Dynamics of Bacteriorhodopsin in the Dark-Adapted State from Solution Nuclear Magnetic Resonance Spectroscopy

To achieve efficient proton pumping in the light-driven proton pump bacteriorhodopsin (bR), the protein must be tightly coupled to the retinal to rapidly convert retinal isomerization into protein structural rearrangements. Methyl group dynamics of bR embedded in lipid nanodiscs were determined in the dark-adapted state, and were found to be mostly well ordered at the cytosolic side. Methyl groups in the M145A mutant of bR, which displays only 10 % residual proton pumping activity, are less well ordered, suggesting a link between side-chain dynamics on the cytosolic side of the bR cavity and proton pumping activity. In addition, slow conformational exchange, attributed to low frequency motions of aromatic rings, was indirectly observed for residues on the extracellular side of the bR cavity. This may be related to reorganization of the water network. These observations provide a detailed picture of previously undescribed equilibrium dynamics on different time scales for ground-state bR.     

Preparation of Highly Stable and Photoluminescent Cadmium-Free InP/GaP/ZnS Core/Shell Quantum Dots and Application to Quantitative Immunoassay

Indium phosphide (InP) quantum dots (QDs) are ideal substitutes for widely used cadmium-based QDs and have great application prospects in biological fields due to their environmentally benign properties and human safety. However, the synthesis of InP core/shell QDs with biocompatibility, high quantum yield (QY), uniform particle size, and high stability is still a challenging subject. Herein, high quality (QY up to 72%) thick shell InP/GaP/ZnS core/shell QDs (12.8 ± 1.4 nm) are synthesized using multiple injections of shell precursor and extension of shell growth time, with GaP serving as the intermediate layer and 1-octanethiol acting as the new S source. The thick shell InP/GaP/ZnS core/shell QDs still keep high QY and photostability after transfer into water. InP/GaP/ZnS core/shell QDs as fluorescence labels to establish QD-based fluorescence-linked immunosorbent assay (QD-FLISA) for quantitative detection of C-reactive protein (CRP), and a calibration curve is established between fluorescence intensity and CRP concentrations (range: 1–800 ng mL⁻¹, correlation coefficient: R² = 0.9992). The limit of detection is 2.9 ng mL⁻¹, which increases twofold compared to previously reported cadmium-free QD-based immunoassays. Thus, InP/GaP/ZnS core/shell QDs as a great promise fluorescence labeling material, provide a new route for cadmium-free sensitive and specific immunoassays in biomedical fields.     

原子力显微镜在生物物理领域的新应用

介绍了原子力显微镜在生物物理领域的最新应用:蛋白质去折叠、DNA拉伸、生物膜受力,通过分析实验得到的力谱,可以获取关于蛋白质、DNA、生物膜结构信息.原子力显微镜不仅能反映测量体系的力学性质,由于其具有独特的时间、空间分辨及实时成像,因而能提供更多信息.     

与HBV X蛋白相互作用的细胞蛋白的筛选及其鉴定

参考GenBank核苷酸序列库中的HBV的核苷酸序列设计合成了一对对应于HBx基因的引物，从原发性肝癌(hepatocellular carcinoma，HCC)患者的血清中提取HBV基因组DNA作为模板，扩增HBx编码区并测定了核苷酸序列．将该编码区克隆到酵母双杂交系统的诱饵蛋白表达载体pGBKT7中，转入酵母细胞AH109，进行表型鉴定．然后通过Mating实验从已制备好用于酵母双杂交系统的肝cDNA表达文库中筛选与HBx相互作用的细胞蛋白，并用体外免疫共沉淀实验进一步验证．研究表明，分离得到的与HBx基因编码的蛋白相互作用的4种新的细胞蛋白，分别是醛缩酶B、C8α亚基、一种丝氨酸蛋白酶Hepsin和一种未知蛋白．     

Intrinsic Fluorescence of Actin

In this work actin is used to illustrate connection of protein fluorescence characteristics with its structure. On one hand, it has been demonstrated what kind of information about the contribution of each tryptophan residues to the bulk fluorescence spectrum can be obtained from the special analysis of protein three-dimensional structure. On the other hand, potentials of intrinsic fluorescence for elucidation of proteins structure, dynamics and processes of folding-unfolding are shown. In particular, using this method a new essentially unfolded kinetic intermediate state of actin was detected and characterized, and the place of inactivated actin and its kinetic predecessor in the process of folding-unfolding was determined. It has been revealed that inactivated actin is not intermediate state between the native and completely unfolded states, as it has been accepted before, but a result of protein misfolding. On the basis of the obtained data a new model of actin folding-unfolding pathway has been proposed.     

Electrophoretic study of the effect of ultradisperse iron powder on cotton seed proteins

The protein composition of seeds treated with a suspension of ultradisperse iron was studied using electrophoresis on PAAG. Changes were noted in the fractions with relative electrophoretic mobility (REM) 0.11 of water-soluble and REM 0.37 of buffer-soluble An-9 cotton seed proteins. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 384–385, July–August, 2006.     

Scanning probe microscopy characterization of gold-chemisorbed poplar plastocyanin mutants

Two poplar plastocyanin mutants adsorbed onto gold electrodes have been characterized at single molecule level by scanning probe microscopy. Immobilization of the two redox metalloprotein mutants on Au(1 1 1) surface was achieved by either a disulphide bridge (PCSS) or a single thiol (PCSH), both the anchoring groups having been introduced by site-directed mutagenesis. Scanning tunneling microscopy (STM) and atomic force microscopy (AFM) analysis gives evidence of a stable and robust binding of both mutants to gold. The lateral dimensions, as estimated by STM, and the height above the gold substrate, as evaluated by AFM, of the two mutants well agree with crystallographic sizes. A narrower height distribution is observed for PCSS compared to PCSH, corresponding to a more homogeneous orientation of the former mutant adsorbed onto gold. Major differences between the mutants are observed by electrochemical STM. In particular, the image contrast of adsorbed PCSS is affected by tuning the external electrochemical potential to the redox levels of the mutant, consistent with some involvement of copper active site in the tunneling process. On the contrary, no contrast variation is observed in electrochemical STM of adsorbed PCSH. Moreover, scanning tunneling spectroscopy experiments reveal asymmetric I–V characteristics for single PCSS proteins, reminiscent of a rectifying-like behaviour, whereas an almost symmetric I–V relation is observed for PCSH.     

Cationic poly(methacryl oxyethyl trimethylammonium) and its poly(ethylene glycol)‐grafted analogue as capillary coating materials in electrophoresis

Cationic polyelectrolytes were synthesized and used as semipermanent coating materials for capillaries in electrophoresis. The polyelectrolytes used were a homopolymer of poly(methacryl oxyethyl trimethylammonium chloride) (PMOTAC) and its poly(ethylene glycol) (PEG)‐grafted analogue. Two PMOTAC polyelectrolytes, with molar masses of 85,000 and 300,000 g/mol, and PEG‐grafted PMOTAC with a molar mass of 280,000 g/mol were synthesized and then characterized by size exclusion chromatography (SEC) and nuclear magnetic resonance (NMR) spectroscopy. Attachment of the polyelectrolytes to the wall of the fused silica capillary for electrophoresis caused the electroosmotic flow (EOF) to reverse. The polyelectrolyte coatings were tested over the pH range 2–11 at different buffer ionic strengths, and the most stable and strongest anodic EOFs were obtained at acidic pH values with low ionic strength buffers. Between runs the capillary is merely rinsed for 2 or 3 min with the background electrolyte solution. With the PMOTAC coatings at pH values ≤5, the RSDs of the EOFs were less than 2.9% after 60 injections. The effects of the molar mass of the polycation and of PEGylation of PMOTAC on the interactions between the polycations and basic proteins were studied at acidic pH values. The differences in the effective electrophoretic mobilities, resolution values, and plate numbers of the proteins with the different coatings were due to the EOF, as demonstrated through calculations of reduced mobilities, relative resolution values, and relative plate numbers. © 2007 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 45: 2655–2663, 2007