Palindromic assembly of the giant muscle protein titin in the sarcomeric Z-disk

The Z-disk of striated and cardiac muscle sarcomeres is one of the most densely packed cellular structures in eukaryotic cells. It provides the architectural framework for assembling and anchoring the largest known muscle filament systems by an extensive network of protein-protein interactions, requiring an extraordinary level of mechanical stability. Here we show, using X-ray crystallography, how the amino terminus of the longest filament component, the giant muscle protein titin, is assembled into an antiparallel (2:1) sandwich complex by the Z-disk ligand telethonin. The pseudosymmetric structure of telethonin mediates a unique palindromic arrangement of two titin filaments, a type of molecular assembly previously found only in protein-DNA complexes. We have confirmed its unique architecture in vivo by protein complementation assays, and in vitro by experiments using fluorescence resonance energy transfer. The model proposed may provide a molecular paradigm of how major sarcomeric filaments are crosslinked, anchored and aligned within complex cytoskeletal networks.     

医疗业务系统改造中的逆向工程方法

医院信息系统可以使用数据交换或者业务过程管理(BPM)的方式实现。它们的差别在于数据交换方式没有将业务逻辑与控制逻辑分离,而当业务过程改变时,需要更改代码适应。为充分利用这种已有的系统代码资源,将其升级、改造成BPM系统,该文分析了现有系统的实现方式,提出了一种基于过程挖掘的改造方法建立过程模型、提取组织知识以配置BPM系统。最后使用湖南省望城县计划生育服务站的站内系统改造的实例验证了该方法,并说明了具体的升级过程。相对于直接由人工进行升级的方法,该文中的方法更加方便快捷。     

散热器面罩边框零件的逆向设计和曲面展开

以散热器面罩边框零件的逆向设计为例,介绍设计中零件表面数据采集方法,研究了点云的数据处理、曲面划分等前处理的关键技术;提出在CATIA软件的逆向设计模块中有关特征曲线的几种创建方法,介绍曲线曲面的重构方法.以Dynaform为平台,总结出零件展料分析的一般步骤和方法,并对曲面展开的关键技术进行探讨.     

Insulin stimulates sugar transport in giant muscle fibres of the barnacle

Insulin stimulates sugar transport in vertebrate skeletal muscle but the mechanism of insulin action is unknown. It has been reported that Na transport in giant muscle fibers of the barnacle (Balanus nubilis) is sensitive to insulin but no one has examined the sensitivity of sugar tansport to insulin in this preparation. We show here that insulin does, indeed, stimulate sugar transport in barnacle muscle. The great advantage of barnacle muscle over all other muscles used so far for investigating the mechanism of insulin action is its large size, which facilitates measurements on single cells and permits the experimenter to control the intracellular environment of the muscle fibre by the technique of internal dialysis. Using single muscle fibres it is possible to show that acceleration of sugar transport by insulin is associated with a fall in ionized Ca, a fall in cyclic AMP and a rise in cyclic GMP. Working with internally dialysed muscle fibres we find that insulin only increases sugar transport when the dialysis solution contains ATP. In the absence of insulin, sugar transport is dialysed muscle is increased by a rise in ionized Ca, a fall in cyclic AMP and, when the internal Ca is elevated, by a rise in cyclic GMP.     

Hidden complexity in the mechanical properties of titin

Individual molecules of the giant protein titin span the A-bands and I-bands that make up striated muscle. The I-band region of titin is responsible for passive elasticity in such muscle, and contains tandem arrays of immunoglobulin domains. One such domain (I27) has been investigated extensively, using dynamic force spectroscopy and simulation. However, the relevance of these studies to the behaviour of the protein under physiological conditions was not established. Force studies reveal a lengthening of I27 without complete unfolding, forming a stable intermediate that has been suggested to be an important component of titin elasticity. To develop a more complete picture of the forced unfolding pathway, we use mutant titins--certain mutations allow the role of the partly unfolded intermediate to be investigated in more depth. Here we show that, under physiological forces, the partly unfolded intermediate does not contribute to mechanical strength. We also propose a unified forced unfolding model of all I27 analogues studied, and conclude that I27 can withstand higher forces in muscle than was predicted previously.     

Expression of a mammalian Na-H exchanger in muscle fibres of the giant barnacle

It is now well established that the internal pH (pHi) of mammalian cells is regulated by means of a plasma membrane transport system that exchanges extracellular Na+ for intracellular H+ (ref. 1). Furthermore, modulation of the activity of the Na-H exchanger seems to have a crucial role in the action of various mitogens and growth factors. The possibility that such a mammalian Na-H exchanger might be efficiently expressed in a giant invertebrate cell was suggested to us by recent results of Barnard and Miledi and colleagues, who demonstrated in frog oocytes the expression of various plasma membrane channels that presumably were encoded by the mammalian messenger RNA wih which the oocytes had been injected. We used muscle fibres of the giant barnacle, which normally have no demonstrable Na-H exchanger activity, and report here that, when injected with poly(A)+ RNA isolated from rabbit liver, the muscle fibres express a Na-H exchanger. No such expression is observed, however, when the injected material is pretreated with ribonuclease A. As hepatocytes are known to possess a Na-H exchanger, the most straightforward interpretation of our data is that a mammalian Na-H exchanger has been expressed in the muscle fibre of an invertebrate.     

Mapping the transition state and pathway of protein folding by protein engineering

In the transition state for unfolding of barnase, the hydrophobic core between the major alpha-helix and beta-sheet is somewhat weakened, the C terminus of the major helix is largely intact but its N terminus is exposed and a major loop has been invaded by solvent.     

Redesign of the coenzyme specificity of a dehydrogenase by protein engineering

Directed mutagenesis and molecular modelling have been used to identify a set of amino-acid side chains in glutathione reductase that confer specificity for the coenzyme NADP+. Systematic replacement of these amino acids, all of which occur in a 'fingerprint' structural motif in the NADP+-binding domain, leaves the substrate specificity unchanged but converts the enzyme into one displaying a marked preference for the coenzyme NAD+.     

Transient folding intermediates characterized by protein engineering

Kinetic experiments on engineered mutants of barnase detect an intermediate on the folding pathway and allow the mapping of the tertiary interactions of the side chains and their energetics. Many of the interactions present in the final folded state tend to be either fully formed or not formed at all in the intermediate or subsequent transition state for folding, but the hydrophobic core becomes progressively consolidated. These methods in combination with NMR provide extensive structural characterization of the folding intermediate and the sequence of events in the folding pathway.     

Prediction of electrostatic effects of engineering of protein charges

Accurate prediction of electrostatic effects on catalytic activity is an essential component of protein design. Site-directed mutagenesis of charged groups in subtilisin of Bacillus amyloliquefaciens has provided experimental measurements of electrostatic interactions which may be used to test such theoretical methods. The pKa of the histidine of the active site has been perturbed by +0.08 to -1.0 units by modifying one or two residues. Electrostatic effects in proteins can be modelled by the algorithm of Warwicker and Watson, which uses classical electrostatics and considers both the charge position and the shape of the molecule. Here we report that the algorithm can model several pKa shifts in subtilisin to fair accuracy.     

Reverse hydrophobic effects relieved by amino-acid substitutions at a protein surface

It is rare for amino-acid substitutions on the surface of proteins to have large stabilizing or destabilizing effects. Nevertheless, one substitution of this type, the Tyr 26----Cys mutation in lambda Cro, increases the melting temperature of the protein by 11 degrees C and the stability by 2.2 kcal mol-1. Here we show that the stability of Cro can be increased by many different amino-acid substitutions at position 26, with increasing stability showing a good correlation with decreasing side-chain hydrophobicity. As Tyr 26 is hyper-exposed to solvent in the Cro crystal structure, we suggest that wild-type and variant proteins with other hydrophobic side chains at position 26 are destabilized as a result of a reverse hydrophobic effect caused by the side chain being more exposed to solvent in the native than in the denatured state.     

包涵体复性研究

包涵体的形成是外源重组蛋白质在大肠杆菌中高效表达的必然结果，也是目前产生重组蛋白质最有效的方法之一、综述了国内外在外源重组蛋白复性研究方面的主要进展，包括包涵体的分离和溶解、外源重组蛋白复性的原则、影响复性的物理因素及各种复性方法．     

Molecular architecture and engineering of spider dragline silk protein

Spider dragline silk, which is produced in spider major ampullate gland, is a composite proteinacious fiber with highly repetitive Ala-Gly-rich domain. The unique combination of both high tensile strength and high elasticity makes spider dragline silk superior to almost any other natural or synthetic fibers. Cloning of the genes reveals that the silk is composed of at least two major proteins. Each protein component contains multiple repeats of modular structures that alternate between Ala-rich domains and Gly-rich domains. Molecular engineering not only opens a door to the production of spidroins but also provides a valuable experimental system to test and further establish the relationship between modular structures and mechanical properties. Here, based on our own studies, we review the latest progress of the modular structure and genetic engineering and outline the future prospects.     

Distal residues in the oxygen binding site of haemoglobin studied by protein engineering

The geometries of the Fe-O2 and Fe-CO bonds in myoglobin and haemoglobin differ significantly from those in free porphyrin model compounds. It has been suggested that steric hindrance by Val-E11 and His-E7 and a hydrogen bond between His-E7 and oxygen affect the geometry and electronic state of the Fe-ligand bond, and that these interactions may be important in controlling oxygen affinity. We have produced mutant haemoglobins in E. coli having Val(67 beta)E11 replaced by Ala, Met, Leu or Ile and His(58 beta)E7 by Gln, Val or Gly. We have studied the effect of these mutations on the equilibrium and kinetics of ligand binding. The conformation of the new side chains and their effect on the protein structure have been examined by X-ray crystallography, and the vibrational properties of the Fe-CO bond observed by resonance Raman spectroscopy. We found that the steric hindrance of ligand binding by the E11 residue and the polarity of the E7 residue in the beta subunit are critical for fine-tuning ligand affinity.     

Hydrogen bonding and biological specificity analysed by protein engineering

The role of complementary hydrogen bonding as a determinant of biological specificity has been examined by protein engineering of the tyrosyl-tRNA synthetase. Deletion of a side chain between enzyme and substrate to leave an unpaired, uncharged hydrogen-bond donor or acceptor weakens binding energy by only 0.5-1.5 kcal mol-1. But the presence of an unpaired and charged donor or acceptor weakens binding by a further approximately 3 kcal mol-1.