A refined calculation of the solution dimensions of the complex between gene 32 protein and single stranded DNA based on estimates of the bending persistence length

The rotation diffusion coefficient of a complex of GP32, the single stranded DNA binding protein of the bacteriophage T4, with a single stranded DNA fragment with about 270 bases was determined to obtain further information on the flexibility of this particle. The rotation diffusion of these molecules is used as a sensitive measure of the flexibility of different DNA protein complexes. Using the theory of Hagerman and Zimm (Biopolymers 20, 1481 (1981)) and assuming a bending persistence length of about 35 nanometer it can be shown that the axial increment for GP32 complexes with single stranded DNA is close to 0.5 nm per base. The value for the bending persistence length is in agreement with values found for much larger DNA protein complexes using light scattering experiments. This value for the persistence length also implies that the complex is thin. The radius is estimated to be around 1.7 nm, which shows a moderate degree of hydration. With this set of parameters we can describe all the hydrodynamic experiments on GP32 complexes from 76 to more than 7000 bases obtained using electric birefringence, quasi-elastic light scattering and sedimentation experiments performed in our group over the last few years.     

Structure and dynamics of the complex of single stranded DNA binding protein of Escherichia coli with circular single stranded DNA of filamentous phages

We have analyzed the equilibrium and nonequilibrium properties of the complex of the single stranded DNA binding protein of Escherichia coli (EcoSSB) and circular single stranded DNA of filamentous phages M13mp8 and F1 using static and dynamic light scattering, analytical ultracentrifugation and electron microscopy. Upon binding to the single stranded DNA the EcoSSB tetramer replaces an equivalent volume of water trapped within the coiled single stranded DNA and hinders the folding of the single stranded DNA into secondary structures at all salt concentrations. The salt dependent compaction of the stoichiometric complex can be described assuming a flexible polyelectrolyte chain. The solution structure of the macromolecular complex is a random coil and in the electron microscope a beaded flexible structure of the complex with a bead diameter of 6 nm appears at all salt concentrations used. The internal motions of the stoichiometric complex can be described by the Rouse-Zimm model of polymer dynamics. The segmental mobility of the complex can be correlated with changes in the binding site size of the EcoSSB tetramer; it indicates the presence of interactions between EcoSSB tetramers bound to single stranded DNA.     

Adsorption of DNA onto positively charged amidine colloidal spheres and the resultant bridging interaction

The complexation behaviour of duplex linear DNA (negatively charged) with amidine functionalised sub-micron latex spheres (positively charged) was studied using dynamic light scattering (DLS) and a PALS interferrometric zeta potential sizer. Four types of DNA-sphere complex were investigated as a function of component concentration by combining amidine functionalised polystyrene microspheres with radii of 10.5 nm and 60 nm, and herring DNA of lengths of 35 nm and 85 nm. At low DNA concentrations (c(DNA)), the undercharged complexes showed a small increase in measured hydrodynamic radius (R(h)) and a decrease in zeta potential with increasing c(DNA). Within a critical DNA concentration range R(h) was seen to peak sharply, and the zeta potentials were approximately 0 mV, corresponding to the formation of unstable neutral complexes. Immediately above this concentration region the measured R(h) values became comparable with those at low c(DNA), and the zeta potential became negative, indicating the formation of stable overcharged complexes. The small and large spheres formed multi-sphere and single sphere overcharged aggregates respectively, which is thought to be determined by the relative magnitude of the chain persistence length (approximately 50 nm) and the sphere radius, switching on or off the DNA bridging interaction.     

Structure of human platelet membrane glycoproteins IIb and IIIa as determined by electron microscopy

The glycoprotein (GP) IIb-IIIa complex was isolated from human platelet membranes and examined for glycoprotein stoichiometry and morphology. To determine the ratio of glycoproteins in the complex, the isolated glycoproteins were solubilized with sodium dodecyl sulfate and separated by high-performance liquid chromatography. Quantitative amino acid analysis of individual glycoproteins showed that the ratio of GP IIb to GP IIIa in the Ca2+-dependent complex was 0.93:1. Morphology was determined by electron microscopy of rotary-shadowed and negatively stained specimens. Individual complexes consisted of two domains: an oblong head of approximately 8 X 10 nm with two rodlike tails extending approximately 14-17 nm from one side of the head. Treatment of the isolated complex with EDTA resulted in the appearance of a mixture of oblong and filamentous structures, which could be separated by a sucrose gradient sedimentation in Triton X-100. As seen by rotary and unidirectional shadowing, GP IIb was a compact structure, approximately 8 X 10 nm in size. Isolated GP IIIa was more heterogeneous but was most often observed in an elongated form, varying in length from 20 to 30 nm and in width from 2 to 3 nm. By comparing these structures to that of the heterodimer complex, it was determined that the oblong domain was GP IIb and the rodlike tails were GP IIIa. Each milligram of isolated GP IIb-IIIa complex bound 0.30 mg of [3H]Triton X-100, indicating that the glycoprotein complex contained limited hydrophobic domains. Upon removal of detergent, GP IIb-IIIa complexes formed aggregates that sedimented in sucrose gradients as a diffuse peak ranging from 14 to 32 s. Examination of these aggregates by electron microscopy showed that they were composed of clusters or "rosettes" of 2 to 20 or more of the GP IIb-IIIa complexes. The orientation of these rosettes was such that the tails were joined in the center, with the head portions directed away from the interacting tails. It thus appears that the primary hydrophobic domains of the GP IIb-IIIa complex exist at the tips of the GP IIIa tails. Because the GP IIb-IIIa complex is an intrinsic membrane glycoprotein, these findings indicate a potential membrane attachment site for the GP IIb-IIIa complexes.     

Effects of Na+ on the persistence length and excluded volume of T7 bacteriophage DNA.

Total intensity, Rayleigh light scattering has been used to measure the rms radius, second virial coefficient, persistence length, and excluded volume of homogeneous T7 bacteriophage DNA as a function of Na+ concentration (0.005 to 3.0 M). All parameters decrease sharply as [Na+] increases, and tend to level off at high Na+. The variation of persistence length with [Na+] is consistent with predictions from counterion condensation theory.     

Intramolecular interference effects in dynamic light scattering: rigid double spirals and superhelical DNAs

A theory is developed for dynamic light scattering (DLS) from rigid double spirals by treating an invisible rigid cylinder with two helical scattering stripes on opposite sides of its cylindrical surface. The exact initial, or first cumulant, diffusion coefficient Dapp (K) is obtained in terms of the translational diffusion coefficients (D parallel and D perpendicular) parallel and perpendicular to the symmetry axis, the rotational diffusion coefficients (DR parallel and DR perpendicular) around the symmetry and transverse axes, the length (L) and radius (b) of the cylindrical surface bearing the stripes, and the pitch (p). Interference effects, namely geometrical antiresonances, between strands, produce deep minima in the static structure factor S (K) and corresponding prominent peaks in Dapp (K). These peaks in Dapp (K) depend sensitively on the rotational dynamics around the symmetry axis, and nearly vanish when DR parallel = 0. Some results for single spirals are also presented. A simpler model in which scattering points are attached at opposite ends of an otherwise invisible thin rigid rod is also treated, and shown to exhibit modest minima in S (K) and corresponding maxima in Dapp (K). Confining this rod to a plane containing K enhances the amplitudes of the oscillations in S (K) and Dapp (K), as expected. Rigid double spirals are employed as crude models for interwound supercoiled DNAs in order to assess the possible occurrence of interference effects. Although native supercoiled DNAs exhibit a cylinder diameter that is much too small to exhibit geometrical antiresonances in the presently accessible range of K2, nearly relaxed supercoiled DNAs are predicted to exhibit their first maximum in Dapp (K) just inside this range. Previously reported data for the effect of Escherichia coli single-strand binding (ssb) protein on the DLS of supercoiled pBR322 DNA cannot be mimicked by a gradual homogeneous reduction of superhelix density with increasing ssb, but instead can be mimicked by inhomogeneous all-or-none binding in which uncomplexed native DNAs and nearly relaxed saturated ssb/DNA complexes coexist in varying proportions. Experimental Dapp (K) and S (K) data for a sample of relaxed pUC8 dimers display, respectively, a broad maximum and a corresponding minimum, in qualitative agreement with rough theoretical predictions.     

Ru(phen)2DPPZ]2+ is in contact with DNA bases when it forms a luminescent complex with single-stranded oligonucleotides

The luminescence intensity of the Delta- and Lambda-enantiomer of [Ru(phen)2DPPZ]2+ ([Ru(phenanthroline)2 dipyrido[3,2-a:2',3'-c]phenazine]2+) complex enhanced upon binding to double stranded DNA, which has been known as "light switch effect". The enhancement of the luminescence required the intercalation of the large ligand between DNA base pairs. In this study, we report the enhancement in the luminescence intensity when the metal complexes bind to single stranded oligonucleotides, indicating that the "light switch effect" does not require intercalation of the large DPPZ ligand. Oligonucleotides may provide a hydrophobic cavity for the [Ru(phen)2DPPZ]2+ complex to prevent the quenching by the water molecule. In the cavity, the metal complex is in contact with DNA bases as is evidenced by the observation that the excited energy of the DNA bases transfer to the bound metal complex. However, the contact of the metal complex with DNA bases is different from the stacking of DPPZ in the intercalation pocket. In addition to the normal two luminescence lifetimes, a short lifetime in the range of 1-2 ns was found for both the delta- and lambda-enantiomer of [Ru(phen)2DPPZ]2+ when complexed with single stranded oligonucleotides, which may be assigned to the metal complex that is outside of the cavity, interacting with phosphate groups of DNA.     

Salt dependent changes in structure and dynamics of circular single stranded DNA of filamentous phages of Escherichia coli

We have analyzed the static and dynamic behaviour of the circular single stranded DNA of the filamentous Escherichia coli phages F1 and M13mp8 in solution as a function of salt concentration using static and dynamic light scattering and sedimentation analysis in the analytical ultracentrifuge. We show by static light scattering that native and denatured single stranded DNA behave like a randomly coiled macromolecule at all salt concentrations used. The size of the native single stranded DNA is governed by the formation of secondary structures. While the radius of gyration decreases with increasing salt concentration the translational diffusion of the center-of-mass of native single stranded DNA and the sedimentation coefficient increase with increasing salt concentration in a biphasic manner. Below 100 mM monovalent cation concentration there is a strong dependence of the hydrodynamic parameters upon salt which is reduced approx. 3-fold at higher salt concentrations. We attribute the compaction of single stranded DNA by salt to electrostatic shielding and, in case of native single stranded DNA, secondary structure formation. Internal motions of the native single stranded DNA are observable at all salt concentrations and can be interpreted with a model of segmental diffusion of the elements of the polymer chain. The observed segmental diffusion coefficient of the native single stranded polynucleotide increases with increasing salt under the conditions investigated.     

A study of the chain stiffness and extension of alginates, in vitro epimerized alginates, and periodate-oxidized alginates using size-exclusion chromatography combined with light scattering and viscosity detectors

A series of alginates isolated from the stem and leaf of a brown algae (Laminaria hyperborea), bacterial mannuronan, in vitro epimerized mannuronans, and periodate oxidized alginates were analyzed by size-exclusion chromatography (SEC) combined with online multiangle laser light scattering (MALS) and viscometry (collectively abbreviated SMV). Selected samples were also analyzed off-line using low-angle laser light scattering and capillary viscometry. Excellent agreement between the two methods was obtained for properly purified samples. In contrast, abnormal results were obtained for some industrial samples due to the presence of particulate material. Naturally occurring alginates and in vitro epimerized mannuronans were found to obey essentially the same RG-M and [eta]-M relations, and hence, the same Mark-Houwink-Sakurada (MHS) equations (valid for I = 0.10 M): 20 000 g/mol < M < 100 000 g/mol, [eta] = 0.0054 .M(1.00); 100 000 g/mol < M < 1 000 000 g/mol, [eta] = 0.071 .M(0.89). Application of the wormlike chain model to the [eta]-M data obtained by SMV yielded persistence lengths (q) of 15 nm for all alginates at an ionic strength of 0.17 M. Intrinsic viscosities corresponding to infinite ionic strength were estimated on the basis of Smidsr?d's B-parameter, and the wormlike chain model then yielded q = 12 nm. Periodate oxidized alginates showed, in contrast, a pronounced decrease in persistence length with increasing degree of oxidation, reaching values below 4 nm at 44% oxidation. Periodate oxidation also resulted in some depolymerization, even in the presence of a free-radical scavenger.     

Change of conformation and internal dynamics of supercoiled DNA upon binding of Escherichia coli single-strand binding protein

The influence of Escherichia coli single-strand binding (SSB) protein on the conformation and internal dynamics of pBR322 and pUC8 supercoiled DNAs has been investigated by using dynamic light scattering at 632.8 and 351.1 nm and time-resolved fluorescence polarization anisotropy of intercalated ethidium. SSB protein binds to both DNAs up to a stoichiometry that is sufficient to almost completely relax the superhelical turns. Upon saturation binding, the translational diffusion coefficients (D0) of both DNAs decrease by approximately 20%. Apparent diffusion coefficients (Dapp) obtained from dynamic light scattering display the well-known increase with K2 (K = scattering vector), leveling off toward a plateau value (Dplat) at high K2. For both DNAs, the difference Dplat - D0 increases upon relaxation of supercoils by SSB protein, which indicates a corresponding enhancement of the subunit mobilities in internal motions. Fluorescence polarization anisotropy measurements on free and complexed pBR322 DNA indicate a (predominantly) uniform torsional rigidity for the saturated DNA/SSB protein complex that is significantly reduced compared to the free DNA. These observations are all consistent with the notion that binding of SSB protein is accompanied by a gradual loss of supercoils and saturates when the superhelical twist is largely removed.     

Label-less fluorescence-based method to detect hybridization with applications to DNA micro-array

By coupling scattered light from DNA to excite fluorescence in a polymer, we describe a quantitative, label-free assay for DNA hybridization detection. Since light scattering is intrinsically proportional to number of molecules, the change in (scattering coupled) fluorescence is highly linear with respect to percent binding of single stranded DNA (ssDNA) target with the immobilized ssDNA probes. The coupling is achieved by immobilizing ssDNA on a fluorescent polymer film at optimum thickness in nanoscale. The fluorescence from the underlining polymer increases due to proportionate increase in scattering from double stranded DNA (dsDNA) (i.e., probe-target binding) compared to ssDNA (i.e., probe). Because the scattering is proportional to fourth power of refractive index, the detection of binding is an order of magnitude more sensitive compared to other label-free optical methods, such as, reflectivity, interference, ellipsometry and surface-plasmon resonance. Remarkably, polystyrene film of optimum thickness 30 nm is the best fluorescent agent since its excitation wavelength matches (within 5 nm) with wavelength for the maximum refractive index difference between ssDNA and dsDNA. A quantitative model (with no fitting parameters) explains the observations. Potential dynamic range is 1 in 10(4) at signal-to-noise ratio of 3:1.     

Chain flexibility and hydrodynamics of the B and Z forms of poly(dG-dC).poly(dG-dC).

The solution properties of the B and Z forms of poly(dG-dC).poly(dG-dC) have been measured by static and dynamic laser light scattering. The radius of gyration, persistence length, translational and segmental diffusion coefficients, and the Rouse-Zimm parameters have been evaluated. The persistence length of the Z form determined at 3 M NaCl is about 200 nm compared to 84 and 61 nm respectively for the B forms of poly(dG-dC).poly(dG-dC), and calf thymus DNA, both determined at 0.1 M NaCl. The data on persistence length, diffusion coefficients and the Rouse-Zimm parameters indicate a large increase in the chain stiffness of Z DNA compared to the B form. These results are opposite to the ionic strength effects on random sequence native DNAs, for which the flexibility increases with ionic strength and levels off at about 1 M NaCl.     

Time resolved structure analysis of growing beta-amyloid fibers

Formation of beta-amyloid plaques is a crucial feature of Alzheimer's disease. In the present work time resolved static light scattering was applied to investigate the size and shape of growing beta-amyloid aggregates preceding plaque formation. The beta-amyloid protein with 40 amino acid residues was used. Salt free buffer solutions and solutions with 0.15M NaCl at 37 degrees C served as the aggregation medium. The focus lay on the first 2h following initiation of the aggregation process which corresponds to the protofibril phase. Addition of the NaCl accelerated the aggregation process considerably. Scattering data from aggregation in saline solutions indicated formation of long fibers which suggest interpretation of data with the worm-like chain model. Two important results were revealed: (i) At the end of the time resolved recordings, the worm-like chain model provided a fully adequate picture for the growing aggregates. Chain stiffness is characterised in terms of the persistence length, which is close to 50 nm. The linear mass density of the growing fibers approached a value of two monomers per nm corresponding to single stranded fibers, which is in accordance with presently existing models for the aggregation of beta-amyloid. The fibers finally reached contour lengths of several thousand nanometers. (ii) The plateau values for the persistence length and linear mass density observed in the final regime are gradually approached from higher values. This observation is inconsistent with simple worm-like chains. Rather does it indicate existence of another species during the initial phase of the aggregation, in addition to monomers and fibers. Aside from further insight into fundamental aspects of beta-amyloid aggregation, time resolved static light scattering provides an appropriate tool for assay tests with drugs designed to interfere with the aggregation process.     

DNA‐doxorubicin interaction: New insights and peculiarities

We have investigated the interaction of the DNA molecule with the anticancer drug doxorubicin (doxo) by using three different experimental techniques: single molecule stretching, single molecule imaging, and dynamic light scattering. Such techniques allowed us to get new insights on the mechanical behavior of the DNA‐doxo complexes as well as on the physical chemistry of the interaction. First, the contour length data obtained from single molecule stretching were used to extract the physicochemical parameters of the DNA‐doxo interaction under different buffer conditions. This analysis has proven that the physical chemistry of such interaction can be modulated by changing the ionic strength of the surrounding buffer. In particular we have found that at low ionc strengths doxo interacts with DNA by simple intercalation (no aggregation) and/or by forming bound dimers. For high ionic strengths, otherwise, doxo‐doxo self‐association is enhanced, giving rise to the formation of bound doxo aggregates composed by 3 to 4 molecules along the double‐helix. On the other hand, the results obtained for the persistence length of the DNA‐doxo complexes is strongly force‐dependent, presenting different behaviors when measured with stretching or non‐stretching techniques.     

Soluble complex formation between low-density lipoprotein and glycosaminoglycans. A 2H and 31P-NMR, and quasi-elastic light scattering study

Soluble complex formation between LDL and heparin (HEP) and chondroitin sulfate (CS) has been studied by 2H- and 31P-NMR and light scattering. The 2H-NMR linewidths of [2H]HEP and [2H]C4S increase substantially upon binding to LDL, with the [2H]HEP linewidths broader at low glycosaminoglycan (GAG)/low density lipoprotein (LDL) ratios. Preliminary analysis of the bound C2H3 group correlation times suggests that the observed linewidths are determined by the complex size, and that both [2H]GAGs have similar motions when bound to LDL. The 31P-NMR data demonstrate that large LDL-HEP complexes (diameter approx. 50 nm) are formed only over a narrow range of HEP concentrations, whereas the size of LDL-CS complexes increases continuously over the range of CS concentrations studied, reaching values of 32-35 nm for both C4S and C6S. At the lower protein concentrations studied by light scattering (less than or equal to 1 mg/ml), the same trends are observed, although the mean diameters are less than those estimated by 31P-NMR. Soluble complex formation was unaffected by the presence of 2 mM Ca2+. Dilution studies demonstrate that complex size varies with protein concentration. The binding of GAGs to LDL was also examined by HEP-CS competition studies. HEP has the higher affinity while no differences in binding could be detected between C4S and C6S.     

Incomplete reversion of double stranded DNA cleavage mediated by Drosophila topoisomerase II: formation of single stranded DNA cleavage complex in the presence of an anti-tumor drug VM26.

Anti-tumor drug VM26 greatly stimulates topoisomerase II mediated DNA cleavage by stabilizing the cleavable complex. Addition of a strong detergent such as SDS to the cleavable complex induces the double stranded DNA cleavage. We demonstrate here that heat treatment can reverse the double stranded DNA cleavage; however, topoisomerase II remains bound to DNA even in the presence of SDS. This reversed complex has been shown to contain single strand DNA breaks with topoisomerase II covalently linked to the nicked DNA. Chelation of Mg++ by EDTA and the addition of salt to a high concentration also reverse the double strand DNA cleavage, and like heat reversion, topoisomerase II remains bound to DNA through single strand DNA break. The reversion complex can be analyzed and isolated by CsCl density gradient centrifugation. We have detected multiple discrete bands from such a gradient, corresponding to protein/DNA complexes with 1, 2, 3, ..... topoisomerase II molecules bound per DNA molecule. Analysis of topoisomerase II/DNA complexes isolated from the CsCl gradient indicates that there are single stranded DNA breaks associated with the CsCl stable complexes. Therefore, topoisomerase II/DNA complex formed in the presence of VM26 cannot be completely reversed to yield free DNA and enzyme. We discuss the possible significance of this finding to the mechanism of action of VM26 in the topoisomerase II reactions.     

A measurement of the fraction of chloroplast DNA transcribed in Euglena

The fraction of the chloroplast DNA transcribed in the single celled alga $\text{Euglena}$ has been determined by RNA-DNA hybridization. A vast excess of total cell RNA from cells which were rapidly dividing in the light was hybridized in liquid to [¹²⁵I] — chloroplast DNA, and the resulting duplexes separated on hydroxyapatite columns. The contribution of DNA-DNA duplex formation was determined separately and was used to calculate that portion of the duplex which was actually a RNA-DNA hybrid. Sixteen percent of the single stranded chloroplast DNA forms a duplex with this RNA suggesting that 32 percent of the double stranded DNA molecule is being transcribed into RNA under these conditions of cell growth.     

Dynamics of superhelical DNA studied by photon correlation spectroscopy

We have conducted photon correlation spectroscopy (PCS) studies on the plasmid pUC8 (2717 bp) in order to elucidate the internal dynamics of this superhelical DNA. We confirm that the first-order autocorrelation function of the scattered light from pUC8 solutions can be separated into two distinct exponential decay components, as first shown by Lewis et al. (R. Lewis, J.H. Huang and P. Pecora, Macromolecules 18 (1985) 944). A thorough analysis of the dependence on scattering vector K of the rates and amplitudes of the two components enables us to assign the slowly relaxing part to the center-of-mass diffusion of the DNA, while the faster component corresponds to rotational, bending and twisting motions of the superhelix. For larger K values the internal motions can be formally expressed in terms of an 'internal diffusion coefficient' Di, whose value of 2.0-2.5 X 10(-11) m2 s-1 is approximately equal to the translational diffusion coefficient predicted for a stiff DNA piece of the persistence length, 65 nm. Comparison of our measured Di values to those predicted from a recent theory of circular worm-like coils (K. Soda, Macromolecules 17 (1984) 2365) shows that the internal motions are faster than the theoretical values. One of the reasons for this discrepancy could be that the theory does not take into account torsional motions, which contribute significantly to the internal dynamics (J.C. Thomas, S.A. Allison, C.J. Appelof and J.M. Schurr, Biophys. Chem. 12 (1980) 177). At low K values, the fast relaxation of superhelical pUC8 is no longer proportional to K2, but reaches a constant value as K approaches zero. This behavior, not seen for the linearized DNA, can be interpreted in terms of rotational diffusion of a flexible rod-like molecule (T. Maeda and S. Fujime, Macromolecules 17 (1984) 2381) and supports an interwound rod-like structure for pUC8 DNA with an average end-to-end distance of 220 nm.     

Dynamic light scattering study of the effect of Mg2+ and ATP on synthetic myosin filaments.

The dynamic light scattering (DLS) method provides us with information about the apparent diffusion coefficient, Dapp, as well as the static scattering intensity, Is, of particles in solution. For long but thin rods with length L and diameter d, the dependence on L and d of Dapp is quite different from that of Is. By means of DLS we studied synthetic myosin filaments of rabbit skeletal muscle in solution at pH 8.3 and 10 degrees C. It appeared that Mg2+ ions induced thickening and lengthening of the filaments, whereas ATP (and ADP) induced thinning and shortening (depolymerization) of the filaments. When ATP was added to the filament preparation in the presence of Mg2+ ions, it was clearly observed that thinning of the filament (or splitting into subfilaments) occurred before shortening (or depolymerization).     

Persistence length of titin from rabbit skeletal muscles measured with scattering and microrheology techniques

The persistence length of titin from rabbit skeletal muscles was measured using a combination of static and dynamic light scattering, and neutron small angle scattering. Values of persistence length in the range 9-16 nm were found for titin-II, which corresponds to mainly physiologically inelastic A-band part of the protein, and for a proteolytic fragment with 100-nm contour length from the physiologically elastic I-band part. The ratio of the hydrodynamic radius to the static radius of gyration indicates that the proteins obey Gaussian statistics typical of a flexible polymer in a -solvent. Furthermore, measurements of the flexibility as a function of temperature demonstrate that titin-II and the I-band titin fragment experience a similar denaturation process; unfolding begins at 318 K and proceeds in two stages: an initial gradual 50% change in persistence length is followed by a sharp unwinding transition at 338 K. Complementary microrheology (video particle tracking) measurements indicate that the viscoelasticity in dilute solution behaves according to the Flory/Fox model, providing a value of the radius of gyration for titin-II (63 +/- 1 nm) in agreement with static light scattering and small angle neutron scattering results.