Movement of microtubules by single kinesin molecules

Kinesin is a motor protein that uses energy derived from ATP hydrolysis to move organelles along microtubules. Using a new technique for measuring the movement produced in vitro by individual kinesin molecules, it is shown that a single kinesin molecule can move a microtubule for several micrometers. New information about the mechanism of force generation by kinesin is presented.     

Single kinesin molecules studied with a molecular force clamp.

Kinesin is a two-headed, ATP-driven motor protein that moves processively along microtubules in discrete steps of 8 nm, probably by advancing each of its heads alternately in sequence. Molecular details of how the chemical energy stored in ATP is coupled to mechanical displacement remain obscure. To shed light on this question, a force clamp was constructed, based on a feedback-driven optical trap capable of maintaining constant loads on single kinesin motors. The instrument provides unprecedented resolution of molecular motion and permits mechanochemical studies under controlled external loads. Analysis of records of kinesin motion under variable ATP concentrations and loads revealed several new features. First, kinesin stepping appears to be tightly coupled to ATP hydrolysis over a wide range of forces, with a single hydrolysis per 8-nm mechanical advance. Second, the kinesin stall force depends on the ATP concentration. Third, increased loads reduce the maximum velocity as expected, but also raise the apparent Michaelis-Menten constant. The kinesin cycle therefore contains at least one load-dependent transition affecting the rate at which ATP molecules bind and subsequently commit to hydrolysis. It is likely that at least one other load-dependent rate exists, affecting turnover number. Together, these findings will necessitate revisions to our understanding of how kinesin motors function.     

单光刀与单光镊激光微束系统

提出了构建一种用于细胞非接触操作的激光微束系统。该系统由 Nd:YAG激光束经声压、热膨胀、汽化等综合效应实现的光刀和 He- Ne激光束经光学动力学效应实现的光镊组成。将两激光束耦合到显微镜中 ,实现了生物细胞的捕获、移动、翻转、打孔等一系列操作。在此基础上 ,分析了形成光镊所必需产生梯度力场的条件和形成光刀对能量的要求 ,进行了系统的总体设计、关键部件设计和选择 ,构建了一套激光微束操作实验系统 ,得到了预期的试验结果。在该系统上成功地实现了非接触细胞操作 ,并对染色体进行了切割。     

Making light work with optical tweezers.

Microscopic objects, including biological material, can be remotely manipulated with tightly focused beams of infrared laser light. The use of optical traps, or 'optical tweezers', holds great promise for noninvasive micromanipulation and mechanical measurement in cell biology. Optical tweezers are the 'tractor beams' of today's technology.     

Compliance of bacterial flagella measured with optical tweezers

The development of the gradient force optical particle trap ('optical tweezers') has made it possible to manipulate biological materials using a single beam of laser light. Optical traps can produce forces in the microdyne range on intact cells without causing overt damage: such forces are sufficient to arrest actively swimming bacteria and can overcome torque generated by the flagellar motor of a bacterium tethered to a glass surface by a flagellar filament. By calibrating the trapping force against Stokes' drag and measuring the twist that is sustained by this force, we determined the torsional compliance of flagella in tethered Escherichia coli and a motile Streptococcus. Flagella behaved as linear torsion springs for roughly half a revolution, but became much more rigid when turned beyond this point in either direction.     

Tying a molecular knot with optical tweezers.

Filamentous structures are abundant in cells. Relatively rigid filaments, such as microtubules and actin, serve as intracellular scaffolds that support movement and force, and their mechanical properties are crucial to their function in the cell. Some aspects of the behaviour of DNA, meanwhile, depend critically on its flexibility-for example, DNA-binding proteins can induce sharp bends in the helix. The mechanical characterization of such filaments has generally been conducted without controlling the filament shape, by the observation of thermal motions or of the response to external forces or flows. Controlled buckling of a microtubule has been reported, but the analysis of the buckled shape was complicated. Here we report the continuous control of the radius of curvature of a molecular strand by tying a knot in it, using optical tweezers to manipulate the strand's ends. We find that actin filaments break at the knot when the knot diameter falls below 0.4 microm. The pulling force at breakage is around 1 pN, two orders of magnitude smaller than the tensile stress of a straight filament. The flexural rigidity of the filament remained unchanged down to this diameter. We have also knotted a single DNA molecule, opening up the possibility of studying curvature-dependent interactions with associated proteins. We find that the knotted DNA is stronger than actin.     

鹅去氧胆酸分子钳对中性分子的识别性能研究

利用差紫外光谱滴定法考察了鹅去氧胆酸分子钳1～5对苯胺、对硝基苯胺、对甲氧基苯胺等中性分子的识别性能,测定了主客体间的结合常数(ka)和自由能变化(ΔG°).结果表明,主体对所考察的客体分子显示良好的识别作用,主客体间形成1∶1型超分子配合物,最大结合常数可达1292.51 L.mol-1,识别作用的主要推动力为氢键,范德华力等的协同作用.讨论了主客体间形状、大小匹配和几何互补等因素,对形成超分子配合物的影响,并利用计算机分子模拟作为辅助手段对实验结果与现象进行了解释.     

手性不对称脲联苯二甲酸分子钳对中性分子的识别性能研究

用差紫外光谱滴定法考察了新型分子钳1～5对苯胺、对甲氧基苯胺、对氨基苯乙酮等中性分子的识别性能,测定了主客体问的结合常数(kg)和自由能变化(△G°).结果 表明,分子钳主体对所考察的客体分子显示良好的识别作用,主客体间形成11型超分子配合物,最大结合常数可达5119.23 L·mol-1,识别作用的主要推动力为氢键,π-π重叠等的协同作用.讨论了主体与客体间形状、大小匹配和几何互补等因素,对形成超分子配合物的影响,并利用计算机分子模拟作为辅助手段对实验结果与现象进行了解释.     

Highly coupled ATP synthesis by F1-ATPase single molecules

F1-ATPase is the smallest known rotary motor, and it rotates in an anticlockwise direction as it hydrolyses ATP. Single-molecule experiments point towards three catalytic events per turn, in agreement with the molecular structure of the complex. The physiological function of F1 is ATP synthesis. In the ubiquitous F0F1 complex, this energetically uphill reaction is driven by F0, the partner motor of F1, which forces the backward (clockwise) rotation of F1, leading to ATP synthesis. Here, we have devised an experiment combining single-molecule manipulation and microfabrication techniques to measure the yield of this mechanochemical transformation. Single F1 molecules were enclosed in femtolitre-sized hermetic chambers and rotated in a clockwise direction using magnetic tweezers. When the magnetic field was switched off, the F1 molecule underwent anticlockwise rotation at a speed proportional to the amount of synthesized ATP. At 10 Hz, the mechanochemical coupling efficiency was low for the alpha3beta3gamma subcomplex (F1-epsilon)), but reached up to 77% after reconstitution with the epsilon-subunit (F1+epsilon)). We provide here direct evidence that F1 is designed to tightly couple its catalytic reactions with the mechanical rotation. Our results suggest that the epsilon-subunit has an essential function during ATP synthesis.     

Two-electron dissociation of single molecules by atomic manipulation at room temperature

Using the tip of a scanning tunnelling microscope (STM) to mechanically manipulate individual atoms and molecules on a surface is now a well established procedure. Similarly, selective vibrational excitation of adsorbed molecules with an STM tip to induce motion or dissociation has been widely demonstrated. Such experiments are usually performed on weakly bound atoms that need to be stabilized by operating at cryogenic temperatures. Analogous experiments at room temperature are more difficult, because they require relatively strongly bound species that are not perturbed by random thermal fluctuations. But manipulation can still be achieved through electronic excitation of the atom or molecule by the electron current tunnelling between STM tip and surface at relatively high bias voltages, typically 1-5 V. Here we use this approach to selectively dissociate chlorine atoms from individual oriented chlorobenzene molecules adsorbed on a Si(111)-7 x 7 surface. We map out the final destination of the chlorine daughter atoms, finding that their radial and angular distributions depend on the tunnelling current and hence excitation rate. In our system, one tunnelling electron has nominally sufficient energy to induce dissociation, yet the process requires two electrons. We explain these observations by a two-electron mechanism that couples vibrational excitation and dissociative electron attachment steps.     

Chi-sequence recognition and DNA translocation by single RecBCD helicase/nuclease molecules

Major pathways of recombinational DNA repair in Escherichia coli require the RecBCD protein--a heterotrimeric, ATP-driven, DNA translocating motor enzyme. RecBCD combines a highly processive and exceptionally fast helicase (DNA-unwinding) activity with a strand-specific nuclease (DNA-cleaving) activity (refs 1, 2 and references therein). Recognition of the DNA sequence 'chi' (5'-GCTGGTGG-3') switches the polarity of DNA cleavage and stimulates recombination at nearby sequences in vivo. Here we attach microscopic polystyrene beads to biotin-tagged RecD protein subunits and use tethered-particle light microscopy to observe translocation of single RecBCD molecules (with a precision of up to approximately 30 nm at 2 Hz) and to examine the mechanism by which chi modifies enzyme activity. Observed translocation is unidirectional, with each molecule moving at a constant velocity corresponding to the population-average DNA unwinding rate. These observations place strong constraints on possible movement mechanisms. Bead release at chi is negligible, showing that the activity modification at chi does not require ejection of the RecD subunit from the enzyme as previously proposed; modification may occur through an unusual, pure conformational switch mechanism.     

紫外可见光谱法研究染料大分子的非线性光学特性

用紫外可见光谱研究了萘基及联苯基氮型染料大分子的三阶非线性光学超极化率。结果表明：对共长度不同的染料分子，γ与Ｎ关系符合幂律，当Ｎ从６增至１６时，标度指数ａ从６趋于宏观值１；这类分子的饱和共轭长度，     

Conformations and adsorption behavior of poly（allylamine hydrochloride） studied by single molecule force spectroscopy

Poly（allylamine hydrochloride） （PAH）, which is frequently used in fabricating polyelectrolyte multilayer films, was studied by single molecule force spectroscopy （SMFS）. Plenty of force-extension curves with a long plateau were obtained in water, indicating that train-like structure was predominant when PAH was adsorbed on the substrate. It was found that the peak-type force-extension curves of PAH in water were not able to be fitted by the modified freely-jointed chain model. Additionally, there was a flat region in the derivative of force-extension curves. Thus, it was inferred that PAH chain in water was in a special conformation and underwent a ＂conformational transition＂ under the stretching of an external force. This phenomenon did not appear in the SMFS experiment in 1 mol/L urea solution, which indicated that urea was able to break the special conformation.     

光学法测量单根纤维接触角的影响因素

采用光学法测量了不同材质单根纤维的接触角,并对测量单根纤维接触角的影响因素进行分析。结果表明:不同的纤维制备方法制取的纤维接触角不同,其中,硝酸-氯酸钾离析的纤维接触角与次氯酸钠和过氧化氢-冰醋酸离析的纤维接触角差异比较显著。同一处理方式得到的不同纤维的接触角不同,苎麻纤维接触角最小。对于单根天然植物纤维,环境的温、湿度都会影响其接触角,湿度的影响更明显;对于单根再生纤维素纤维,温度对接触角几乎没有影响,湿度的影响较大;对于单根化学合成纤维,环境的温、湿度对其影响较小,湿度几乎没有影响。     

1,3取代方酸分子非线性光学性质的有限场方法研究

为了进一步研究方酸及其衍生物的非线性光学性质 ,设计了给电子和吸电子基团取代的 1,3位取代方酸小分子 ,用FF AM1方法对其非线性光学性质进行了计算 .结果表明 ,与典型D -π -A结构的分子对硝基苯胺 (PNA)相比 ,多数方酸衍生物具有更强的光学非线性 .     

基于第一性原理的KMgF_3晶体电子结构和光学性质研究

应用第一性原理研究了完整KMgF3晶体的态密度、介电函数、吸收光谱、复折射率和复数光电导谱.结果表明,介电函数虚部、吸收光谱、复折射率和复数光电导谱的峰值位置与KMgF3晶体的电子结构存在一一对应关系,它们都与KMgF3晶体的的电子结构有关.计算结果与实验结果相吻合.     

单透射峰Faraday反常色散原子滤光器中检偏器角度的影响

本文研究了工作在Rb原子D2线（5S1/2→5P3/2）780nm单透射峰的法拉第反常色散原子滤光器（Faraday Anomalous Dispersion Optical Filter,FADOF）中检偏器角度变化对单透射峰的影响,在室温时,发现检偏器角度每增加1°,峰值透射率平均增加0.21%,从相对值来说,从90°-89°时,等效透射率增加一倍.同时测量了检偏器角度变化对传统多透射峰FADOF的影响,以及铷泡温度、长度变化对透射率的影响.