Mechanical unfolding intermediates in titin modules

The modular protein titin, which is responsible for the passive elasticity of muscle, is subjected to stretching forces. Previous work on the experimental elongation of single titin molecules has suggested that force causes consecutive unfolding of each domain in an all-or-none fashion. To avoid problems associated with the heterogeneity of the modular, naturally occurring titin, we engineered single proteins to have multiple copies of single immunoglobulin domains of human cardiac titin. Here we report the elongation of these molecules using the atomic force microscope. We find an abrupt extension of each domain by approximately 7 A before the first unfolding event. This fast initial extension before a full unfolding event produces a reversible 'unfolding intermediate' Steered molecular dynamics simulations show that the rupture of a pair of hydrogen bonds near the amino terminus of the protein domain causes an extension of about 6 A, which is in good agreement with our observations. Disruption of these hydrogen bonds by site-directed mutagenesis eliminates the unfolding intermediate. The unfolding intermediate extends titin domains by approximately 15% of their slack length, and is therefore likely to be an important previously unrecognized component of titin elasticity.     

Reverse engineering of the giant muscle protein titin

Through the study of single molecules it has become possible to explain the function of many of the complex molecular assemblies found in cells. The protein titin provides muscle with its passive elasticity. Each titin molecule extends over half a sarcomere, and its extensibility has been studied both in situ and at the level of single molecules. These studies suggested that titin is not a simple entropic spring but has a complex structure-dependent elasticity. Here we use protein engineering and single-molecule atomic force microscopy to examine the mechanical components that form the elastic region of human cardiac titin. We show that when these mechanical elements are combined, they explain the macroscopic behaviour of titin in intact muscle. Our studies show the functional reconstitution of a protein from the sum of its parts.     

从高分子构象与动力学到蛋白质折叠

针对作用在聚合物刷上的键拉力研究表明作用在接枝基面上的力随着聚合物刷接枝密度的增大反而减小,然而尾端单体上的拉伸张力并没有消失.高分子的构象和动力学转变决定了其物性和多种多样的应用,而生物大分子蛋白质作为由二十种不同属性的氨基酸构成的序列,更是具有由其序列所决定的特别的三维自然结构.本文就聚合物刷、聚合物纳米复合材料、聚合物网络等几种高分子体系的构象与动力学过程,及蛋白质构象和其折叠与去折叠的动力学过程做了介绍.特别是蛋白质的折叠与去折叠速率在单分子操纵实验中受到拉力的调控,通过测量这种拉力依赖的动力学过程、蛋白质的自由能曲面和折叠去折叠路径可以得到系统全面的研究.本文以肌肉蛋白titin的免疫球蛋白结构域I27为例对蛋白质折叠研究进行了阐述.     

Characteristics of Two Intermediates Trapped in the Unfolding Pathway of Arginine Kinase Induced by Guanidinium Chloride

Equilibrium guanidinium chloride （GdmCl）-induced unfolding of arginine kinase （AK） was investigated by enzymatic activity, intrinsic fluorescence, 8-anilinonaphthalene-1-sulfonic acid （ANS） fluorescence, circular dichroism （CD） spectrum, and size-exclusion chromatography. The measurements showed that AK unfolded through two equilibrium intermediates： the molten globule state and the partly folded state. Both intermediates have no enzyme activity. The molten globule state exists at 0.4-0.8 mol/L GdmCi, perhaps after the N-terminal domain has unfolded but the C-terminal domain is still intact. The partly folded state occurs at 1.1-1.5 mol/L GdmCI with a hydrodynamic volume no more than 1.6-fold larger than the native state and a pronounced far UV-CD signal. Its ANS fluorescence intensity is about 50% of the molten globule state. This partly folded state shares similarities with the “burst” kinetic intermediate of protein folding.     

Sr^2＋诱导尖吻蝮蛇毒中抗凝血因子I的结构稳定性及重新折叠

尖吻蝮蛇毒抗凝血因子I（ACFI）是活化凝血因子X（FXa）结合蛋白，具有显著的抗凝血活性．用荧光光谱研究了Sr^＋诱导ACF I的结构稳定性及重新折叠．结果表明，脱钙ACF I（apo-ACFI）可结合两个Sr^＋．ACF I与FXa的结合反应不是绝对依赖于Ca^2＋，Sr^2＋也可以诱导ACF I与FXa的结合反应．盐酸胍诱导的Sr^2＋重组ACFI（Sr^2＋-ACF I）去折叠过程是一个三态过程，有一个稳定的中间态．像Ca^2＋一样，Sr^2＋不仅能显著增加ACF I的结构稳定性，而且在不改变变性剂浓度条件下，能诱导去折叠的脱钙ACF I重新折叠成Sr^2＋-ACF I的中间态．Sr^2＋诱导apo-ACF I重新折叠过程包含快慢两步反应，Sr^2＋取代Ca^2＋只降低快折叠反应速度，而不影响慢折叠反应速度．这说明，金属离子影响快折叠过程，而慢折叠过程只决定于蛋白质本身性质．     

Multiple conformations of a protein demonstrated by magnetization transfer NMR spectroscopy

It is generally accepted that a globular protein in its native state adopts a single, well-defined conformation. However, there have been several reports that some proteins may exist in more than one distinct folded form in equilibrium. In the case of staphylococcal nuclease, evidence for multiple conformations has come from electrophoretic and NMR studies, although there has been some controversy as to whether these are actually interconvertible forms of the same molecular species. Recently, magnetization transfer (MT)-NMR has been developed as a means of studying the kinetics of conformational transitions in proteins. In the study reported here, this approach has been extended and used to demonstrate the presence of at least two native forms of nuclease in equilibrium and to study their interconversion with the unfolded state under the conditions of the thermal unfolding transition. The experiments reveal that two distinct native forms of the protein fold and unfold independently and that these can interconvert directly as well as via the unfolded state. The spectra of the different forms suggest that they are structurally similar but the MT experiments show that the kinetics of folding and unfolding are quite different. Characterization of this behaviour will, therefore, have important implications for our understanding of the relationship between structure and folding kinetics.     

Solution structure of a protein denatured state and folding intermediate

The most controversial area in protein folding concerns its earliest stages. Questions such as whether there are genuine folding intermediates, and whether the events at the earliest stages are just rearrangements of the denatured state or progress from populated transition states, remain unresolved. The problem is that there is a lack of experimental high-resolution structural information about early folding intermediates and denatured states under conditions that favour folding because competent states spontaneously fold rapidly. Here we have solved directly the solution structure of a true denatured state by nuclear magnetic resonance under conditions that would normally favour folding, and directly studied its equilibrium and kinetic behaviour. We engineered a mutant of Drosophila melanogaster Engrailed homeodomain that folds and unfolds reversibly just by changing ionic strength. At high ionic strength, the mutant L16A is an ultra-fast folding native protein, just like the wild-type protein; however, at physiological ionic strength it is denatured. The denatured state is a well-ordered folding intermediate, poised to fold by docking helices and breaking some non-native interactions. It unfolds relatively progressively with increasingly denaturing conditions, and so superficially resembles a denatured state with properties that vary with conditions. Such ill-defined unfolding is a common feature of early folding intermediate states and accounts for why there are so many controversies about intermediates versus compact denatured states in protein folding.     

Mimicking unfolding motion of a beetle hind wing

This paper presents an experimental research aiming to realize an artificial hind wing that can mimic the wing unfolding motion of AIIomyrina dichotoma, an insect in coleopteran order. Based on the understanding of working principles of beetle wing folding/unfolding mechanisms, the hind wing unfolding motion is mimicked by a combination of creative ideas and state-of-art artificial muscle actuator. In this work, we devise two types of artificial wings and the successfully demonstrate that they can be unfolded by actuation of shape memory alloy wires to provide actuation force at the wing base and along the leading edge vein. The folding/unfolding mechanisms may provide an insight for portable nano/micro air vehicles with morphing wings.     

Activity and Structure Changes of Arginine Kinase from Shrimp Feneropenaeus chinensis Muscle in Trifluoroethanol Solutions

Trifluoroethanol has often been used in protein folding studies. The changes in activity and unfolding of arginine kinase from shrimp Feneropenaeus Ghinensis muscle during denaturation in different concentrations of trifuoroethanol were investigated using far-ultraviolet circular dichroism and fluorescence emission spectra. Arginine kinase was inactivated in trifluoroethanol solutions. The tertiary and secondary structures of arginine kinase were also destroyed in the trifluoroethanol solutions. The unfolding and inactivation courses were measured and compared. Inactivation occurred prior to unfolding, which suggests that the arginine kinase active site is more easily damaged by the denaturant than the enzyme as a whole. The result also indicates that the arginine kinase active site is situated in a limited and flexible region of the enzymemolecule.     

A regular pattern of two types of 100-residue motif in the sequence of titin

Titin is the largest polypeptide yet described (relative molecular mass approximately 3 x 10(6); refs 1, 2) and an abundant protein of striated muscle. Its molecules are string-like and in vivo span from the M to Z-lines. I-band regions of titin are thought to make elastic connections between the thick filament and the Z-line, thereby forming a third type of sarcomere filament. These would centre the A-band in the sarcomere and provide structural continuity in relaxed myofibrils. The A-band region of titin seems to be bound to the thick filament, where it has been proposed to act as a 'molecular ruler' regulating filament length and assembly. Here, we show that partial titin complementary DNAs encode a regular pattern of two types of 100-residue motif, each of which probably folds into a separate domain type. Such motifs are present in several evolutionarily divergent muscle proteins, all of which are likely to interact with myosin. One or both of the domain types is therefore likely to bind to myosin.     

Demonstration by NMR of folding domains in lysozyme

Although there has been much speculation on the pathways of protein folding, only recently have experimental data on the topic been available. The study of proteins under conditions where species intermediate between the fully folded and unfolded states are stable has provided important information, for example about the disulphide intermediates in BPTI, cis/trans proline isomers of RNase A3 and the molten globule state of alpha-lactalbumin. An alternative approach to investigating folding pathways has involved detection and characterization of transient conformers in refolding studies using stopped-flow methods coupled with NMR measurements of hydrogen exchange. The formation of intermediate structures has been detected in the early stages of folding of cytochrome c, RNaseA and barnase. For alpha-lactalbumin, hydrogen exchange kinetics monitored by NMR proved to be crucial for identifying native-like structural features in the stable molten globule state. An analogous partially folded protein stable under equilibrium conditions has not been observed for the structurally homologous protein hen egg-white lysozyme, although there is evidence that a similar but transient state is formed during refolding. Here we describe NMR experiments based on competition between hydrogen exchange and the refolding process which not only support the existence of such a transient species for lysozyme, but enable its structural characteristics to be defined. The results indicate that the two structural domains of lysozyme are distinct folding domains, in that they differ significantly in the extent to which compact, probably native-like, structure is present in the early stages of folding.     

还原型牛胰岛素在疏水界面上的折叠

用疏水色谱（ＨＰＨＩＣ）对还原变性的牛胰岛素在疏水界面上的折叠规律进行了探讨，对于未来原的胍变和脲变ｉｎｓｕｌｉｎ来说，胍变ｉｎｓｕｌｉｎ用ＨＰＨＩＣ法可以完全复性，而脲变ｉｎｓｕｌｉｎ只能部分复性。对于还原变性的ｉｎｓｕｌｉｎＨＰＨＩＣ对胍变、脲变ｉｎｓｕｌｉｎ只能部分复性。在非氧化型流动相中，它们的复性情况较差，而采用氧化型流动相，有助于二硫键的正确对接，可大大提高胍变和脲变ｉｎｓｕｌｉｎ的复     

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.     

Characterization of the Partially Folded Monomeric Intermediate of Creatine Kinase

IntroductionRecent studies of the protein folding pathway andintermediate states in vitro and in vivo haveinduced much interest in the importance ofunderstanding the propertiesof partially structuredintermediates[1 7] . Studies have suggested thatintermed…     

Single-molecule fluorescence reveals sequence-specific misfolding in multidomain proteins

A large range of debilitating medical conditions is linked to protein misfolding, which may compete with productive folding particularly in proteins containing multiple domains. Seventy-five per cent of the eukaryotic proteome consists of multidomain proteins, yet it is not understood how interdomain misfolding is avoided. It has been proposed that maintaining low sequence identity between covalently linked domains is a mechanism to avoid misfolding. Here we use single-molecule F?rster resonance energy transfer to detect and quantify rare misfolding events in tandem immunoglobulin domains from the I band of titin under native conditions. About 5.5 per cent of molecules with identical domains misfold during refolding in vitro and form an unexpectedly stable state with an unfolding half-time of several days. Tandem arrays of immunoglobulin-like domains in humans show significantly lower sequence identity between neighbouring domains than between non-adjacent domains. In particular, the sequence identity of neighbouring domains has been found to be preferentially below 40 per cent. We observe no misfolding for a tandem of naturally neighbouring domains with low sequence identity (24 per cent), whereas misfolding occurs between domains that are 42 per cent identical. Coarse-grained molecular simulations predict the formation of domain-swapped structures that are in excellent agreement with the observed transfer efficiency of the misfolded species. We infer that the interactions underlying misfolding are very specific and result in a sequence-specific domain-swapping mechanism. Diversifying the sequence between neighbouring domains seems to be a successful evolutionary strategy to avoid misfolding in multidomain proteins.     

Verification of the Crooks fluctuation theorem and recovery of RNA folding free energies

Atomic force microscopes and optical tweezers are widely used to probe the mechanical properties of individual molecules and molecular interactions, by exerting mechanical forces that induce transitions such as unfolding or dissociation. These transitions often occur under nonequilibrium conditions and are associated with hysteresis effects-features usually taken to preclude the extraction of equilibrium information from the experimental data. But fluctuation theorems allow us to relate the work along nonequilibrium trajectories to thermodynamic free-energy differences. They have been shown to be applicable to single-molecule force measurements and have already provided information on the folding free energy of a RNA hairpin. Here we show that the Crooks fluctuation theorem can be used to determine folding free energies for folding and unfolding processes occurring in weak as well as strong nonequilibrium regimes, thereby providing a test of its validity under such conditions. We use optical tweezers to measure repeatedly the mechanical work associated with the unfolding and refolding of a small RNA hairpin and an RNA three-helix junction. The resultant work distributions are then analysed according to the theorem and allow us to determine the difference in folding free energy between an RNA molecule and a mutant differing only by one base pair, and the thermodynamic stabilizing effect of magnesium ions on the RNA structure.     

Effects of Glycerol in the Refolding and Unfolding of Creatine Kinase

IntroductionProtein folding is the process by which the aminoacid sequence of a protein determines the three-dimensional conformation of the functionalprotein[1 ] . The elucidation of the molecularmechanism of protein folding from a disorderedpolypeptide chain to the specific native stateremains one of the major challenges inbiochemistry[2 ,3] ,namely,deciphering the secondhalf of the genetic code[4 ] .Molecular chaperonesplay an importantrole in protein folding in vivo aswell asin vitro.A cha…     

机电集成静电谐波微型传动系统的Casimir力和强迫响应

提出一种机电集成静电谐波微型传动系统。随着传动尺寸的减小,分子力的影响变大,应该予以考虑。本文给出了同时考虑静电力和Casimir力时机电集成静电谐波微型传动系统对于电压扰动的强迫响应方程。运用该方程,研究了Casimir力对系统强迫响应的影响规律。结果表明:Casimir力对于系统强迫响应具有重要影响,随着柔轮与定子间初始间隙的减小和电压的增大,Casimir力对系统强迫响应的影响变大。研究结果对于该种传动系统的进一步小型化具有指导意义。     

Independent evolution of striated muscles in cnidarians and bilaterians

Striated muscles are present in bilaterian animals (for example, vertebrates, insects and annelids) and some non-bilaterian eumetazoans (that is, cnidarians and ctenophores). The considerable ultrastructural similarity of striated muscles between these animal groups is thought to reflect a common evolutionary origin. Here we show that a muscle protein core set, including a type II myosin heavy chain (MyHC) motor protein characteristic of striated muscles in vertebrates, was already present in unicellular organisms before the origin of multicellular animals. Furthermore, 'striated muscle' and 'non-muscle' myhc orthologues are expressed differentially in two sponges, compatible with a functional diversification before the origin of true muscles and the subsequent use of striated muscle MyHC in fast-contracting smooth and striated muscle. Cnidarians and ctenophores possess striated muscle myhc orthologues but lack crucial components of bilaterian striated muscles, such as genes that code for titin and the troponin complex, suggesting the convergent evolution of striated muscles. Consistently, jellyfish orthologues of a shared set of bilaterian Z-disc proteins are not associated with striated muscles, but are instead expressed elsewhere or ubiquitously. The independent evolution of eumetazoan striated muscles through the addition of new proteins to a pre-existing, ancestral contractile apparatus may serve as a model for the evolution of complex animal cell types.     

Nanomechanics of individual amyloid fibrils using atomic force microscopy

Compression elasticity of glucagon amyloid fibrils in the transverse direction was investigated by a nanoindentation approach based on atomic force microscopy (AFM).With force-volume mapping, we obtained the correlations between radially applied force and compression of amyloid fibrils, from which the radial compressive elasticity can be deduced.The estimated elastic modulus at three typical locations of fibrils varied from (0.72±0.80) GPa to (1.26±0.62) GPa under small external forces, imply-ing the struct...