lacking the binding site of the NG domain. The FRET results suggest that in the SRP complex 4.5S RNA is present in a bent, rather than extended, conformation. The domain rearrangement of Ffh that takes place upon formation of the SRP is probably important for subsequent steps of membrane targeting, including interactions with the translating ribosome and the SRP receptor.     

FLIM on a wide field fluorescence microscope

Summary FLIM (Fluorescence Lifetime Imaging Microscopy) is a new tool to detect interaction between proteins. The proteins under investigation are fused with fluorescent donor and acceptor molecules. Interaction between the two proteins is accompanied by direct energy transfer from donor to acceptor (FRET), resulting in a shorter lifetime of the fluorescence emitted by the donor molecule. This change in lifetime is detected by FLIM. Fluorescence lifetime imaging can now be done on a widefield fluorescence microscope by using an attachment that is easy to install and simple to operate. The new LIFA attachment is equipped to use different excitation sources. High brightness modulated LEDs as well as lasers modulated by an Accousto Optical Modulator can be used as excitation light source. A modulated image intensifier with digital camera is used as a detector. Power supplies and signal generator are integrated in one control unit that is connected to the light source, detector and computer. All parameters for image acquisition, processing and viewing are easy accessible in the user interface of the software package that uses a modular structure. Lifetime images showing FRET in MCF7 cells with ErbB1-GFP as donor and Py72/Cy3 as acceptor that were taken at EMBL, Heidelberg are shown.     

Dissecting the binding mechanism of the linker histone in live cells: an integrated FRAP analysis

The linker histone H1 has a fundamental role in DNA compaction. Although models for H1 binding generally involve the H1 C‐terminal tail and sites S1 and S2 within the H1 globular domain, there is debate about the importance of these binding regions and almost nothing is known about how they work together. Using a novel fluorescence recovery after photobleaching (FRAP) procedure, we have measured the affinities of these regions individually, in pairs, and in the full molecule to demonstrate for the first time that binding among several combinations is cooperative in live cells. Our analysis reveals two preferred H1 binding pathways and we find evidence for a novel conformational change required by both. These results paint a complex, highly dynamic picture of H1–chromatin binding, with a significant fraction of H1 molecules only partially bound in metastable states that can be readily competed against. We anticipate the methods we have developed here will be broadly applicable, particularly for deciphering the binding kinetics of other nuclear proteins that, similar to H1, interact with and modify chromatin.     

3个不同品系小鼠ES细胞的绿色荧光蛋白标记

用电穿孔法将线性化的质粒pEGFP—N3分别导入来自129／ter、C57BL/6J和BALB／c3个品系的小鼠胚胎干细胞：MESPU—13、MESPU—35和MESPU—62中,经G418筛选、荧光显微镜镜检、阳性克隆扩增、流式细胞仪分选、再扩增以及核型分析等过程,分别得到核型大于85％的被EGFP稳定标记的细胞株5个(129／ter2个、C57BL/6J1个、BALB／c2个),分别命名为MESPU—13／G1和MESPU—13／G2、MESPU—35／G1、MESPU—62／G1和MESPU—62／G2。从不同品系中各选一个增殖生长快、形态典型的标记细胞株,进行碱性磷酸酶染色、oct4群基因产物的表达检测、类胚形成和体内分化鉴定,结果表明所得到的核型正常的、稳定标记的ES细胞系具有原ES细胞的典型特征。     

含RGD修饰的病毒样颗粒递送ICG靶向肿瘤的研究

目的:获取含RGD靶向肽的乙肝核心病毒样颗粒,为药物靶向纳米递送系统提供一种新型载体。方法:将实验室前期构建测序正确的含RGD修饰的乙肝核心病毒重组质粒转化入大肠杆菌BL21(DE3)中,单因素分析及正交试验探究重组蛋白最适表达条件。在最适表达条件下扩培,收集菌体超声破碎后离心,采用凝胶过滤层析、离子交换和蔗糖密度梯度离心进行纯化,利用透射电镜对形成的RGD-HBc VLPs的形态及稳定性进行鉴定。纯化的RGD-HBc VLPs利用其体外自组装的特性,将光敏剂ICG装载到颗粒的内部,通过静脉注射到4T1乳腺癌荷瘤小鼠,探究重组RGD-HBc VLPs作为纳米递送系统的靶向性。结果:RGD-HBc VLPs在温度32℃、IPTG0.5mmol/L、诱导4h时以可溶性蛋白的形式得到高效表达。经蔗糖密度梯度离心纯化后纯度到达95%以上。透射电镜下观察纯化的RGD-HBc VLPs形态、大小均一,直径约为32nm,通过近红外荧光活体成像证实了RGD-HBc作为纳米载体的靶向性。结论:经表达和纯化后,RGD-HBc VLPs具有较高的表达量和大小均一的形态外貌,近红外荧光活体成像证实具有较好的靶向性,这不仅为肿瘤的可视化诊断提供一种快速、精准、方便的方法,而且为今后靶向免疫治疗提供一种新型载体。