Dynamic Strength of Titin's Z-Disk End

Titin is a giant filamentous protein traversing the half sarcomere of striated muscle with putative functions as diverse as providing structural template, generating elastic response, and sensing and relaying mechanical information. The Z-disk region of titin, which corresponds to the N-terminal end of the molecule, has been thought to be a hot spot for mechanosensing while also serving as anchorage for its sarcomeric attachment. Understanding the mechanics of titin's Z-disk region, particularly under the effect of binding proteins, is of great interest. Here we briefly review recent findings on the structure, molecular associations, and mechanics of titin's Z-disk region. In addition, we report experimental results on the dynamic strength of titin's Z1Z2 domains measured by nanomechanical manipulation of the chemical dimer of a recombinant protein fragment

Lokalna promjenljivost (nestalnost) mehaničkih svojstava tandemskih Ig/segmenata titina

The functionally elastic, I-band part of the myofibrillar protein titin (connectin) contains differentially expressed arrays of serially linked immunoglobulin (Ig)-like domains, the length and composition of which vary among the titin isoforms. The biological rationale of the differential expression as well as the contribution of the Ig domain mechanical characteristics to the overall mechanical behavior of titin are not exactly known. The paper briefly reviews the relevant works that have addressed the Ig-domain mechanics problems and presents the authors’ experimental approach to studying the mechanical behavior of Ig domains. The mechanics of an eight domain segment from the differentially expressed tandem Ig region of titin (I55-62) was studied with an atomic force microscope specially used for stretching single molecules, and the results were compared to known mechanical properties of other domains and segments.Funkcionalno elastična I-vrpca miofibrilarnoga proteina titina (konektina) sadrži razlicito izražene vrste serijski povezanih domena sličnih imunoglobulinu (Ig) čija se duljina i sastav razlikuju u pojedinim titinskim izoformama. Biološki razlozi diferencijalne ekspresije kao i doprinos mehaničkih svojstava domena koje su slične Ig ponašanju titina nisu u cjelosti poznati. U ovom su članku pregledno prikazani dosadašnji relevantni radovi koji se bave problemima mehaničkih svojstava Ig-domena te autorski eksperimentalni pristupi u istraživanju toga problema. U radu su također prikazani rezultati istraživanja mehaničkih svojstava diferencijalno eksprimiranoga tandemskoga Ig-područja titina (155-62), koji se sastoji od osam domena, pomoću mikroskopa atomske snage razlučivanja specijaliziranoga za rastezanje pojedinačne molekule. Ovim postupkom utvrđena mehanička svojstva ispitivanoga dijela titinske molekule uspoređena su s poznatim mehaničkim svojstvima drugih domena i segmenata

Muscle thixotropy: more than just cross-bridges?

AbstractAlthough Campbell and Lakie in a Comment to the Editor in this issue of Biophysical Journal suggested that exclusive cross-bridge action is behind muscle thixotropy, recent findings and our preliminary observations suggest that additional mechanisms could also be involved

A Quantitative Theory of Mechanical Unfolding of a Homopolymer Globule

We propose the quantitative mean-field theory of mechanical unfolding of a globule formed by long flexible homopolymer chain collapsed in poor solvent and subjected to extensional deformation. We demonstrate that depending on the degree of polymerization and solvent quality (quantified by the Flory-Huggins $\chi$ parameter) the mechanical unfolding of the collapsed chain may either occur continuously (by passing a sequence of uniformly elongated configurations) or involves intra-molecular micro-phase coexistence of a collapsed and a stretched segment followed by an abrupt unraveling transition. The force-extension curves are obtained and quantitatively compared to our recent results of numerical self-consistent field (SCF) simulations. The phase diagrams for extended homopolymer chains in poor solvent comprising one- and two-phase regions are calculated for different chain length or/and solvent quality.Comment: 24 pages, 18 figure

Effective Area-Elasticity and Tension of Micro-manipulated Membranes

We evaluate the effective Hamiltonian governing, at the optically resolved scale, the elastic properties of micro-manipulated membranes. We identify floppy, entropic-tense and stretched-tense regimes, representing different behaviors of the effective area-elasticity of the membrane. The corresponding effective tension depends on the microscopic parameters (total area, bending rigidity) and on the optically visible area, which is controlled by the imposed external constraints. We successfully compare our predictions with recent data on micropipette experiments.Comment: To be published in Phys. Rev. Let

Dynamics of folding in Semiflexible filaments

We investigate the dynamics of a single semiflexible filament, under the action of a compressing force, using numerical simulations and scaling arguments. The force is applied along the end to end vector at one extremity of the filament, while the other end is held fixed. We find that, unlike in elastic rods the filament folds asymmetrically with a folding length which depends only on the bending stiffness and the applied force. It is shown that this behavior can be attributed to the exponentially falling tension profile in the filament. While the folding time depends on the initial configuration, at late time, the distance moved by the terminal point of the filament and the length of the fold shows a power law dependence on time with an exponent 1/2.Comment: 13 pages, Late

Stretching of a polymer below the Theta point

The unfolding of a polymer below the $\theta$ point when pulled by an external force is studied both in d=2 on the lattice and in $d=3$ off lattice. A ground state analysis of finite length chains shows that the globule unfolds via multiple steps, corresponding to transitions between different minima, in both cases. In the infinite length limit, these intermediate minima have a qualitative effect only in $d=2$. The phase diagram in d=2 is determined using transfer matrix techniques. Energy-entropy and renormalization group arguments are given which predict a qualitatively correct phase diagram and a change of the order of the transition from d=2 to d=3.Comment: 4 pages, 3 eps figure

Single Molecule Statistics and the Polynucleotide Unzipping Transition

We present an extensive theoretical investigation of the mechanical unzipping of double-stranded DNA under the influence of an applied force. In the limit of long polymers, there is a thermodynamic unzipping transition at a critical force value of order 10 pN, with different critical behavior for homopolymers and for random heteropolymers. We extend results on the disorder-averaged behavior of DNA's with random sequences to the more experimentally accessible problem of unzipping a single DNA molecule. As the applied force approaches the critical value, the double-stranded DNA unravels in a series of discrete, sequence-dependent steps that allow it to reach successively deeper energy minima. Plots of extension versus force thus take the striking form of a series of plateaus separated by sharp jumps. Similar qualitative features should reappear in micromanipulation experiments on proteins and on folded RNA molecules. Despite their unusual form, the extension versus force curves for single molecules still reveal remnants of the disorder-averaged critical behavior. Above the transition, the dynamics of the unzipping fork is related to that of a particle diffusing in a random force field; anomalous, disorder-dominated behavior is expected until the applied force exceeds the critical value for unzipping by roughly 5 pN.Comment: 40 pages, 18 figure

Manipulating Protein Conformations By Single-molecule Afm-fret Nanoscopy

Combining atomic force microscopy and fluorescence resonance energy transfer spectroscopy (AFM-FRET), we have developed a single-molecule AFM-FRET nanoscopy approach capable of effectively pinpointing and mechanically manipulating a targeted dye-labeled single protein in a large sampling area and simultaneously monitoring the conformational changes of the targeted protein by recording single-molecule FRET time trajectories. We have further demonstrated an application of using this nanoscopy on manipulation of single-molecule protein conformation and simultaneous single-molecule FRET measurement of a Cy3-Cy5-labeled kinase enzyme, HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase). By analyzing time-resolved FRET trajectories and correlated AFM force pulling curves of the targeted single-molecule enzyme, we are able to observe the protein conformational changes of a specific coordination by AFM mechanic force pulling

Single-molecule experiments in biological physics: methods and applications

I review single-molecule experiments (SME) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SME it is possible to: manipulate molecules one at a time and measure distributions describing molecular properties; characterize the kinetics of biomolecular reactions and; detect molecular intermediates. SME provide the additional information about thermodynamics and kinetics of biomolecular processes. This complements information obtained in traditional bulk assays. In SME it is also possible to measure small energies and detect large Brownian deviations in biomolecular reactions, thereby offering new methods and systems to scrutinize the basic foundations of statistical mechanics. This review is written at a very introductory level emphasizing the importance of SME to scientists interested in knowing the common playground of ideas and the interdisciplinary topics accessible by these techniques. The review discusses SME from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied. I briefly discuss experimental techniques such as atomic-force microscopy (AFM), laser optical tweezers (LOT), magnetic tweezers (MT), biomembrane force probe (BFP) and single-molecule fluorescence (SMF). I then present several applications of SME to the study of nucleic acids (DNA, RNA and DNA condensation), proteins (protein-protein interactions, protein folding and molecular motors). Finally, I discuss applications of SME to the study of the nonequilibrium thermodynamics of small systems and the experimental verification of fluctuation theorems. I conclude with a discussion of open questions and future perspectives.Comment: Latex, 60 pages, 12 figures, Topical Review for J. Phys. C (Cond. Matt