果蝇非常规肌球蛋白的结构与功能

肌球蛋白是一类重要的分子马达,可以将ATP水解产生的能量转化成动能,沿由肌动蛋白组成的细丝运动。肌球蛋白构成一个大的基因家族,在许多细胞活动中起着重要作用,包括肌肉收缩、胞内转运、听觉、视觉等。果蝇基因组有13种肌球蛋白基因,包括2种常规肌球蛋白和11种非常规肌球蛋白。本文综述了近年来果蝇非常规肌球蛋白的研究进展。     

肌球蛋白动力冲程分布研究

通过建立肌球蛋白工作循环的四态模型,利用化学动力学方法,讨论了动力冲程过程的几率和无机磷酸盐的释放率是影响动力冲程分布的两个重要因素,得到动力冲程的大小近似呈均值为（8-10）nm的高斯分布。     

肌球蛋白工作循环的一个新模型

分析总结关于分子马达肌球蛋白的最新研究结果,给出一个新的肌球蛋白工作循环的机械化学偶联模型.从新模型出发,用一组化学动力学方程描述肌肉中大量肌球蛋白的集体工作行为.利用动力学方程的非平衡定态解,并结合Pate和Cooke的实验结果得到了力作为变量的肌肉态方程.理论结果同热力学原理一致,与传统的肌肉收缩理论有一定区别.根据肌肉的特殊结构,对肌肉态方程做了进一步讨论.     

肌球蛋白Ⅵ定向运动的蒙特卡罗模拟

根据myosin Ⅵ分子马达头部核苷的变化建立一个四态循环,然后利用蒙特卡罗方法模拟分子马达的动力学性质,讨论了myosin Ⅵ速度、滞留时间与溶液中ATP浓度、ADP浓度和负载力F的关系。模拟得到：myosin Ⅵ会在轨道微丝上进行梯跳运动并且每次在微丝上的滞留时间并不相同;另外,myosin Ⅵ的速度会随着ATP浓度的增加而增大,随着ADP浓度和负载力的增加而减小;负载力对myosin Ⅵ在胞内执行输运还是连接功能起一定的调节作用。     

平滑肌肌球蛋白轻链激酶N端删除载体的构建

目的:构建重组平滑肌肌球蛋白轻链激酶(myosin light chain kinase,MLCK)N端删除栽体,为研究平滑肌MLCK的分子机制提供研究模型.方法:以重组质粒pCoid/155为模板,根据其待删除序列(N端1-41个氨基酸)设计上下游引物,行PCR扩增.将扩增片段以NdeI/EeoRl双酶切,产物行琼脂糖凝胶电泳回收得到目的基因.将目的基因与栽体连接,转化至大肠杆菌.筛选阳性克隆,并对阳性克隆进行测序.结果:用NdeI和EcoRI双酶切重组质粒pCold/155,琼脂糖凝胶电泳显示得到约4.4kb栽体和约3.4kb的MLCK片段.阳性克隆经测序证实MLCK的N端41个氨基酸序列已被成功删除.结论:成功构建了重组MLCK N端删除栽体 pCold/155/D41.     

洋葱鳞茎中肌球蛋白和肌动蛋白的免疫化学鉴定

制备了粘菌肌动蛋白抗血清和鸡骨骼肌肌球蛋白抗血清。用这两种抗血清,经对流免疫电泳,火箭电泳及酶联免疫吸附分析,证明了高等植物洋葱鳞茎中肌球蛋白和肌动蛋白的存在。     

人心肌肌球蛋白轻链1与重链和肌动蛋白的结合

在测得中国人心肌肌球蛋白轻链 1cDNA的核苷酸序列 ,并获得一株单克隆抗体 (HCMLC1 8)的基础上 ,用PCR方法 ,以中国人心肌肌球蛋白轻链 1的cDNA为模板 ,分别获得中国人心肌肌球蛋白轻链 1的各为 98个氨基酸的N端和C端片段cDNA的克隆并进行了表达。同时进行了其表达产物和大鼠心肌肌球蛋白重链和人心肌肌动蛋白以及单克隆抗体结合的研究 ,发现三者均和轻链 1的N端相结合 ,结合位点各不相同。这些结合位点可能均位于轻链 1的分子表面 ,而且如果轻链 1在实验状态下先与肌动蛋白结合 ,则有可能影响轻链与重链间的彼此结合。肌动蛋白在体外能以不同位点结合肌球蛋白重链和轻链 ,可能在肌肉收缩过程中具有重要的生理意义     

肌肉收缩Hill特性式的理论研究

从肌球蛋白工作循环的机械化学偶联模型出发,利用化学动力学方法和生物化学热力学原理,结合肌球蛋白单分子实验结果,从能量转化的观点给出了肌肉收缩的Hill特性式,加深了对Hill特性式及肌肉收缩过程中能量转化的理解,在整合肌球蛋白单分子性质与肌肉收缩宏观性质的信息方面做了尝试。     

肌肉收缩Hill特性式的理论研究

从肌球蛋白工作循环的机械化学偶联模型出发,利用化学动力学方法和生化热力学原理,结合肌球蛋白单分子实验结果,从能量转化的角度,研究了肌肉收缩过程中的力与速度关系,发现结果与Hill特性式基本一致。     

肌肉力量的微观起源

人体实现机械运动所需的力量是由肌肉产生的。理解肌肉的力产生机理是目前科学研究的最前沿课题之一。肌肉的最基本单元是肌小节。肌小节是一个设计极为精巧的力学装置,其中负责产生力的是一种叫做肌球蛋白的分子马达。通过介绍肌小节和肌球蛋白的结构和工作原理,可以看到生命如何利用蛋白质分子巧妙地实现了复杂的力学功能。此外,对于肌球蛋白马达研究中有待理解的相关问题也进行了简要介绍。     

Does 2,3-butanedione monoxime inhibit nonmuscle myosin?

Summary. BDM (2,3-butanedione monoxime) has been used extensively to inhibit nonmuscle myosin. However, recent articles raise the question of what BDM actually does, because of experiments in which BDM did not affect the actin-activated ATPase of nonmuscle myosins. We describe results that indicate that BDM indeed inhibits motility due to nonmuscle myosins: in many different cells BDM has the same effects as anti-actin agents and/or as other anti-myosin agents, and BDM slows or stops the sliding between actin filaments and myosin in vitro. We discuss how the two sets of apparently contradictory results might be resolved, and we suggest possible experiments that might clarify the contradictory interpretations. Correspondence and reprints: Biology Department, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.     

Mouse Myosin-19 Is a Plus-end-directed,High-duty Ratio Molecular Motor

Class XIX myosin (Myo19) is a vertebrate-specific unconventional myosin, responsible for the transport of mitochondria. To characterize biochemical properties of Myo19, we prepared recombinant mouse Myo19-truncated constructs containing the motor domain and the IQ motifs using the baculovirus/Sf9 expression system. We identified regulatory light chain (RLC) of smooth muscle/non-muscle myosin-2 as the light chain of Myo19. The actin-activated ATPase activity and the actin-gliding velocity of Myo19-truncated constructs were about one-third and one-sixth as those of myosin-5a, respectively. The apparent affinity of Myo19 to actin was about the same as that of myosin-5a. The RLCs bound to Myo19 could be phosphorylated by myosin light chain kinase, but this phosphorylation had little effect on the actin-activated ATPase activity and the actin-gliding activity of Myo19-truncated constructs. Using dual fluorescence-labeled actin filaments, we determined that Myo19 is a plus-end-directed molecular motor. We found that, similar to that of the high-duty ratio myosin, such as myosin-5a, ADP release rate was comparable with the maximal actin-activated ATPase activity of Myo19, indicating that ADP release is a rate-limiting step for the ATPase cycle of acto-Myo19. ADP strongly inhibited the actin-activated ATPase activity and actin-gliding activity of Myo19-truncated constructs. Based on the above results, we concluded that Myo19 is a high-duty ratio molecular motor moving to the plus-end of the actin filament.     

Using Fluorescent Myosin to Directly Visualize Cooperative Activation of Thin Filaments

Contraction of striated muscle is tightly regulated by the release and sequestration of calcium within myocytes. At the molecular level, calcium modulates myosin''s access to the thin filament. Once bound, myosin is hypothesized to potentiate the binding of further myosins. Here, we directly image single molecules of myosin binding to and activating thin filaments. Using this approach, the cooperative binding of myosin along thin filaments has been quantified. We have found that two myosin heads are required to laterally activate a regulatory unit of thin filament. The regulatory unit is found to be capable of accommodating 11 additional myosins. Three thin filament activation states possessing differential myosin binding capacities are also visible. To describe this system, we have formulated a simple chemical kinetic model of cooperative activation that holds across a wide range of solution conditions. The stochastic nature of activation is strongly highlighted by data obtained in sub-optimal activation conditions where the generation of activation waves and their catastrophic collapse can be observed. This suggests that the thin filament has the potential to be turned fully on or off in a binary fashion.     

A Toxoplasma gondii Class XIV Myosin,Expressed in Sf9 Cells with a Parasite Co-chaperone,Requires Two Light Chains for Fast Motility

Many diverse myosin classes can be expressed using the baculovirus/Sf9 insect cell expression system, whereas others have been recalcitrant. We hypothesized that most myosins utilize Sf9 cell chaperones, but others require an organism-specific co-chaperone. TgMyoA, a class XIVa myosin from the parasite Toxoplasma gondii, is required for the parasite to efficiently move and invade host cells. The T. gondii genome contains one UCS family myosin co-chaperone (TgUNC). TgMyoA expressed in Sf9 cells was soluble and functional only if the heavy and light chain(s) were co-expressed with TgUNC. The tetratricopeptide repeat domain of TgUNC was not essential to obtain functional myosin, implying that there are other mechanisms to recruit Hsp90. Purified TgMyoA heavy chain complexed with its regulatory light chain (TgMLC1) moved actin in a motility assay at a speed of ∼1.5 μm/s. When a putative essential light chain (TgELC1) was also bound, TgMyoA moved actin at more than twice that speed (∼3.4 μm/s). This result implies that two light chains bind to and stabilize the lever arm, the domain that amplifies small motions at the active site into the larger motions that propel actin at fast speeds. Our results show that the TgMyoA domain structure is more similar to other myosins than previously appreciated and provide a molecular explanation for how it moves actin at fast speeds. The ability to express milligram quantities of a class XIV myosin in a heterologous system paves the way for detailed structure-function analysis of TgMyoA and identification of small molecule inhibitors.     

Recombinant motor domain constructs of Chara corallina myosin display fast motility and high ATPase activity

The mechanism and structural features that are responsible for the fast motility of Chara corallina myosin (CCM) have not been elucidated, so far. The low yields of native CCM that can be purified to homogeneity were the major reason for this. Here, we describe the expression of recombinant CCM motor domains, which support the fast movement of actin filaments in an in vitro motility assay. A CCM motor domain without light chain binding site moved actin filaments at a velocity of 8.8 microm/s at 30 degrees C and a CCM motor domain with an artificial lever arm consisting of two alpha-actinin repeats moved actin filaments at 16.2 microm/s. Both constructs displayed high actin-activated ATPase activities ( approximately 500 Pi/s/head), which is indicative of a very fast hydrolysis step. Our results provide an excellent system to dissect the specific structural and functional features that distinguish the myosin responsible for fast cytoplasmic streaming.     

Kinetic Characterization of Nonmuscle Myosin IIB at the Single Molecule Level

Nonmuscle myosin IIB (NMIIB) is a cytoplasmic myosin, which plays an important role in cell motility by maintaining cortical tension. It forms bipolar thick filaments with ∼14 myosin molecule dimers on each side of the bare zone. Our previous studies showed that the NMIIB is a moderately high duty ratio (∼20–25%) motor. The ADP release step (∼0.35 s⁻¹) of NMIIB is only ∼3 times faster than the rate-limiting phosphate release (0.13 ± 0.01 s⁻¹). The aim of this study was to relate the known in vitro kinetic parameters to the results of single molecule experiments and to compare the kinetic and mechanical properties of single- and double-headed myosin fragments and nonmuscle IIB thick filaments. Examination of the kinetics of NMIIB interaction with actin at the single molecule level was accomplished using total internal reflection fluorescence (TIRF) with fluorescence imaging with 1-nm accuracy (FIONA) and dual-beam optical trapping. At a physiological ATP concentration (1 mm), the rate of detachment of the single-headed and double-headed molecules was similar (∼0.4 s⁻¹). Using optical tweezers we found that the power stroke sizes of single- and double-headed heavy meromyosin (HMM) were each ∼6 nm. No signs of processive stepping at the single molecule level were observed in the case of NMIIB-HMM in optical tweezers or TIRF/in vitro motility experiments. In contrast, robust motility of individual fluorescently labeled thick filaments of full-length NMIIB was observed on actin filaments. Our results are in good agreement with the previous steady-state and transient kinetic studies and show that the individual nonprocessive nonmuscle myosin IIB molecules form a highly processive unit when polymerized into filaments.     

Bulkiness or aromatic nature of tyrosine-143 of actin is important for the weak binding between F-actin and myosin-ADP-phosphate

Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60 Å² to the solvent in F-actin. Because tyrosine-143 flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostelium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (V_max) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller K_app) than that of the wild-type actin, with the V_max being almost unchanged. The K_app and V_max of the Tyr143Phe-actin were similar to those of the wild-type actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of K_app was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas (ASA) of the replaced amino acid molecule. Because 1/K_app reflects the affinity of F-actin for the myosin–ADP-phosphate intermediate (M.ADP.Pi) through the weak binding, these data suggest that the bulkiness or the aromatic nature of the tyrosin-143 is important for the initial binding of the M.ADP.Pi intermediate with F-actin but not for later processes such as the phosphate release.     

Pollen tube extract supports the movement of actin filaments in vitro

Summary Muscle actin filaments labeled with rhodamine-phalloidin were observed to move on the surface coated with a crude extract of pollen tubes ofLilium longiflorum with an average velocity of 1.99±0.55 m/sec. The movement required both Mg²⁺ and ATP. These results indicate that the extract of pollen tubes contains a myosin-like translocatorAbbreviations ATP adenosine-5-triphosphate - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethylether)N,N,N,N-tetraacetic acid - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - PMSF phenylmethylsulfonyl fluoride