Methods for Studying Motor-Driven Viral DNA Packaging in Bacteriophages phi29, Lambda, and T4 via Single DNA Molecule Manipulation and Rapid Solution Exchange.

Viral DNA packaging is a required step in the assembly of many dsDNA viruses. A molecular motor fueled by ATP hydrolysis packages the viral genome to near crystalline density inside a pre-formed prohead shell in ~5 min at room temperature in vitro. We describe procedures for measuring the packaging of single DNA molecules into single viral proheads with optical tweezers.

Role of DNA-DNA sliding friction and nonequilibrium dynamics in viral genome ejection and packaging.

Many viruses eject their DNA via a nanochannel in the viral shell, driven by internal forces arising from the high-density genome packing. The speed of DNA exit is controlled by friction forces that limit the molecular mobility, but the nature of this friction is unknown. We introduce a method to probe the mobility of the tightly confined DNA by measuring DNA exit from phage phi29 capsids with optical tweezers.

Role of DNA-DNA sliding friction and non-equilibrium dynamics in viral genome ejection and packaging.

Many viruses eject their DNA via a nanochannel in the viral shell, driven by internal forces arising from the high-density genome packing. The speed of DNA exit is controlled by friction forces that limit the molecular mobility, but the nature of this friction is unknown. We introduce a method to probe the mobility of the tightly confined DNA by measuring DNA exit from phage phi29 capsids with optical tweezers.

Power-efficientbrain-machine interfaces via brain-state estimation.

Advances in brain-machine interfaces (BMIs) can potentially improve the quality of life of millions of users with spinal cord injury or other neurological disorders by allowing them to interact with the physical environment at their will.To reduce the power consumption of the brain-implanted interface, this article presents the first hardware realization of anintention-aware interface via brain-state estimation.It is shown that incorporating brain-state estimation reduces thepower consumption and reduces total energy dissipation by over 1.

Function of a viral genome packaging motor from bacteriophage T4 is insensitive to DNA sequence.

Many viruses employ ATP-powered motors during assembly to translocate DNA into procapsid shells. Previous reports raise the question if motor function is modulated by substrate DNA sequence: (i) the phage T4 motor exhibits large translocation rate fluctuations and pauses and slips; (ii) evidence suggests that the phage phi29 motor contacts DNA bases during translocation; and (iii) one theoretical model, the 'B-A scrunchworm', predicts that 'A-philic' sequences that transition more easily to A-form would alter motor function. Here, we use single-molecule optical tweezers measurements to compare translocation of phage, plasmid, and synthetic A-philic, GC rich sequences by the T4 motor.

Single-Molecule Measurements of Motor-Driven Viral DNA Packaging in Bacteriophages Phi29, Lambda, and T4 with Optical Tweezers.

Viral DNA packaging is a required step in the assembly of many dsDNA viruses. A molecular motor fueled by ATP hydrolysis packages the viral genome to near crystalline density inside a preformed prohead shell in ~5 min at room temperature. We describe procedures for measuring the packaging of single DNA molecules into single viral proheads with optical tweezers.

Experimental comparison of forces resisting viral DNA packaging and driving DNA ejection.

We compare forces resisting DNA packaging and forces driving DNA ejection in bacteriophage phi29 with theoretical predictions. Ejection of DNA from prohead-motor complexes is triggered by heating complexes after in vitro packaging and force is inferred from the suppression of ejection by applied osmotic pressure. Ejection force from 0% to 80% filling is found to be in quantitative agreement with predictions of a continuum mechanics model that assumes a repulsive DNA-DNA interaction potential based on DNA condensation studies and predicts an inverse-spool conformation.

Single DNA molecule jamming and history-dependent dynamics during motor-driven viral packaging.

In many viruses molecular motors forcibly pack single DNA molecules to near-crystalline density into ~50-100 nm prohead shells. Unexpectedly, we found that packaging frequently stalls in conditions that induce net attractive DNA-DNA interactions. Here, we present findings suggesting that this stalling occurs because the DNA undergoes a nonequilibrium jamming transition analogous to that observed in many soft-matter systems, such as colloidal and granular systems.

Walker-A Motif Acts to Coordinate ATP Hydrolysis with Motor Output in Viral DNA Packaging.

During the assembly of many viruses, a powerful ATP-driven motor translocates DNA into a preformed procapsid. A Walker-A "P-loop" motif is proposed to coordinate ATP binding and hydrolysis with DNA translocation. We use genetic, biochemical, and biophysical techniques to survey the roles of P-loop residues in bacteriophage lambda motor function.

Continuous allosteric regulation of a viral packaging motor by a sensor that detects the density and conformation of packaged DNA.

We report evidence for an unconventional type of allosteric regulation of a biomotor. We show that the genome-packaging motor of phage ϕ29 is regulated by a sensor that detects the density and conformation of the DNA packaged inside the viral capsid, and slows the motor by a mechanism distinct from the effect of a direct load force on the motor. Specifically, we show that motor-ATP interactions are regulated by a signal that is propagated allosterically from inside the viral shell to the motor mounted on the outside.

Repulsive DNA-DNA interactions accelerate viral DNA packaging in phage Phi29.

We use optical tweezers to study the effect of attractive versus repulsive DNA-DNA interactions on motor-driven viral packaging. Screening of repulsive interactions accelerates packaging, but induction of attractive interactions by spermidine(3+) causes heterogeneous dynamics. Acceleration is observed in a fraction of complexes, but most exhibit slowing and stalling, suggesting that attractive interactions promote nonequilibrium DNA conformations that impede the motor.

Evidence for an electrostatic mechanism of force generation by the bacteriophage T4 DNA packaging motor.

How viral packaging motors generate enormous forces to translocate DNA into viral capsids remains unknown. Recent structural studies of the bacteriophage T4 packaging motor have led to a proposed mechanism wherein the gp17 motor protein translocates DNA by transitioning between extended and compact states, orchestrated by electrostatic interactions between complimentarily charged residues across the interface between the N- and C-terminal subdomains. Here we show that site-directed alterations in these residues cause force dependent impairments of motor function including lower translocation velocity, lower stall force and higher frequency of pauses and slips.

Nonequilibrium dynamics and ultraslow relaxation of confined DNA during viral packaging.

Many viruses use molecular motors that generate large forces to package DNA to near-crystalline densities inside preformed viral proheads. Besides being a key step in viral assembly, this process is of interest as a model for understanding the physics of charged polymers under tight 3D confinement. A large number of theoretical studies have modeled DNA packaging, and the nature of the molecular dynamics and the forces resisting the tight confinement is a subject of wide debate.