Quantitative study of polymer conformation and dynamics by single-particle tracking

We present a new method for analyzing the dynamics of conformational fluctuations of individual flexible polymer molecules. In single-particle tracking (SPT), one end of the polymer molecule is tethered to an immobile substratum. A microsphere attached to the other end serves as an optical marker. The conformational fluctuations of the polymer molecule can be measured by optical microscopy via the motion of the microsphere. The bead-and-spring theory for polymer dynamics is further developed to account for the microsphere, and together the measurement and the theory yield quantitative information about molecular conformations and dynamics under nonperturbing conditions. Applying the method to measurements carried out on DNA molecules provides information complementary to recent studies of single DNA molecules under extensional force. Combining high precision measurements with the theoretical analysis presented here creates a powerful tool for studying conformational dynamics of biological and synthetic macromolecules at the single-molecule level.     

Flexibility of DNA in complex with proteins deduced from the distribution of bending angles observed by scanning force microscopy

Flexibility and dynamics of DNA are important for DNA-binding and recognition by proteins. Here the flexibility of DNA is calculated from the distribution of DNA-bending angles of single DNA molecules as observed by scanning force microscopy by applying an equation that links the force constant of DNA-bending (f) to the variance of the distribution of bending angles (sigma): f=RT/sigma(2). Using published data, f is calculated to be 3-5 J/degree(2) for free DNA. Thus, bending DNA by 20 degrees requires approx. 0.5-1 kJ/mol. This result shows that DNA is very flexible and readily can be bent by thermal motion. DNA-flexibility is not altered in some protein-DNA complexes (HhaI methyltransferase, EcoRV restriction endonuclease). In contrast, DNA-binding by EcoRI endonuclease increases DNA-flexibility and binding by EcoRI methyltransferase restricts the flexibility of DNA. During the transition of the RNA polymerase-sigma(54)-DNA complex from the closed to the open form and of cro repressor from a non-specific to a specific binding mode the flexibility of the DNA is strongly reduced.     

Inhibition of microtubule formation by DNA

In vitro assembly of porcine brain microtubular protein to microtubules is affected by calf thymus DNA. Dependent on mass ratio of DNA/MTP microtubule formation is partly inhibited or blocked. Microtubules formed in presence of DNA are not to be distinguished from those assembled without DNA by electron microscopy. Addition of DNA to microtubules in assembly buffer causes their disassembly.     

The interaction of E. coli integration host factor and lambda cos DNA: multiple complex formation and protein-induced bending.

The interaction of E. coli's integration Host Factor (IHF) with fragments of lambda DNA containing the cos site has been studied by gel-mobility retardation and electron microscopy. The cos fragment used in the mobility assays is 398 bp and spans a region from 48,298 to 194 on the lambda chromosome. Several different complexes of IHF with this fragment can be distinguished by their differential mobility on polyacrylamide gels. Relative band intensities indicate that the formation of a complex between IHF and this DNA fragment has an equilibrium binding constant of the same magnitude as DNA fragments containing lambda's attP site. Gel-mobility retardation and electron microscopy have been employed to show that IHF sharply bends DNA near cos and to map the bending site. The protein-induced bend is near an intrinsic bend due to DNA sequence. The position of the bend suggests that IHF's role in lambda DNA packaging may be the enhancement of terminase binding/cos cutting by manipulating DNA structure.     

Dynamic and Structural Changes in the Minimally Restructuring EcoRI Bound to a Minimally Mutated DNA Chain

The dynamics of a protein plays an important role in protein functionality. Here, we examine the differences in the dynamics of a minimally restructuring protein, EcoRI, when it is bound to its cognate DNA and to a noncognate sequence which differs by just a single basepair. Molecular dynamics simulations of the complexes and essential dynamics analyses reveal that the overall dynamics of the protein subunits change from a coordinated motion in the cognate complex to a scrambled motion in the noncognate complex. This dynamical difference extends to the protein-DNA interface where EcoRI tries to constrict the DNA in the cognate complex. In the noncognate complex, absence of the constricting motion of interfacial residues, overall change in backbone dynamics and structural relaxation of the arms enfolding the DNA leave the DNA less-kinked relative to the situation in the cognate complex, thus indicating that the protein is poised for linear diffusion along the DNA rather than for catalytic action. In a larger context, the results imply that the DNA sequences dictate protein dynamics and that when a protein chances upon the recognition sequence some of the key domains of the protein undergo dynamical changes that prepare the protein for eventual catalytic action.     

Real-time imaging of single DNA molecules with fluorescence microscopy.

A fluorescence microscopy technique was used to image the dynamics of individual DNA molecules. Lambda, calf thymus, cosmid (circular), and T4 DNA were studied with the fluorescent dye acridine orange. Experiments with DNAase I were conducted, and the results indicate that these observations correspond to DNA molecules. The results of experiments with circular DNA provide strong evidence that these were single DNA molecules. Molecules were observed free in solution or attached to a glass or copper surface at one or several points. The Brownian motion of these molecules was observed, indicating that DNA in solution exists in a partially supercoiled state. Some molecules appeared stretched and were attached to the surface by their termini; the lengths of these molecules were measured. Such molecules also exhibited elastic behavior upon breaking. The power of this technique is demonstrated in images of cosmid DNA molecules, catenanes, and DNA extending from T4 phage particles. These results suggest immediate applications to molecular biology, such as examining the dynamics of protein-DNA interactions. Areas of ongoing research are discussed.     

Characterizing locus specific chromatin structure and dynamics with correlative conventional and super-resolution imaging in living cells

The dynamic rearrangement of chromatin is critical for gene regulation, but mapping both the spatial organization of chromatin and its dynamics remains a challenge. Many structural conformations are too small to be resolved via conventional fluorescence microscopy and the long acquisition time of super-resolution photoactivated localization microscopy (PALM) precludes the structural characterization of chromatin below the optical diffraction limit in living cells due to chromatin motion. Here we develop a correlative conventional fluorescence and PALM imaging approach to quantitatively map time-averaged chromatin structure and dynamics below the optical diffraction limit in living cells. By assigning localizations to a locus as it moves, we reliably discriminate between bound and unbound dCas9 molecules, whose mobilities overlap. Our approach accounts for changes in DNA mobility and relates local chromatin motion to larger scale domain movement. In our experimental system, we show that compacted telomeres move faster and have a higher density of bound dCas9 molecules, but the relative motion of those molecules is more restricted than in less compacted telomeres. Correlative conventional and PALM imaging therefore improves the ability to analyze the mobility and time-averaged nanoscopic structural features of locus specific chromatin with single molecule sensitivity and yields unprecedented insights across length and time scales.     

Dynamic and Structural Changes in the Minimally Restructuring EcoRI Bound to a Minimally Mutated DNA Chain

Abstract

The dynamics of a protein plays an important role in protein functionality. Here, we examine the differences in the dynamics of a minimally restructuring protein, EcoRI, when it is bound to its cognate DNA and to a noncognate sequence which differs by just a single basepair. Molecular dynamics simulations of the complexes and essential dynamics analyses reveal that the overall dynamics of the protein subunits change from a coordinated motion in the cognate complex to a scrambled motion in the noncognate complex. This dynamical difference extends to the protein-DNA interface where EcoRI tries to constrict the DNA in the cognate complex. In the noncognate complex, absence of the constricting motion of interfacial residues, overall change in backbone dynamics and structural relaxation of the arms enfolding the DNA leave the DNA less-kinked relative to the situation in the cognate complex, thus indicating that the protein is poised for linear diffusion along the DNA rather than for catalytic action. In a larger context, the results imply that the DNA sequences dictate protein dynamics and that when a protein chances upon the recognition sequence some of the key domains of the protein undergo dynamical changes that prepare the protein for eventual catalytic action.     

The Connection between Chromatin Motion on the 100 nm Length Scale and Core Histone Dynamics in Live XTC-2 Cells and Isolated Nuclei

The diffusive motion of DNA-containing chromatin in live cells and isolated nuclei is investigated using a two-photon standing wave fluorescence photobleaching experiment with 100 nm spatial resolution. The chromatin is labeled using the minor groove binding dye Hoechst 33342. In live cells, the mean diffusion rate is 5 × 10⁻⁴ μm²/s, with considerable cell-to-cell variation. This diffusion is highly constrained and cannot be observed in a standard, single beam fluorescence recovery after photobleaching experiment. To determine the chemical origin of the diffusion, we study motion in isolated nuclei and vary the strength of the histone-DNA interactions by changing the ionic strength and using chemical and photocross-linking experiments. At higher NaCl concentrations, we see increased chromatin diffusion as the histone-DNA interaction is weakened due to ionic screening, whereas photocross-linking the core histones to the DNA results in a complete absence of diffusive motion. These trends are consistent with the 100 nm scale motion being correlated with the interactions of histone proteins with the DNA. If chromatin diffusion is connected to the nucleosomal dynamics on much smaller length scales, this may provide a way to assay biochemical activity in vivo based on larger scale macromolecular dynamics observed via fluorescence microscopy.     

Functionally important correlated motions in the single-stranded DNA-binding protein encoded by filamentous phage Pf3

To elucidate the interplay between different parts of dimeric single-stranded DNA-binding proteins we have studied the correlated motions in the protein encoded by filamentous phage Pf3 via the combined use of 15N-NMR relaxation experiments, molecular dynamics simulations and essential dynamics calculations. These studies provide insight into the mechanism underlying the protein-DNA binding reaction. The most important motions can be described by a few essential modes. Most outstanding is the correlated symmetric motion of the DNA-binding wings, which are far apart in the structure. This motion determines the access of DNA to the DNA-binding domain. A correlation between the motion of the DNA-binding wing and the complex loop is indicated to play a role in the cooperative binding of the protein to DNA. These motions are in the nanosecond regime in correspondence with the 15N-NMR relaxation experiments.     

Fluorescence Microscopy of Single Viral Capsids

To build a foundation for the single-molecule fluorescence microscopy of protein complexes, the present study achieved fluorescence microscopy of single, nucleic acid-free protein capsids of bacteriophage T7. The capsids were stained with Alexa 488 (green emission). Manipulation of the capsids' thermal motion was achieved in three dimensions. The procedure for manipulation included embedding the capsids in an agarose gel. The data indicate that the thermal motion of capsids is reduced by the sieving of the gel. The thermal motion can be reduced to any desired level. A semilogarithmic plot of an effective diffusion constant as a function of gel concentration is linear. Single, diffusing T7 capsids were also visualized in the presence of single DNA molecules that had been both stretched and immobilized by gel-embedding. The DNA molecules were stained with ethidium (orange emission). This study shows that single-molecule (protein and DNA) analysis is possible for both packaging of DNA in a bacteriophage capsid and other events of DNA metabolism. The major problem is the maintenance of biochemical activity.     

Dissecting protein-induced DNA looping dynamics in real time

Many proteins that interact with DNA perform or enhance their specific functions by binding simultaneously to multiple target sites, thereby inducing a loop in the DNA. The dynamics and energies involved in this loop formation influence the reaction mechanism. Tethered particle motion has proven a powerful technique to study in real time protein-induced DNA looping dynamics while minimally perturbing the DNA–protein interactions. In addition, it permits many single-molecule experiments to be performed in parallel. Using as a model system the tetrameric Type II restriction enzyme SfiI, that binds two copies of its recognition site, we show here that we can determine the DNA–protein association and dissociation steps as well as the actual process of protein-induced loop capture and release on a single DNA molecule. The result of these experiments is a quantitative reaction scheme for DNA looping by SfiI that is rigorously compared to detailed biochemical studies of SfiI looping dynamics. We also present novel methods for data analysis and compare and discuss these with existing methods. The general applicability of the introduced techniques will further enhance tethered particle motion as a tool to follow DNA–protein dynamics in real time.     

Charge transport in DNA model with vibrational and rotational coupling motions

The dynamics of the Peyrard-Bishop model for vibrational motion of DNA dynamics, which has been extended by taking into account the rotational motion for the nucleotides (Silva et al., J. Biol. Phys. 34, 511–519, 2018) is studied. We report on the presence of the modulational instability (MI) of a plane wave for charge migration in DNA and the generation of soliton-like excitations in DNA nucleotides. We show that the original differential-difference equation for the DNA dynamics can be reduced in the continuum approximation to a set of three coupled nonlinear equations. The linear stability analysis of continuous wave solutions of the coupled systems is performed and the growth rate of instability is found numerically. Numerical simulations show the validity of the analytical approach with the generation of wave packets provided that the wave numbers fall in the instability domain.     

Electron Microscope and Autoradiographic Study of Ultrastructural Aspects of Competence and Deoxyribonucleic Acid Absorption in Bacillus subtilis: Localization of Uptake and of Transport of Transforming Deoxyribonucleic Acid in Competent Cells

With the aid of serial-section electron microscopy two types of mesosomes can be distinguished in cells of competent cultures of Bacillus subtilis: (i) mesosomes connected to the plasma membrane only (plasma membrane mesosomes) and (ii) mesosomes which extend from the plasma membrane into the nuclear bodies (nuclear mesosomes). Contrary to plasma membrane mesosomes, nuclear mesosomes are absent from the tip zones. Electron microscopic autoradiography of sections of Bacillus subtilis cells exposed to [(3)H]thymidine-labeled transforming deoxyribonucleic acid (DNA) for a short period of time shows that the DNA becomes associated with mesosomes. As a function of time the DNA migrates towards the nucleoids. Transport of DNA is completed within 15 to 60 min after termination of DNA uptake. During its migration the DNA continues to be associated with mesosomes, presumably with nuclear mesosomes. DNA initially associated with plasma membrane mesosomes of the tip zones is probably transported first towards the middle zones peripherally and from there towards the nucleoids.     

Hydration dependence of chain dynamics and local diffusion in L-alpha-dipalmitoylphosphtidylcholine multilayers studied by incoherent quasi-elastic neutron scattering.

Incoherent quasi-elastic neutron scattering is applied to study the local diffusion and chain dynamics of L-alpha-diplamiotylphosphatidylcholine molecules in oriented model membranes. Different motions are distinguished by changing the hydration of the multilayers as well as by measuring below and above the gel-to-liquid crystalline phase transition. The time range of the utilized time-of-flight spectrometer permits to observe two types of motion to be observed more closely: chain defect motions and the local diffusion of the whole molecule in its solvation cage. Oriented lipid membranes are a useful system for the observation of chain defects, as they can be macroscopically oriented, in contrast to most polymers. As a representative model for a chain defect a kink is chosen and the corresponding scattering functions are derived. The kink motion can explain the entire dynamics seen in the gel phase, and the lifetime of such a defect was found to be 10-15 ps, in good agreement with theoretical predictions. On the other hand the dynamics in the liquid crystalline phase cannot be explained even by a superposition of several kinks and thus requires the consideration of an additional motion: the local diffusion of the molecule in its solvation cage. The size of the solvation cage is increasing with multilayer hydration and reduced temperature. Particularly interesting in view of recent discussions about the origin of the short-range repulsive forces between membranes is the experimental finding of an out-of-plane motion with an amplitude of 1-1.5 A, which cannot be explained by the undulation of the whole membrane.     

Dynamics and consequences of DNA looping by the FokI restriction endonuclease

Genetic events often require proteins to be activated by interacting with two DNA sites, trapping the intervening DNA in a loop. While much is known about looping equilibria, only a few studies have examined DNA-looping dynamics experimentally. The restriction enzymes that cut DNA after interacting with two recognition sites, such as FokI, can be used to exemplify looping reactions. The reaction pathway for FokI on a supercoiled DNA with two sites was dissected by fast kinetics to reveal, in turn: the initial binding of a protein monomer to each site; the protein–protein association to form the dimer, trapping the loop; the subsequent phosphodiester hydrolysis step. The DNA motion that juxtaposes the sites ought on the basis of Brownian dynamics to take ~2 ms, but loop capture by FokI took 230 ms. Hence, DNA looping by FokI is rate limited by protein association rather than DNA dynamics. The FokI endonuclease also illustrated activation by looping: it cut looped DNA 400 times faster than unlooped DNA.     

Real-time observation of a single DNA digestion by lambda exonuclease under a fluorescence microscope field

A fluorescence microscopy technique has been developed to visualize the behavior of individual DNA and protein molecules. Real-time direct observation of a single DNA molecule can be used to investigate the dynamics of DNA-protein interactions, such as the DNA digestion reaction by lambda exonuclease. In conventional methods it is impossible to analyze the dynamics of an individual lambda exonuclease molecule on a DNA because they can only observe the average behavior of a number of exonuclease molecules. Observation of a single molecule, on the other hand, can reveal processivity and binding rate of an individual exonuclease molecule. To evaluate the dynamics of lambda exonuclease, a stained lambda DNA molecule with one biotinylated terminal was fixed on an avidin-coated coverslip and straightened using a d.c. electric field. Microscopic observation of digestion of a straightened DNA molecule by lambda exonuclease revealed that the DNA digestion rate was approximately 1000 bases/s and also demonstrated high processivity.     

Dynamics of leading lamellae of living fibroblasts visualized by high-speed scanning probe microscopy

In this study, we aimed at improving the temporal resolution of scanning probe microscopy (SPM) for observing living cells by introducing soft cantilevers, low feedback-gain operations, and cantilever deflection imaging. We achieved visualization of the mechanical architecture in leading lamellae of living fibroblasts at a temporal resolution of around 10 s, which is higher than that of conventional contact-mode SPM. Time-lapse SPM could be used to monitor not only cytoskeletal dynamics but also the dynamics of numerous microgranules. Statistical analysis of microgranular motion revealed that the microgranules have superdiffusive behaviors and significant directional order of motion. We also found that the direction of their motion is correlated with the direction of growing actin stress fibers. The combination of SPM with fluorescence microscopy showed that vinculin, a component of cell-substratum adhesion sites, localizes at the microgranules. Our experimental data provides a new insight into the intracellular mechanical architecture and its structural dynamics, suggesting that high-speed live-cell SPM has great potential for investigating the structural origin of cellular dynamics.