Reversible unfolding of single RNA molecules by mechanical force

Here we use mechanical force to induce the unfolding and refolding of single RNA molecules: a simple RNA hairpin, a molecule containing a three-helix junction, and the P5abc domain of the Tetrahymena thermophila ribozyme. All three molecules (P5abc only in the absence of Mg2+) can be mechanically unfolded at equilibrium, and when kept at constant force within a critical force range, are bi-stable and hop between folded and unfolded states. We determine the force-dependent equilibrium constants for folding/unfolding these single RNA molecules and the positions of their transition states along the reaction coordinate.     

Direct observation of the three-state folding of a single protein molecule

We used force-measuring optical tweezers to induce complete mechanical unfolding and refolding of individual Escherichia coli ribonuclease H (RNase H) molecules. The protein unfolds in a two-state manner and refolds through an intermediate that correlates with the transient molten globule-like intermediate observed in bulk studies. This intermediate displays unusual mechanical compliance and unfolds at substantially lower forces than the native state. In a narrow range of forces, the molecule hops between the unfolded and intermediate states in real time. Occasionally, hopping was observed to stop as the molecule crossed the folding barrier directly from the intermediate, demonstrating that the intermediate is on-pathway. These studies allow us to map the energy landscape of RNase H.     

Experimental identification of downhill protein folding

Theory predicts the existence of barrierless protein folding. Without barriers, folding should be noncooperative and the degree of native structure should be coupled to overall protein stability. We investigated the thermal unfolding of the peripheral subunit binding domain from Escherichia coli's 2-oxoglutarate dehydrogenase multienzyme complex (termed BBL) with a combination of spectroscopic techniques and calorimetry. Each technique probed a different feature of protein structure. BBL has a defined three-dimensional structure at low temperatures. However, each technique showed a distinct unfolding transition. Global analysis with a statistical mechanical model identified BBL as a downhill-folding protein. Because of BBL's biological function, we propose that downhill folders may be molecular rheostats, in which effects could be modulated by altering the distribution of an ensemble of structures.     

Protein-RNA interactions in an icosahedral virus at 3.0 A resolution

Nearly 20 percent of the packaged RNA in bean-pod mottle virus (BPMV) binds to the capsid interior in a symmetric fashion and is clearly visible in the electron density map. The RNA displaying icosahedral symmetry is single-stranded with well-defined polarity and stereochemical properties. Interactions with protein are dominated by nonbonding forces with few specific contacts. The tertiary and quaternary structures of the BPMV capsid proteins are similar to those observed in animal picornaviruses, supporting the close relation between plant comoviruses and animal picornaviruses established by previous biological studies.     

Scaling in tensile "skeletons": structures with scale-independent length dimensions

Skeletal structures that resist only tensile forces can scale differently than compression resisting structures that fail in bending or buckling. The tensile structures examined scalelike simple ropes: length and diameter of the structure are not correlated, and in three of four cases, length is independent of scale or load, but diameter is dependent on scale. These relations suggest that similarity in stress rather than strain, or deformational behavior, is the basis for mechanical adaptation in the gross dimensions of these tensile structures.     

一类简化的竹编水泥沼气池力学模型分析

针对目前在滇南一带开发推广的竹编水泥沼气池，在实地测试和实验室测试研究的基础上，建立一类简化的三次超静定力学模型，并采用力法对赘余力进行严格的数学，力学分析与计算，构造与实际结构接近的理论模型，针对壁厚3,5cm的情形进行数值计算，计算与实测结果基本一致，说明所讨论的模型及力学算法分析是正确的，计算程度是可靠的。     

Fluorescence-force spectroscopy maps two-dimensional reaction landscape of the holliday junction

Despite the recent advances in single-molecule manipulation techniques, purely mechanical approaches cannot detect subtle conformational changes in the biologically important regime of weak forces. We developed a hybrid scheme combining force and fluorescence that allowed us to examine the effect of subpiconewton forces on the nanometer scale motion of the Holliday junction (HJ) at 100-hertz bandwidth. The HJ is an exquisitely sensitive force sensor whose force response is amplified with an increase in its arm lengths, demonstrating a lever-arm effect at the nanometer-length scale. Mechanical interrogation of the HJ in three different directions helped elucidate the structures of the transient species populated during its conformational changes. This method of mapping two-dimensional reaction landscapes at low forces is readily applicable to other nucleic acid systems and their interactions with proteins and enzymes.     

Chaperonin function: folding by forced unfolding

The ability of the GroEL chaperonin to unfold a protein trapped in a misfolded condition was detected and studied by hydrogen exchange. The GroEL-induced unfolding of its substrate protein is only partial, requires the complete chaperonin system, and is accomplished within the 13 seconds required for a single system turnover. The binding of nucleoside triphosphate provides the energy for a single unfolding event; multiple turnovers require adenosine triphosphate hydrolysis. The substrate protein is released on each turnover even if it has not yet refolded to the native state. These results suggest that GroEL helps partly folded but blocked proteins to fold by causing them first to partially unfold. The structure of GroEL seems well suited to generate the nonspecific mechanical stretching force required for forceful protein unfolding.     

Coherent sensing of a mechanical resonator with a single-spin qubit

Mechanical systems can be influenced by a wide variety of small forces, ranging from gravitational to optical, electrical, and magnetic. When mechanical resonators are scaled down to nanometer-scale dimensions, these forces can be harnessed to enable coupling to individual quantum systems. We demonstrate that the coherent evolution of a single electronic spin associated with a nitrogen vacancy center in diamond can be coupled to the motion of a magnetized mechanical resonator. Coherent manipulation of the spin is used to sense driven and Brownian motion of the resonator under ambient conditions with a precision below 6 picometers. With future improvements, this technique could be used to detect mechanical zero-point fluctuations, realize strong spin-phonon coupling at a single quantum level, and implement quantum spin transducers.     

Complexity and the economy

After two centuries of studying equilibria-static patterns that call for no further behavioral adjustments-economists are beginning to study the general emergence of structures and the unfolding of patterns in the economy. When viewed in out-of-equilibrium formation, economic patterns sometimes simplify into the simple static equilibria of standard economics. More often they are ever changing, showing perpetually novel behavior and emergent phenomena. Complexity portrays the economy not as deterministic, predictable, and mechanistic, but as process dependent, organic, and always evolving.     

Structural basis of glmS ribozyme activation by glucosamine-6-phosphate

The glmS ribozyme is the only natural catalytic RNA known to require a small-molecule activator for catalysis. This catalytic RNA functions as a riboswitch, with activator-dependent RNA cleavage regulating glmS messenger RNA expression. We report crystal structures of the glmS ribozyme in precleavage states that are unliganded or bound to the competitive inhibitor glucose-6-phosphate and in the postcleavage state. All structures superimpose closely, revealing a remarkably rigid RNA that contains a preformed active and coenzyme-binding site. Unlike other riboswitches, the glmS ribozyme binds its activator in an open, solvent-accessible pocket. Our structures suggest that the amine group of the glmS ribozyme-bound coenzyme performs general acid-base and electrostatic catalysis.     

硬质合金丝锥在铸铁高速攻丝中的切削力分析

发动机汽缸生产线上需求大量的高速加工用硬质合金丝锥.攻丝过程中,由于切削力的大小直接影响了加工精度及丝锥寿命,丝锥的参数优化也与切削力变化紧密相关,因此切削力分析在新型硬质合金丝锥的研制过程中显得尤为重要.通过立式加工中心和Kistler测力系统,对一批新型丝锥进行了切削性能评价,比较了不同的硬质合金丝锥加工灰铸铁和球墨铸铁过程中的切削力在旋入和旋出两个阶段的变化情况.指出了在铸铁攻丝中切削力的变化几乎不受丝锥磨损的影响,而与刀具夹持方式紧密相关.此外,攻丝力还与螺旋角、丝锥槽数以及试件材料的塑性均有不同程度的联系.     

Unfolding pathways of individual bacteriorhodopsins

Atomic force microscopy and single-molecule force spectroscopy were combined to image and manipulate purple membrane patches from Halobacterium salinarum. Individual bacteriorhodopsin molecules were first localized and then extracted from the membrane; the remaining vacancies were imaged again. Anchoring forces between 100 and 200 piconewtons for the different helices were found. Upon extraction, the helices were found to unfold. The force spectra revealed the individuality of the unfolding pathways. Helices G and F as well as helices E and D always unfolded pairwise, whereas helices B and C occasionally unfolded one after the other. Experiments with cleaved loops revealed the origin of the individuality: stabilization of helix B by neighboring helices.     

Cytoplasmic extraction: polyribosomes and heterogenous ribonucleoproteins without associated DNA

Two methods are described for preparing cytoplasmic extracts from sea urchin embryos. One method, involving homogenization, yields DNA structures that cosediment with polyribosomes and subribosomal ribonucleoproteins. In addition this method also yields extraneous structures containing RNA that cosediment with polyribosomes. The DNA is not associated with polyribosomes, as shown by buoyant density analysis. Furthermore, this DNA appears to be spurious, because its release into a cytoplasmic extract does not occur when a different method of cell disruption, involving passage of embryos through a hypodermic needle, is used. With this second method, polyribosomes are obtained without extraneous cosedimenting RNA structures and subribosomal ribonucleo-proteins are obtained in the virtual absence of DNA.     

Nonequilibrium self-assembly of long chains of polar molecules in superfluid helium

It is shown that in the low-temperature (0.37 kelvin) environment of superfluid helium droplets, long-range dipole-dipole forces acting between two polar molecules can result in the self-assembly of noncovalently bonded linear chains. At this temperature the effective range of these forces is on the nanometer scale, making them important in the growth of nanoscale structures. In particular, the self-assembly of exclusively linear hydrogen cyanide chains is observed, even when the folded structures are energetically favored. This suggests a design strategy for the growth of new nanoscale oligomers composed of monomers with defined dipole (or higher order) moment directions.     

Detection of molecular alignment in confined films

Optical second harmonic generation was used to study the in-plane alignment of self-assembled silane monolayers attached to a glass surface under mechanical loading. The measurements allow correlation of the macroscopic forces acting on the monolayer with the average orientation and the azimuthal molecular alignment of the terminal molecular entity. Compression and shear forces lead to an alignment of the initially randomly oriented molecules on a macroscopic length scale. The change in azimuthal alignment of molecules under mechanical stress was found to be irreversible on the time scale of 12 hours, whereas changes of the molecular tilt angle were reversible.     

Membranous structures associated with translation and transcription of poliovirus RNA

Poliovirus RNA and proteins are synthesized in association with distinct membranous structures that were separated by means of Isopycnic centrifugation of cytoplasmic extracts in discontinuous sucrose-density gradients. Viral RNA is replicated in a structure that contains rapidly labeled replicative intermediate RNA and viral RNA polymerase associated with the smooth membrane fraction. In sucrose gradients this viral RNA replication complex is distributed at densities in the range of 1.12 to 1.18 grams per cubic centimeter. Viral proteins are synthesized on polyribosomes bound to membranes and sediment with polyribosomes at densities of less than 1.25 grams per cubic centimeter.     

Water-inserted alpha-helical segments implicate reverse turns as folding intermediates

Information relevant to the folding and unfolding of alpha helices has been extracted from an analysis of protein structures. The alpha helices in protein crystal structures have been found to be hydrated, either externally by a water molecule hydrogen bonding to the backbone carbonyl oxygen atom, or internally by inserting into the helix hydrogen bond and forming a hydrogen-bonded bridge between the backbone carbonyl oxygen and the amide nitrogen atoms. The water-inserted alpha-helical segments display a variety of reverse-turn conformations, such as type III, type II, type I, and opened out, that can be considered as folding intermediates that are trapped in the folding-unfolding process of alpha helices. Since the alpha helix, most turns, and the extended beta strand occupy contiguous regions in the conformational space of phi, psi dihedral angles, a plausible pathway can be proposed for the folding-unfolding process of alpha helices in aqueous solution.