肌红蛋白力致去折叠的全原子分析

位于脊椎动物骨骼肌中的肌红蛋白在生命过程中扮演着重要角色，其折叠过程依赖于血红素的绑定.本文着重于肌红蛋白在力的作用下去折叠过程的全原子统计分析.结果表明血红素不仅具有生物学上的功能，而且决定其去折叠的动力学过程.在血红素不存在的情况下，肌红蛋白的力致去折叠过程存在一个中间态，其构象不同于化学变性剂所导致的去折叠中间态，发现新的中间态.结论与相关实验结果相符，揭示了肌红蛋白力致去折叠的一般机制.     

肌红蛋白力致去折叠的全原子分析（英文）

位于脊椎动物骨骼肌中的肌红蛋白在生命过程中扮演着重要角色,其折叠过程依赖于血红素的绑定.本文着重于肌红蛋白在力的作用下去折叠过程的全原子统计分析.结果表明血红素不仅具有生物学上的功能,而且决定其去折叠的动力学过程.在血红素不存在的情况下,肌红蛋白的力致去折叠过程存在一个中间态,其构象不同于化学变性剂所导致的去折叠中间态,发现新的中间态.结论与相关实验结果相符,揭示了肌红蛋白力致去折叠的一般机制.     

肌动蛋白纤维的组装动力学

以细胞骨架中肌动蛋白纤维为例,针对肌动蛋白亚基结合核苷酸状态简化为单态和双态两种模型,介绍了肌动蛋白纤维的组装动力学,并着重阐述了肌动蛋白纤维稳态时出现的"踏车现象"和长度扩散行为.     

基于光谱法研究盐酸胍诱导的人血红蛋白去折叠过程

借助于紫外-可见吸收光谱法、荧光光谱法以及停流-荧光光谱法研究了盐酸胍(GdmHcl)诱导人血红蛋白的去折叠过程。实验发现,盐酸胍诱导的血红蛋白去折叠有两个不同的过程,即随着GdmHcl浓度增加到1.0 mol·L-1左右时,血红蛋白亚基发生解聚,形成中间态;持续增加其浓度时,各亚基发生内部去折叠,最终导致血红素发生崩解。加入还原剂(β-巯基乙醇)对血红蛋白亚基解聚、血红素崩解有协同作用且直接引起亚基和全分子同步变构。血红蛋白去折叠过程从“三态模型”转变为“二态模型”。     

新天花粉蛋白的盐酸胍去折叠过程

本文利用荧光光谱和园二色光谱研究了新天花粉蛋白的盐酸胍去折叠过程.结果显示:新天花粉蛋白的盐酸胍去折叠是一个只包含天然蛋白和变性终态的二态过程,与已经报道的天花粉蛋白的盐酸胍去折叠过程不同.     

弹性蛋白力学特性的单分子力谱

弹性蛋白是一类有着特殊力学特性的蛋白.在生物体内它们是承受和传递力的主要媒介;在生物体外,它们更是被广泛地用作高强度的生物材料.根据其功能不同,弹性蛋白的力学特性也各异.有些具有比较高的力学强度,有些则具有较大的延展性和弹性.科学家们很早就采用多种手段来人工合成弹性蛋白用于材料和纳米领域,但对于弹性蛋白的力学特性和序列结构之间的关系还不甚明晰.本综述介绍通过单分子力谱的实验方法来直接表征单根蛋白质在受力下结构的变化,研究其力学特性.基于Bell模型,推导出了蛋白质解折所需力与拉伸速率之间的关系,揭示了蛋白质力学强度的动力学特性,当拉伸速率较低时,解折叠力将正比于拉伸速率,对于较大的拉伸速率,解折叠力与拉伸速率成指数关系;探讨了决定蛋白质力学特性的结构因素和调控蛋白质力学特性的实验方法;介绍了单分子力谱测量的实验方法,包括基于光镊、磁镊和原子力显微镜的单分子力谱技术,着重介绍了原子力显微镜单分子力谱,并特别介绍了多聚蛋白技术来提供单分子测量的"指纹谱"和提高测量效率;论述了基于原子力显微镜单分子力谱研究蛋白质力学特性的最新进展,包括提高原子力显微镜的稳定性和力分辨率的方法,与荧光标记法相结合来提高实验效率的技术和高速扫描原子力显微镜;阐述了如何通过单分子力谱实验来理性设计蛋白质材料的力学特性,并对未来的研究热点做了展望.     

光镊与差动共焦显示术整合系统在蛋白质弹性研究中的应用

由于光镊对被操纵物的轴向位移检测技术尚不成熟,因此引入差动共焦显微术与光镊相结合,并以肌凝蛋白与肌动蛋白为样本,测量蛋白质对微球在缓冲液中自然扰动的影响.结果表明,微球在蛋白质环境中有较大的扰动现象,但此扰动在肌凝蛋白-肌动蛋白的结合影响下随轴向抬升而明显降低.此外,还即时检测到肌凝蛋白-肌动蛋白的结合会延迟微球的轴向运动的特性.本系统的动态测量范围可达3μm以上,结合其纳米解析度,适合应用于蛋白质的弹性研究中.     

全内反射瞬逝场照明高精度磁镊及其在DNA解旋酶研究中的应用

传统磁镊的测量精度受限于磁球的布朗涨落, 当磁力小于约10 pN时, 磁球的布朗涨落明显增大, 对应磁镊的空间分辨率显著下降. 为了提高传统磁镊在小力条件下的测量精度, 本文将全内反射荧光技术引入到磁镊技术中, 并建立相适应的“磁球-手柄-荧光微球-待测生物分子”单分子连接系统, 在小力条件下(小于10 pN)获得纳米量级的测量精度. 应用改进的磁镊对DNA发卡的折叠-去折叠态的转变过程进行了研究, 依据DNA发卡的折叠-去折叠态转变的性质对全内反射场的穿透深度进行了校正, 并结合实验结果对改进后的磁镊的测量精度进行分析. 观察了Bloom解旋酶的解旋动力学过程, 获得初步实验结果, 证实了改进的磁镊在单分子研究中的实用性. 关键词： 磁镊 全内反射荧光 DNA发卡 解旋酶     

非晶丝端部磁场效应的模拟

实验表明,不同长度的非晶丝以及非晶丝端部不同的位置都具有不同的磁特性.为了研究这种端部磁场效应,基于磁荷分布的假设,采用数值计算方法获得了非晶丝端部的磁场分布.然后,基于数值计算结果,采用拟合方法获得非晶丝内部磁场分布的一般性计算形式.由于非晶丝内部磁场强度不可能超出外磁场强度,提出由中间的均匀磁区以及两端的入磁区和出磁区所构成的非晶丝三磁区模型,并由此获得端部磁场效应的临界长度计算公式.该理论模型对端部磁场效应的模拟计算结果与已有实验现象能够符合很好.     

光镊与差动共焦显示术整合系统在蛋白质弹性研究中的应用

由于光镊对被操纵物的轴向位移检测技术发展并未不成熟,因此引入差动共焦显微术与光镊相结合,并以肌凝蛋白与肌动蛋白为样本,测量蛋白质对微球在缓冲液中自然扰动的影响。结果表明,微球在蛋白质环境中有较大的扰动现象,但此扰动在肌凝蛋白-肌动蛋白的结合影响下随轴向抬升而明显降低。此外,还即时检测到肌凝蛋白-肌动蛋白的结合会延迟微球的轴向运动的特性。本系统的动态测量范围可达3μm以上,结合其纳米解析度,适合应用于蛋白质的弹性研究中。     

Growth of attached actin filaments

In several studies of actin-based cellular motility, the barbed ends of actin filaments have been observed to be attached to moving obstacles. Filament growth in the presence of such filament-obstacle interactions is studied via Brownian dynamics simulations of a three-dimensional energy-based model. We find that with a binding energy greater than 24k _B T and a highly directional force field, a single actin filament is able to push a small obstacle for over a second at a speed of half of the free filament elongation rate. These results are consistent with experimental observations of plastic beads in cell extracts. Calculations of an external force acting on a single-filament-pushed obstacle show that for typical in vitro free-actin concentrations, a 3pN pulling force maximizes the obstacle speed, while a 4pN pushing force almost stops the obstacle. Extension of the model to treat beads propelled by many filaments suggests that most of the propulsive force could be generated by attached filaments.     

Buckling of actin-coated membranes under application of a local force

The mechanical properties of composite membranes obtained by self-assembly of actin filaments with giant fluid vesicles are studied by micromanipulation with optical tweezers. These complexes exhibit typical mechanical features of a solid shell, including a finite in-plane shear elastic modulus ( approximately 10(-6) N/m). A buckling instability is observed when a localized force of the order of 0.5 pN is applied perpendicular to the membrane plane. Although predicted for polymerized vesicles, this is the first evidence of such an instability.     

Ultrasound enhanced the binding ability of chitinase onto chitin: From an AFM insight

In order to evaluate the effect of ultrasound to chitinase from a molecular level, atomic force microscopy (AFM) was employed to investigate the interaction force of chitinase binding onto chitin surface. In the measurement of force-distance curve, a series of pull-off events were discovered using the immobilized AFM tips with chitinase either treated by ultrasound or not, whereas no interaction peak was observed by the AFM tips without chitinase, indicating that the obtained adhesion forces were coming from the binding functions between chitinase and chitin. Through the analysis of these force curves, at the loading velocity of 0.3 μm/s, the maximum binding force of the chitinase treated by ultrasound for 20 min onto chitin was measured to be 105.33 ± 23.51 pN, while the untreated onto chitin was 71.05 ± 12.73 pN, suggesting the stronger binding force between ultrasonic treated chitinase and chitin substrate. Therefore, AFM has provided a useful method to directly and quantitatively characterize the interactions between chitinase and chitin, and successfully proved that ultrasound could activate chitinase by enhancing the binding ability of chitinase onto chitin.     

Mechanism of the calcium-regulation of muscle contraction — In pursuit of its structural basis —

The author reviewed the research that led to establish the structural basis for the mechanism of the calcium-regulation of the contraction of striated muscles. The target of calcium ions is troponin on the thin filaments, of which the main component is the double-stranded helix of actin. A model of thin filament was generated by adding tropomyosin and troponin. During the process to provide the structural evidence for the model, the troponin arm was found to protrude from the calcium-depleted troponin and binds to the carboxyl-terminal region of actin. As a result, the carboxyl-terminal region of tropomyosin shifts and covers the myosin-binding sites of actin to block the binding of myosin. At higher calcium concentrations, the troponin arm changes its partner from actin to the main body of calcium-loaded troponin. Then, tropomyosin shifts back to the position near the grooves of actin double helix, and the myosin-binding sites of actin becomes available to myosin resulting in force generation through actin-myosin interactions.     

应用分子梳技术对DNA与组蛋白相互作用的研究

利用分子梳技术对λ DNA和组蛋白的相互作用进行了研究. 通过这种简单有效的方法，我们将λ DNA分子拉伸到26—28 μm，相当于其原长（约162 μm）的16—17倍. 当组蛋白与DNA结合后，DNA分子发生凝聚现象，复合体的拉伸长度明显变短，其峰值分布在10—14 μm之间. DNA 组蛋白复合体的拉伸长度与组蛋白的浓度、与碱基对和荧光染料的比例有显著的关系. 关键词： 分子梳 组蛋白 DNA 荧光显微     

Measurement of interaction force between RGD-peptide and Hela cell surface by optical tweezers

Since RGD peptides (R: arginine; G: glycine; D: aspartic acid) are found to promote cell adhesion, they are modified at numerous materials surface for medical applications such as drug delivery and regenerative medicine. Peptide-cell surface interactions play a key role in the above applications. In this letter, we study the adhesion force between the RGD-coated bead and Hela cell surface by optical tweezes. The adhesion is dominated by the binding of α5β1 and RGD-peptide with higher adhesion probability and stronger adhesion strength compared with the adhesion of bare bead and cell surface. The binding force for a single α5β1 -GRGDSP pair is determined to be 16.8 pN at a loading rate of 1.5 nN/s. The unstressed off-rate is 1.65 × 10-2s-1 and the distance of transition state for the rigid binding model is 3.0 nm.     

Collapse of spherical polyelectrolyte brushes in the presence of multivalent counterions

We consider the interaction of multivalent counterions with spherical polyelectrolyte brushes (SPB). The SPB result if linear polyelectrolyte (PE) chains (contour length: 60 nm) are densely grafted to colloidal spheres of 116 nm in diameter. Dispersed in water, the surface layer consisting of chains of the strong PE poly(styrene sulfonic acid) (PSS) will swell. We demonstrate that successive addition of trivalent ions (La3+) leads to a collapse in which the surface layer is shrinking drastically. All findings are discussed on the base of a theoretical mean-field approach using the Donnan equilibrium. The ion exchange and a strong binding of trivalent ions by PE chains is followed up by a drop in the osmotic pressure inside the brush. This reduction is the driving force for the collapse. The strong ion-chain correlation is discussed with results obtained from molecular dynamics simulations.     

Mapping the phase diagram of single DNA molecule force-induced melting in the presence of ethidium

When a single DNA molecule is stretched beyond its normal contour length, a force-induced melting transition is observed. Ethidium binding increases the DNA contour length, decreases the elongation upon melting, and increases the DNA melting force in a manner that is consistent with the ethidium-induced changes in duplex DNA stability known from thermal melting studies. The DNA stretching curves map out a phase diagram and critical point in the force-extension-ethidium concentration space. Intercalation occurs between alternate base pairs at low forces and between every base pair at high forces.     

Measurement of the evanescent field using noncontact mode atomic force microscope

Using the noncontact mode atomic force microscope (AFM) with frequency modulation detection method, force gradient acting on the AFM tip induced by the evanescent field was measured in a high vacuum. Exponential distance dependence of the force gradient by the evanescent field was successfully measured for the first time. Decay lengths of the force gradient were estimated to be 40±3 nm and 43±3 nm for Ar and He-Ne lasers, respectively, and independent of wavelength within the experimental error. The minimum detectable force was estimated to be about 0.1 pN. There was a tendency for the measured decay length to become shorter at a distance less than z=10 nm in many cases. The force gradient induced by the evanescent field inp-polarization was larger than that ins-polarization. This paper was originally presented at the first Asia-Pacific Workshop on Near Field Optics, which was held on August 17 and 18, 1996 at Seoul Education and Culture Center, Seoul, Korea, organized by the Condensed Matter Research Institute, Seoul National University.