Equilibrium and Kinetics of DNA Overstretching Modeled with a Quartic Energy Landscape

It is well known that the dsDNA molecule undergoes a phase transition from B-DNA into an overstretched state at high forces. For some time, the structure of the overstretched state remained unknown and highly debated, but recent advances in experimental techniques have presented evidence of more than one possible phase (or even a mixed phase) depending on ionic conditions, temperature, and basepair sequence. Here, we present a theoretical model to study the overstretching transition with the possibility that the overstretched state is a mixture of two phases: a structure with portions of inner strand separation (melted or M-DNA), and an extended phase that retains the basepair structure (S-DNA). We model the double-stranded DNA as a chain composed of n segments of length l, where the transition is studied by means of a Landau quartic potential with statistical fluctuations. The length l is a measure of cooperativity of the transition and is key to characterizing the overstretched phase. By analyzing the different values of l corresponding to a wide spectrum of experiments, we find that for a range of temperatures and ionic conditions, the overstretched form is likely to be a mix of M-DNA and S-DNA. For a transition close to a pure S-DNA state, where the change in extension is close to 1.7 times the original B-DNA length, we find l ≈ 25 basepairs regardless of temperature and ionic concentration. Our model is fully analytical, yet it accurately reproduces the force-extension curves, as well as the transient kinetic behavior, seen in DNA overstretching experiments.     

Transition dynamics and selection of the distinct S-DNA and strand unpeeling modes of double helix overstretching

Recent studies have revealed two distinct pathways for the DNA overstretching transition near 65 pN: 'unpeeling' of one strand from the other, and a transition from B-DNA to an elongated double-stranded 'S-DNA' form. However, basic questions concerning the dynamics of these transitions, relative stability of the two competing overstretched states, and effects of nicks and free DNA ends on overstretching, remain open. In this study we report that: (i) stepwise extension changes caused by sequence-defined barriers occur during the strand-unpeeling transition, whereas rapid, sequence-independent extension fluctuations occur during the B to S transition; (ii) the secondary transition that often occurs following the overstretching transition is strand-unpeeling, during which the extension increases by 0.01-0.02 nm per base pair of S-DNA converted to single-stranded DNA at forces between 75 and 110 pN; (iii) even in the presence of nicks or free ends, S-DNA can be stable under physiological solution conditions; (iv) distribution of small GC-rich islands in a large DNA plays a key role in determining the transition pathways; and (v) in the absence of nicks or free ends, torsion-unconstrained DNA undergoes the overstretching transition via creation of S-DNA. Our study provides a new, high-resolution understanding of the competition between unpeeling and formation of S-DNA.     

DNA overstretching in the presence of glyoxal: structural evidence of force-induced DNA melting

When a long DNA molecule is stretched beyond its B-form contour length, a transition occurs in which its length increases by a factor of 1.7, with very little force increase. A quantitative model was proposed to describe this transition as force-induced melting, where double-stranded DNA is converted into single-stranded DNA. The force-induced melting model accurately describes the thermodynamics of DNA overstretching as a function of solution conditions and in the presence of DNA binding ligands. An alternative explanation suggests a transformation into S-DNA, a double-stranded form which preserves the interstrand base pairing. To determine the extent to which DNA base pairs are exposed to solution during the transition, we held DNA overstretched to different lengths within the transition in the presence of glyoxal. If overstretching involved strand separation, then force-melted basepairs would be glyoxal-modified, thus essentially permanently single-stranded. Subsequent stretches confirm that a significant fraction of the DNA melted by force is permanently melted. This result demonstrates that DNA overstretching is accompanied by a disruption of the DNA helical structure, including a loss of hydrogen bonding.     

Force spectroscopy and fluorescence microscopy of dsDNA–YOYO-1 complexes: implications for the structure of dsDNA in the overstretching region

When individual dsDNA molecules are stretched beyond their B-form contour length, they reveal a structural transition in which the molecule extends 1.7 times its contour length. The nature of this transition is still a subject of debate. In the first model, the DNA helix unwinds and combined with the tilting of the base pairs (which remain intact), results in a stretched form of DNA (also known as S-DNA). In the second model the base pairs break resulting effectively in two single-strands, which is referred to as force-induced melting. Here a combination of optical tweezers force spectroscopy with fluorescence microscopy was used to study the structure of dsDNA in the overstretching regime. When dsDNA was stretched in the presence of 10 nM YOYO-1 an initial increase in total fluorescence intensity of the dye–DNA complex was observed and at an extension where the dsDNA started to overstretch the fluorescence intensity leveled off and ultimately decreased when stretched further into the overstretching region. Simultaneous force spectroscopy and fluorescence polarization microscopy revealed that the orientation of dye molecules did not change significantly in the overstretching region (78.0°± 3.2°). These results presented here clearly suggest that, the structure of overstretched dsDNA can be explained accurately by force induced melting.     

There and (slowly) back again: entropy-driven hysteresis in a model of DNA overstretching

When pulled along its axis, double-stranded DNA elongates abruptly at a force of ∼65 pN. Two physical pictures have been developed to describe this overstretched state. The first proposes that strong forces induce a phase transition to a molten state consisting of unhybridized single strands. The second picture introduces an elongated hybridized phase called S-DNA. Little thermodynamic evidence exists to discriminate directly between these competing pictures. Here we show that within a microscopic model of DNA we can distinguish between the dynamics associated with each. In experiment, considerable hysteresis in a cycle of stretching and shortening develops as temperature is increased. Since there are few possible causes of hysteresis in a system whose extent is appreciable in only one dimension, such behavior offers a discriminating test of the two pictures of overstretching. Most experiments are performed upon nicked DNA, permitting the detachment (unpeeling) of strands. We show that the long-wavelength progression of the unpeeled front generates hysteresis, the character of which agrees with experiment only if we assume the existence of S-DNA. We also show that internal melting can generate hysteresis, the degree of which depends upon the nonextensive loop entropy of single-stranded DNA.     

91 Kinetics of DNA overstretching: melting vs. B-to-S transition

Single B-form DNA molecules undergo an overstretching transition at force F_ov to a ～1.7-fold longer form when stretched. The nature of overstretched DNA has been debated for over 10?years. Either peeled (PL DNA), internally melted (M DNA), or unwound double-helical (S DNA) forms of overstretched DNA have been suggested. Here, we characterize the kinetics of the overstretching transition in polymeric torsionally unconstrained double-stranded (ds) DNA molecules. We pull ～50?Kbp λ–DNA molecules using optical tweezers with rates ν ～10?nm/s to 5?×?10⁴?nm/s, (overstretching time between 0.2 and 10³?s). The F_ov(ν, [Na⁺]) dependence measured over a broad range of rates and solution ionic strength suggests the existence of all three forms of the overstretched DNA. Thus, at [Na⁺]?>?50?mM and the stretching time >>1?s, internal melting dominates overstretching. This B-to-M transition is highly cooperative (involves ～100?bp), and slow (on/off time ～1000?s). Faster overstretching during ?1?s leads to B-to-S DNA transition, which is less cooperative (involves ～10?bp) and faster (on/off time ～1?s). In contrast, in lower salt ([Na⁺]?<?50?mM), the overstretching during >1?s leads to DNA peeling. However, on the faster time scale of 0.2–1?s, even in low salt, the DNA overstretches into S DNA, as peeling becomes kinetically prohibited. Our conclusions are supported by several independent lines of evidence, including the salt and rate dependence of both the slope of the overstretched DNA force-extension curve and the value of the second transition force (from M or PL DNA into S DNA).     

Force-induced melting of the DNA double helix. 2. Effect of solution conditions

In this paper, we consider the implications of the general theory developed in the accompanying paper, to interpret experiments on DNA overstretching that involve variables such as solution temperature, pH, and ionic strength. We find the DNA helix-coil phase boundary in the force-temperature space. At temperatures significantly below the regular (zero force) DNA melting temperature, the overstretching force, f(ov)(T), is predicted to decrease nearly linearly with temperature. We calculate the slope of this dependence as a function of entropy and heat-capacity changes upon DNA melting. Fitting of the experimental f(ov)(T) dependence allows determination of both of these quantities in very good agreement with their calorimetric values. At temperatures slightly above the regular DNA melting temperature, we predict stabilization of dsDNA by moderate forces, and destabilization by higher forces. Thus the DNA stretching curves, f(b), should exhibit two rather than one overstretching transitions: from single stranded (ss) to double stranded (ds) and then back at the higher force. We also predict that any change in DNA solution conditions that affects its melting temperature should have a similar effect on DNA overstretching force. This result is used to calculate the dependence of DNA overstretching force on solution pH, f(ov)(pH), from the known dependence of DNA melting temperature on pH. The calculated f(ov)(pH) is in excellent agreement with its experimental determination (M. C. Williams, J. R. Wenner, I. Rouzina, and V. A. Bloomfield, Biophys. J., accepted for publication). Finally, we quantitatively explain the measured dependence of DNA overstretching force on solution ionic strength for crosslinked and noncrosslinked DNA. The much stronger salt dependence of f(ov) in noncrosslinked DNA results from its lower linear charge density in the melted state, compared to crosslinked or double-stranded overstretched S-DNA.     

B-S transition in short oligonucleotides

Stretching experiments with long double-stranded DNA molecules in physiological ambient revealed a force-induced transition at a force of 65 pN. During this transition between B-DNA and highly overstretched S-DNA the DNA lengthens by a factor of 1.7 of its B-form contour length. Here, we report the occurrence of this so-called B-S transition in short duplexes consisting of 30 basepairs. We employed atomic-force-microscope-based single molecule force spectroscopy to explore the unbinding mechanism of two short duplexes containing 30 or 20 basepairs by pulling at the opposite 5' termini. For a 30-basepair-long DNA duplex the B-S transition is expected to cause a length increase of 6.3 nm and should therefore be detectable. Indeed 30% of the measured force-extension curves exhibit a region of constant force (plateau) at 65 pN, which corresponds to the B-S transition. The observed plateaus show a length between 3 and 7 nm. This plateau length distribution indicates that the dissociation of a 30-basepair duplex mainly occurs during the B-S transition. In contrast, the measured force-extension curves for a 20-basepair DNA duplex exhibited rupture forces below 65 pN and did not show any evidence of a B-S transition.     

A Three-State Model with Loop Entropy for the Overstretching Transition of DNA

We introduce a three-state model for a single DNA chain under tension that distinguishes among B-DNA, S-DNA, and M (molten or denatured) segments and at the same time correctly accounts for the entropy of molten loops, characterized by the exponent c in the asymptotic expression S ∼ -c ln n for the entropy of a loop of length n. Force extension curves are derived exactly by employing a generalized Poland-Scheraga approach and then compared to experimental data. Simultaneous fitting to force-extension data at room temperature and to the denaturation phase transition at zero force is possible and allows us to establish a global phase diagram in the force-temperature plane. Under a stretching force, the effects of the stacking energy (entering as a domain-wall energy between paired and unpaired bases) and the loop entropy are separated. Therefore, we can estimate the loop exponent c independently from the precise value of the stacking energy. The fitted value for c is small, suggesting that nicks dominate the experimental force extension traces of natural DNA.     

M-DNA is stabilised in G*C tracts or by incorporation of 5-fluorouracil

M-DNA is a complex between the divalent metal ions Zn²⁺, Ni²⁺ and Co²⁺ and duplex DNA which forms at a pH of ~8.5. The stability and formation of M-DNA was monitored with an ethidium fluorescence assay in order to assess the relationship between pH, metal ion concentration, DNA concentration and the base composition. The dismutation of calf thymus DNA exhibits hysteresis with the formation of M-DNA occurring at a higher pH than the reconversion of M-DNA back to B-DNA. Hysteresis is most prominent with the Ni form of M-DNA where complete reconversion to B-DNA takes several hours even in the presence of EDTA. Increasing the DNA concentration leads to an increase in the metal ion concentration required for M-DNA formation. Both poly(dG)•poly(dC) and poly(dA)•poly(dT) formed M-DNA more readily than the corresponding mixed sequence DNAs. For poly(dG)•(poly(dC) M-DNA formation was observed at pH 7.4 with 0.5 mM ZnCl₂. Modified bases were incorporated into a 500 bp fragment of phage λ DNA by polymerase chain reaction. DNAs in which guanine was replaced with hypoxanthine or thymine with 5-fluorouracil formed M-DNA at pHs below 8 whereas substitutions such as 2-aminoadenine and 5-methylcytosine had little effect. Poly[d(A5FU)] also formed a very stable M-DNA duplex as judged from T_m measurements. It is evident that the lower the pK_a of the imino proton of the base, the lower the pH at which M-DNA will form; a finding that is consistent with the replacement of the imino proton with the metal ion.     

Salt dependence of the elasticity and overstretching transition of single DNA molecules

As double-stranded DNA is stretched to its B-form contour length, models of polymer elasticity can describe the dramatic increase in measured force. When the molecule is stretched beyond this contour length, it shows a highly cooperative overstretching transition. We have measured the elasticity and overstretching transition as a function of monovalent salt concentration by stretching single DNA molecules in an optical tweezers apparatus. As the sodium ion concentration was decreased from 1000 to 2.57 mM, the persistence length of DNA increased from 46 to 59 nm, while the elastic stretch modulus remained approximately constant. These results are consistent with the model of Podgornik, et al. (2000, J. Chem. Phys. 113:9343-9350) using an effective DNA length per charge of 0.67 nm. As the monovalent salt concentration was decreased over the same range, the overstretching transition force decreased from 68 to 52 pN. This reduction in force is attributed to a decrease in the stability of the DNA double helix with decreasing salt concentration. Although, as was shown previously, the hydrogen bonds holding DNA strands in a helical conformation break as DNA is overstretched, these data indicate that both DNA strands remain close together during the transition.     

Reaction path ensemble of the B–Z-DNA transition: a comprehensive atomistic study

Since its discovery in 1979, left-handed Z-DNA has evolved from an in vitro curiosity to a challenging DNA structure with crucial roles in gene expression, regulation and recombination. A fundamental question that has puzzled researchers for decades is how the transition from B-DNA, the prevalent right-handed form of DNA, to Z-DNA is accomplished. Due to the complexity of the B–Z-DNA transition, experimental and computational studies have resulted in several different, apparently contradictory models. Here, we use molecular dynamics simulations coupled with state-of-the-art enhanced sampling techniques operating through non-conventional reaction coordinates, to investigate the B–Z-DNA transition at the atomic level. Our results show a complex free energy landscape, where several phenomena such as over-stretching, unpeeling, base pair extrusion and base pair flipping are observed resulting in interconversions between different DNA conformations such as B-DNA, Z-DNA and S-DNA. In particular, different minimum free energy paths allow for the coexistence of different mechanisms (such as zipper and stretch–collapse mechanisms) that previously had been proposed as independent, disconnected models. We find that the B–Z-DNA transition—in absence of other molecular partners—can encompass more than one mechanism of comparable free energy, and is therefore better described in terms of a reaction path ensemble.     

The Influence of Vesicle Size and Composition on α-Synuclein Structure and Stability

Monomeric α-synuclein (αSN), which has no persistent structure in aqueous solution, is known to bind to anionic lipids with a resulting increase in α-helix structure. Here we show that at physiological pH and ionic strength, αSN incubated with different anionic lipid vesicles undergoes a marked increase in α-helical content at a temperature dictated either by the temperature of the lipid phase transition, or (in 1,2-DilauroylSN-Glycero-3-[Phospho-rac-(1-glycerol)] (DLPG), which is fluid down to 0°C) by an intrinsic cold denaturation that occurs around 10-20°C. This structure is subsequently lost in a thermal transition around 60°C. Remarkably, this phenomenon is only observed for vesicles >100 nm in diameter and is sensitive to lipid chain length, longer chain lengths, and larger vesicles giving more cooperative unfolding transitions and a greater degree of structure. For both vesicle size and chain length, a higher degree of compressibility or permeability in the lipid thermal transition region is associated with a higher degree of αSN folding. Furthermore, the degree of structural change is strongly reduced by an increase in ionic strength or a decrease in the amount of anionic lipid. A simple binding-and-folding model that includes the lipid phase transition, exclusive binding of αSN to the liquid disordered phase, the thermodynamics of unfolding, and the electrostatics of binding of αSN to lipids is able to reproduce the two thermal transitions as well as the effect of ionic strength and anionic lipid. Thus the nature of αSN's binding to phospholipid membranes is intimately tied to the lipids' physico-chemical properties.     

Electron injection from the side of an M-DNA duplex

M-DNA is a complex formed between duplex DNA and divalent metal ions (Zn²⁺, Cu²⁺ or Ni²⁺) at pHs above 8. Previous results showed that the fluorescence of an electron donor fluorophore was quenched when an acceptor flourophore was placed in the opposite end of an M-DNA duplex suggesting electron transfer through the duplex and indicating M-DNA may operate as a better conductor than B-DNA. To further investigate the properties of M-DNA, oligodeoxynucleotides were prepared with fluorescein (Fl) as an electron donor placed at different positions along the helix. An internal position of the chromophore was made possible by attaching it to the extra hydroxyl arm in the branched monomer 4′-C-hydroxymethylthymidine. Upon excitation of the donor fluorophore, it was demonstrated that electrons could be injected into the side of an M-DNA helix thereby extending the range of nanoelectronic structures that can be prepared from DNA. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Interaction of nucleic acids with a non-intercalative anti-leukemic compound containing bisquarternary heterocycles

The binding of an antitumour drug with bisquarternary ammonium heterocyclic structure, NSC-101327, to nucleic acids has been examined by using ultraviolet absorption and CD measurements. Like the minor groove-binding oligopeptides, netropsin and distamycin A, the optically inactive chromophoric system of NSC-101327 shows induced Cotton effects in the CD spectra of complexes with various DNAs, RNA and single-stranded polynucleotides. This property directly reflects interaction of NSC-101327 with different types of nucleic acids at moderate ionic strength, which contrasts with previous findings of a higher selective binding of netropsin to B-DNA. However, an efficient interactin of NSC-101327 with dA·dT basepair sequences is demonstrated by a large melting temperature increase of dA·dT-rich DNAs. NSC-101327 also reacts with dG·dC base pairs of B-DNA and forms a complex with Z-DNA of poly(br⁸dG-dC)·poly(br⁸DG-dC). The affinity of NSC-101327 to poly(dG-dC)·poly(dG-dC) is, however, lower, and the CD spectral binding effect depends on the ionic strength. The CD results of the complex with poly(dA-dT)·poly(dA-dT) suggests at least two binding modes, in accordance with previous conclusions. This is indicated by a clear-cut initial increase of the CD signal and a subsequent large decrease to negative CD signals. Competition experiments with netropsin suggest that binding of NSC-101327 occurs preferentially in the minor groove without intercalation. NSC-101327 also tends to interact with lower binding affinity to dG-dC pairs in B-DNA, with rA·rU pairs of RNA and with single-stranded polynucleotides. Thus our results suggest that NSC-101327 represents a DNA groove-binding ligand of lower basepair specificity and lower conformational selectivity compared to the B-specific netropsin probe.     

Stretching and overstretching of DNA in pulsed field gel electrophoresis. I. A quantitative study from the steady state birefringence decay

Using a sensitive birefringence instrument, the birefringence arising from the orientation of the DNA chain during electrophoretic transport has been recorded. This birefringence is shown to proceed both from the alignment (stretching) of the molecule in the direction of the electric field and from the extension of the length of its primitive path (overstretching). The contribution of these two processes can be separated in the decay of the birefringence after the end of the application of the electric field. The fast relaxation of the overstretching occurs first and is demonstrated to be the main contribution to the birefringence. The orientation factor of the remaining stretched state and its decay can be quantitatively understood using the biased reptation model. It provides, in addition, a high value for the tube diameter or gel pore size a (4500 ± 450 Å for a 0.7% agarose gel with a c^?0.6_g dependence in the agarose concentration c_g) and a low value for the effective charge per base pair (0.2e as compared to 0.5e using the condensation hypothesis). The contribution of overstretching to the birefringence is also quantitatively interpreted in term of the change in the mean length l of DNA inside a pore size a. The dynamics of decay of this overstretching is well represented by a stretched exponential with a stretching exponent α = 0.44. The mean decay time decreases slightly with increasing fields and scales with the overall DNA length close to N²₀. © 1993 John Wiley & Sons, Inc.     

Characteristic differences in the X-ray photoelectron spectrum between B-DNA and M-DNA monolayers on gold

Duplex DNA monolayers were self-assembled on gold through a disulfide linkage and both B- and M-DNA conformations were studied using X-ray photoelectron spectroscopy (XPS). The film thickness, density, elemental composition and ratios for samples were analyzed and compared. The DNA surface coverage, calculated from both XPS and electrochemical measurements, was approximately 1.2 × 10¹³ molecules/cm² for B-DNA. All samples showed distinct peaks for C 1s, O 1s, N 1s, P 2p and S 2p as expected for a thiol-linked DNA. On addition of Zn²⁺ to form M-DNA the C 1s, P 2p and S 2p showed only small changes while both the N 1s and O 1s spectra changed considerably. This result is consistent with Zn²⁺ interacting with oxygen on the phosphate backbone as well as replacing the imino protons of thymine (T) and guanine (G) in M-DNA. Analysis of the Zn 2p spectra also demonstrated that the concentration of Zn²⁺ present under M-DNA conditions is consistent with Zn²⁺ binding to both the phosphate backbone as well as replacing the imino protons of T or G in each base pair. After the M-DNA monolayer is washed with a buffer containing only Na⁺ the Zn²⁺ bound to the phosphate backbone is removed while the Zn²⁺ bound internally still remains.     

Nanomechanics of Diaminopurine-Substituted DNA

2,6-diaminopurine (DAP) is a nucleobase analog of adenine. When incorporated into double-stranded DNA (dsDNA), it forms three hydrogen bonds with thymine. Rare in nature, DAP substitution alters the physical characteristics of a DNA molecule without sacrificing sequence specificity. Here, we show that in addition to stabilizing double-strand hybridization, DAP substitution also changes the mechanical and conformational properties of dsDNA. Thermal melting experiments reveal that DAP substitution raises melting temperatures without diminishing sequence-dependent effects. Using a combination of atomic force microscopy (AFM), magnetic tweezer (MT) nanomechanical assays, and circular dichroism spectroscopy, we demonstrate that DAP substitution increases the flexural rigidity of dsDNA yet also facilitates conformational shifts, which manifest as changes in molecule length. DAP substitution increases both the static and dynamic persistence length of DNA (measured by AFM and MT, respectively). In the static case (AFM), in which tension is not applied to the molecule, the contour length of DAP-DNA appears shorter than wild-type (WT)-DNA; under tension (MT), they have similar dynamic contour lengths. At tensions above 60 pN, WT-DNA undergoes characteristic overstretching because of strand separation (tension-induced melting) and spontaneous adoption of a conformation termed S-DNA. Cyclic overstretching and relaxation of WT-DNA at near-zero loading rates typically yields hysteresis, indicative of tension-induced melting; conversely, cyclic stretching of DAP-DNA showed little or no hysteresis, consistent with the adoption of the S-form, similar to what has been reported for GC-rich sequences. However, DAP-DNA overstretching is distinct from GC-rich overstretching in that it happens at a significantly lower tension. In physiological salt conditions, evenly mixed AT/GC DNA typically overstretches around 60 pN. GC-rich sequences overstretch at similar if not slightly higher tensions. Here, we show that DAP-DNA overstretches at 52 pN. In summary, DAP substitution decreases the overall stability of the B-form double helix, biasing toward non-B-form DNA helix conformations at zero tension and facilitating the B-to-S transition at high tension.     

M-DNA: A complex between divalent metal ions and DNA which behaves as a molecular wire

M-DNA is a complex of DNA with divalent metal ions (Zn(2+), Co(2+), or Ni(2+)) which forms at pH conditions above 8. Upon addition of these metal ions to B-DNA at pH 8.5, the pH decreases such that one proton is released per base-pair per metal ion. Together with previous NMR data, this result demonstrated that the imino proton in each base-pair of the duplex was substituted by a metal ion and that M-DNA might possess unusual conductive properties. Duplexes of 20 base-pairs were constructed with fluorescein (donor) at one end and rhodamine (acceptor) at the other. Upon formation of M-DNA (with Zn(2+)) the fluorescence of the donor was 95 % quenched. Fluorescence lifetime measurements showed the presence of a very fast component in the decay kinetics with tau相似文献   

Individual Globular Domains and Domain Unfolding Visualized in Overstretched Titin Molecules with Atomic Force Microscopy

Titin is a giant elastomeric protein responsible for the generation of passive muscle force. Mechanical force unfolds titin’s globular domains, but the exact structure of the overstretched titin molecule is not known. Here we analyzed, by using high-resolution atomic force microscopy, the structure of titin molecules overstretched with receding meniscus. The axial contour of the molecules was interrupted by topographical gaps with a mean width of 27.7 nm that corresponds well to the length of an unfolded globular (immunoglobulin and fibronectin) domain. The wide gap-width distribution suggests, however, that additional mechanisms such as partial domain unfolding and the unfolding of neighboring domain multimers may also be present. In the folded regions we resolved globules with an average spacing of 5.9 nm, which is consistent with a titin chain composed globular domains with extended interdomain linker regions. Topographical analysis allowed us to allocate the most distal unfolded titin region to the kinase domain, suggesting that this domain systematically unfolds when the molecule is exposed to overstretching forces. The observations support the prediction that upon the action of stretching forces the N-terminal ß-sheet of the titin kinase unfolds, thus exposing the enzyme’s ATP-binding site and hence contributing to the molecule’s mechanosensory function.