Optical tweezers are a powerful tool in biophysics due to the ability to manipulate single biomolecules [1,2]. For dual-trap tweezers with a steerable beam and force detection via back focal plane interferometry and a position sensitive detector [3,4], calibration can be done by stretching single DNA molecules [5,6]. Typically, DNA stretching provides accurate values for 𝛼, the force scale factor that converts from bead displacement from the trap to force, and 𝛽, the conversion between rotation of the piezoelectric controlled mirror and the linear displacement of the movable trap. Determining trap compliance using this method relies on nonlinear fitting that may be sensitive to noise and drift
Many viruses employ motors to translocate DNA into capsids. Previous reports raise questions if motor function depends on DNA sequence and a “B-A scrunchworm” model predicts that “A-philic” sequences that transition more easily to A-form would alter function. We use optical tweezers to measure translocation of phage, plasmid, and synthetic A-philic, GC rich sequences by the T4 motor. We observed no differences in motor velocities, even with A-philic sequences predicted to show higher rate, no changes in motor pausing, and only modest changes in slipping. To test more generally for sequence dependence, we conducted correlation analyses across pairs of events. No significant correlations in packaging rate, pausing, or slipping versus position were detected in repeated measurements with different DNA sequences. These studies suggest that the viral motor insensitive to DNA sequence and fluctuations in packaging velocity, pausing, and slipping are primarily stochastic temporal events.
Many viruses contain DNA packed to such a high density that the mobility of the DNA inside the viral capsid is severely restricted, affecting the processes of DNA packaging and ejection. We study phage phi29, which uses an ATP-powered molecular motor to package DNA, by using optical tweezers to measure DNA ejection through the phi29 portal-motor channel after the removal of ATP during DNA packaging. We find that when initiated at low capsid filling levels, DNA exits faster than 10 kbp/s. When initiated at high filling levels, exit occurs with a dramatically reduced average velocity that decreases with increasing initial prohead filling. In individual exit measurements, complex dynamics and transient pausing are seen, which we attribute to the nonequilibrium DNA conformations thought to arise during DNA packaging. We also show that high concentration of Mg2+ slows exit dynamics, suggesting that the internal pressure of the confined DNA is the driving force for the ejection process.
In many viruses molecular motors generate large forces to package DNA to high densities. The dynamics
and energetics of this process is a subject of wide debate and is of interest as a model for studying confined polymer
physics in general. Here we present preliminary results showing that DNA in bacteriophage phi29 undergoes
nonequilibrium conformational dynamics during packaging with a relaxation time >60,000x longer than for free
DNA and >3000x longer than reported for DNA confined in nanochannels. Nonequilibrium dynamics significantly
increases the load on the motor, causes heterogeneity in the rates of packaging, and causes frequent pausing in motor
translocation.
Many double-stranded DNA viruses employ a molecular motor to package DNA into preformed capsid
shells. Based on structures of phage T4 motor proteins determined by X-ray crystallography and cryo-electron
microscopy, Rao, Rossmann and coworkers recently proposed a structural model for motor function. They proposed
that DNA is ratcheted by a large conformational change driven by electrostatic interactions between charged
residues at an interface between two globular domains of the motor protein. We have conducted experiments to test
this model by studying the effect on packaging under applied load of site-directed changes altering these residues.
We observe significant impairment of packaging activity including reductions in packaging rate, percent time
packaging, and time active under high load. We show that these measured impairments correlate well with
alterations in free energies associated with the conformational change predicted by molecular dynamics simulations.
In previous work we developed methods using optical tweezers to measure
protein-mediated formation of loops in DNA structures that can play an
important role in regulating gene expression. We previously applied this method
to study two-site restriction endonucleases, which were convenient model
systems for studying this phenomenon. Here we report preliminary work in which
we have applied this method to study p53, a human tumor suppressor protein,
and show that we can measure formation of loops. Previous biophysical evidence
for loops comes from relatively limited qualitative studies of fixed complexes by
electron microscopy4. Our results provide independent corroboration and future
opportunities for more quantitative studies investigating structure and mechanics.
Bacteriophage T4 is a double stranded DNA virus that infects E.coli by injecting the viral genome through the cellular wall of a host cell. The T4 genome must be ejected from the viral capsid with sufficient force to ensure infection. To generate high ejection forces, the genome is packaged to high density within the viral capsid. A DNA translocation motor, in which the protein gp17 hydrolyzes ATP and binds to the DNA, is responsible for translocating the genome into the capsid during viral maturation of T4. This motor generates forces in excess of 60 pN and packages DNA at rates exceeding 2000 base pairs/second (bp/s)1. Understanding these small yet powerful motors is important, as they have many potential applications. Though much is known about the activity of these motors from bulk and single molecule biophysical techniques, little is known about their detailed molecular mechanism. Recently, two structures of gp17 have been obtained: a high-resolution X-ray crystallographic structure showing a monomeric compacted form of the enzyme, and a cryo-electron microscopic structure of the extended form of gp17 in complex with actively packaging prohead complexes. Comparison of these two structures indicates several key differences, and a model has been proposed to explain the translocation action of the motor2. Key to this model are a set of residues forming ion pairs across two domains of the gp17 molecule that are proposed to be involved in force generation by causing the collapse of the extended form of gp17. Using a dual optical trap to measure the rates of DNA packaging and the generated forces, we present preliminary mutational data showing that these several of these ion pairs are important to motor function. We have also performed preliminary free energy calculations on the extended and collapsed state of gp17, to confirm that these interdomain ion pairs have large contributions to the change in free energy that occurs upon the collapse of gp17 during the proposed ratcheting mechanism.
The genomes of many dsDNA viruses are replicated by a mechanism that produces a long concatemer of multiple
genomes. These viruses utilize multifunctional molecular motor complexes referred to as "terminases" that can excise a
unit genome length of DNA and package it into preformed viral shells. Remarkably, the terminase motor can initiate
packaging at the appropriate start point, translocate DNA, sense when a sufficient length has been packaged, and then
switch into a mode where it arrests and cleaves the DNA to release a filled virus particle. We have recently developed an
improved method to measure single phage lambda DNA packaging using dual-trap optical tweezers and pre-stalled
motor-DNA-procapsid complexes. We are applying this method to test proposed mechanisms for the sensor that triggers
termination; specifically a velocity-monitor model vs. energy-monitor model vs. capsid-filling monitor model.
Polyamine ions such as spermidine3+, along with monovalent and divalent salt ions, screen the negatively charged backbone of dsDNA and thereby facilitate processes in which DNA is confined in small spaces, such as viral DNA packaging. We use optical tweezers to directly manipulate single DNA molecules and have made preliminary measurements of the effect of spermidine on DNA elasticity, condensation, and viral packaging. We determine the concentration of spermidine3+ at which dsDNA condenses in the presence of Mg2+ and Na+ and report a monotonic increase in stretch modulus and decrease in persistence length at incremental spermidine concentrations up to the concentration at which dsDNA condenses. We also discuss the effect of spermidine on DNA packaging in bacteriophage phi29.
Optical tweezers measurements were employed to directly observe viral DNA packaging in
wild type and packaging mutants of bacteriophage lambda. Several key findings are reported here:
DNA packaging by purified wild type lambda motors was measured for the first time, showing
nearly identical behavior in packaging DNA to crude extracts of terminase components. A slow
packaging lambda mutant, T194M, was found to package DNA at ~10× slower velocity than wild
type. Meanwhile another packaging mutant Y46F was found to package DNA slower than the wild
type (60-70% the velocity of the wild type velocity) as well as slipping >10x more frequently (per
length of DNA) than wild type. Another mutant (K84A) showed slower packaging (60-70% the
velocity of wildtype), but displayed slipping and pausing behavior similar to wild type. Finally the
pausing and slipping dependence on length of DNA packaged of the various terminases studied was
discovered, suggesting further structural defects of the mutants that are detrimental to translocation.
These studies confirm the location of an ATPase center in the N-terminal portion of gpA which is
responsible for translocation of dsDNA.
A key step in the assembly of many viruses is the packaging of double-stranded DNA into a viral procapsid (an
empty protein shell) by the action of an ATP-powered portal motor complex. We have developed methods to
measure the packaging of single DNA molecules into single viral proheads in real time using optical tweezers. We
can measure DNA binding and initiation of translocation, the DNA translocation dynamics, and the filling of the
capsid against resisting forces. In addition to studying bacteriophage φ29, we have recently extended these methods
to study the E. coli bacteriophages λ and T4, two important model systems in molecular biology. The three systems
have different capsid sizes/shapes, genome lengths, and biochemical and structural differences in their packaging
motors. Here, we compare and contrast these three systems. We find that all three motors translocate DNA
processively and generate very large forces, each exceeding 50 piconewtons, ~20x higher force than generated by
the skeletal muscle myosin 2 motor. This high force generation is required to overcome the forces resisting the
confinement of the stiff, highly charged DNA at high density within the viral capsids. However, there are also
striking differences between the three motors: they exhibit different DNA translocation rates, degrees of static and
dynamic disorder, responses to load, and pausing and slipping dynamics.
Concentrated solutions of long polymer molecules exhibit reduced molecular diffusivity and striking non-Newtonian fluid properties that arise due to intermolecular entanglements. The most successful theories for describing these properties are based on the notion that on short time scales each polymer is confined to move within a tube-shaped region following its contour. Such a confining field is proposed to arise due to collective intermolecular interactions, yet this has remained a rather vague concept since the confining forces have never been directly measured. Here, we directly measure these forces by using optical tweezers to manipulate single entangled DNA molecules. We found that the forces opposing displacement of a molecule parallel to its local contour were negligible compared with those opposing transverse displacement. A time-dependent harmonic potential opposed transverse displacement. Work per unit length of order 1 kT was required to displace the molecule by a distance roughly equal to the theoretically predicted tube radius in the calculated thermal equilibration time. The required work also decreased gradually with the rate of displacement, in accord with predictions of a recent simulation study that found that the tube radius expands with time. Following the displacement, we measured the relaxation of force acting on the molecule and observed three distinct exponential decay times of ~0.4, 5, and 34 s, which are consistent with theoretically proposed molecular relaxation mechanisms. These measurements quantify the notion of a tube-like molecular confining field assumed in reptation theories.
The bacteriophage φ29 portal motor is capable of packaging the φ29, 19.3 Kbp, genome to high density into its preformed capsid. The packaging process must overcome the forces due to confining the highly negative charge of the DNA to a small volume, as well as the forces due to bending the DNA on length scales smaller than one persistence length. Both of these energetic considerations can be modulated by the ionic nature of the buffer DNA packaging occurs in. To measure the effects of DNA charge shielding on the packaging process, we studied the dynamics of DNA packaging by optical tweezers in a variety of different ionic conditions. We looked at the effects monovalent, divalent, and trivalent cations have on the motor function and its dependence on external force and, we observed the rate of DNA packaging at nominal force as a function of capsid filling. Specifically, we varied the concentrations of Na+, Mg+2, and cobalt hexamine in the solution bathing the bacteriophage during packaging to see what effects, if any, these cations have. From these measurements, we present an inferred internal force as a function of percent filling of the bacteriophage capsid in a variety of ionic environments. Preliminary analysis suggests the ionic environment can modulate internal pressure, with the presence of higher valence cations better shielding the packaged DNA resulting in lower internal pressures.
Looping and cleavage of single DNA molecules by the two-site restriction endonuclease Sau3AI were measured with optical tweezers. A DNA template containing many recognition sites was used, permitting loop sizes from ~10 to 10,000 basepairs. At high enzyme concentration cleavage events were detected within 5 seconds and nearly all molecules were cleaved within 5 minutes. Activity decreased ~10-fold as the DNA tension was increased from 0.03 to 0.7 pN. Substituting Ca2+ for Mg2+ blocked cleavage, permitting measurement of stable loops. At low tension, the initial rates of cleavage and looping were similar (~0.025 s-1 at 0.1 pN), suggesting that looping is rate limiting. Short loops formed more rapidly than long loops. The optimum size decreased from ~250 to 45 bp and the average number of loops (in 1 minute) from 4.2 to 0.75 as tension was increased from 0.03 to 0.7 pN. No looping was detected at 5 pN. These findings are in qualitative agreement with recent theoretical predictions considering only DNA mechanics, but we observed weaker suppression with tension and smaller loop sizes. Our results suggest that the span and elasticity of the protein complex and protein-induced DNA bending and wrapping play an important role.
Optical tweezers have broad applications in studies of structures and processes in molecular and cellular biophysics. Use of optical tweezers for quantitative molecular-scale measurement requires careful calibration in physical units. Here we show that DNA molecules may be used as metrology standards for force and length measurements. Analysis of DNA molecules of two specific lengths allows simultaneous determination of all essential measurement parameters. We validate this "biological calibration" method experimentally and with simulated data, and show that precisions in determining length scale factor (~0.2%), length offset (~0.03%), force scale factor (~2%), and compliance of the traps (~3%) are limited only by current measurement variation, much of which arises from polydispersity of the microspheres (~2%). We find this procedure to be simpler and more convenient than previous methods, and suggest that it provides an easily replicated standard that can insure uniformity of measurements made in different laboratories.
A key step in the life cycle of many viruses, including bacteriophages, adenoviruses, and herpesviruses, is the packaging of replicated viral genomes into pre-assembled proheads by the action of ATP-dependent portal motor complexes. Here we present a method that allows the initiation of packaging by single complexes to be studied using optical tweezers. A procedure is developed for assembling phage Φ29 prohead-motor complexes, which are demonstrated to bind and begin translocation of a target DNA molecule within only a few seconds. We show that the Φ29 DNA terminal protein (gene product 3), which functions to prime DNA replication, also has a dramatic effect on packaging. The DNA tether length measured immediately after binding varied from ~30-100% of the full length, yet shortened monotonically, indicating that packaging does not strictly begin at the terminal end of the DNA. Removal of the terminal protein eliminated this variability, causing packaging to initiate at or very near the end of the DNA. These findings, taken together with electron microscopy data, suggest that rather than simply threading into the portal, the motor captures and dynamically tensions a DNA loop, and that the function of the terminal protein is to load DNA segments on both sides of the loop junction onto separate DNA translocating units.
Here we describe and characterize a method for manipulating desired DNA sequences from any organism with optical tweezers. Molecules are produced from either genomic or cloned DNA by PCR using labeled primers and are tethered between two optically trapped microspheres. We demonstrate that human, insect, plant, bacterial, and viral sequences ranging from ~10 to 40 kbp can be manipulated. Force-extension measurements show that these constructs exhibit uniform elastic properties in accord with the expected contour lengths for the targeted sequences. Detailed protocols for preparing and manipulating these molecules are presented, and tethering efficiency is characterized as a function of DNA concentration, ionic strength, and pH. Attachment strength is characterized by measuring the unbinding time distribution as a function of applied force.
Mechanical manipulation of single DNA molecules can provide novel information about protein-DNA interactions. Here we review two examples studied by our group. First, we have studied the forced unraveling of nucleosomes assembled on heterogeneous DNA using core histones, the histone chaperone NAP-1, and ATP-dependent chromatin assembly and remodeling factor (ACF). We measure abrupt events releasing ~55 to 95 base pairs of DNA, which are attributable to non-equilibrium unraveling of individual nucleosomes. Wide variations observed in the unraveling force and sudden DNA re-wrapping events may have an important regulatory influence on DNA directed biochemical processes. Second, we have studied the mechanics and dynamics of single DNA looping and cleavage by "two-site" restriction enzymes. Cleavage is measured as a function of DNA tension, incubation time, and enzyme concentration, distinguishing enzymes that require DNA looping from ones that do not. Forced disruption of fixed DNA loops formed in the absence of Mg2+ is observed, allowing the distribution of number of loops, loop length, and disruption force to be measured as a function of time, DNA tension, and ionic conditions.
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