Single Molecule manipulations 3

As part of their infection cycle, many viruses must package their newly replicated genomes inside a protein capsid to insure its proper transport and delivery to other host cells. Bacteriophage 29 packages its 6.6mm long double-stranded DNA into a 42 nm dia. X 54 nm high capsid via a portal complex that hydrolyses ATP. This process is remarkable because entropic, electrostatic, and bending energies of the DNA must be overcome to package the DNA to near-crystalline density.

In a recent work by Dr Bustamante, optical tweezers have been used to pull on single DNA molecules as they are packaged, thus demonstrating that the portal complex is a force generating motor. They have shown that this motor can work against loads of up to ~57 picoNewtons on average, making it one of the strongest molecular motors ever reported. Interestingly, the packaging rate decreases as the prohead is filled, indicating that an internal
pressure builds up due to DNA compression. It is estimated that at the end of the packaging the capsid pressure is ~6 MegaPascals, corresponding to an internal force of ~50 pN acting on the motor.

Reference

Single Molecule Manipulations in Biophysics 2

Torque Measurements on single DNA Molecules

The physical properties of the DNA double helix are unlike those of any other natural or synthetic polymer. The molecule’s characteristic base stacking and braided architecture lend it unusual stiffness: It takes about 50 times more energy to bend a double-stranded DNA molecule into a circle than to perform the same operation on single-stranded DNA. Moreover, the phosphates in DNA’s backbone make it one of the most highly charged polymers known.

To perform dynamic torque measurements on single DNA molecules, molecular constructs were made.This kind of experiments have been performed in the Prof. Bustamante's laboratory at Berkeley University.

The use of three distinct chemical modifications of DNA allows for oriented tethering of the ends of the molecule and the subsequent attachment of a rotor to a third, internal position (shown on the figure). A site-specific nick in the duplex DNA is engineered adjacent to the rotor attachment point; this design allows covalent bonds in the intact strand to serve as free swivels, preventing torque from accumulating in the "lower" DNA segment. Thus, torque stored in the "upper" segment can drive the rotation of a submicron object on a low-friction molecular bearing. At low Reynolds numbers, the magnitude of the torque can be measured by multiplying the observed angular velocity by the rotational drag of the rotor.

To be continued

reference: Bustamante, C.Of torques, forces, and protein machines(2004) Protein Science, 13 (11), pp. 3061-3065.doi: 10.1110/ps.041064504

Single Molecules Manipulations in Biophysics

Until very recently, chemists and biochemists have had to rely on bulk methods to investigate the properties of molecules and their reactions. These methods did not make it possible to directly investigate the nature, strength, and direction of intermolecular forces and torques.

During the last few years, however, the advent of novel methods of single-molecule manipulation have begun to offer researchers, for the first time, the opportunity to measure directly the forces holding molecular structures together, to measure the stresses and strains generated in the course of chemical and biochemical reactions, to exert external forces to alter the fate of these reactions, and to reveal the rules that govern the interconversion of mechanical and chemical energy in these reactions. This area of research can be rightly called mechanochemistry.

Biochemical processes as diverse as protein folding, DNA elasticity, the protein-induced bending of DNA, the stress-induced catalysis of enzymes, the mechanical properties of protein motors, and even the ubiquitous process of induced-fit molecular recognition of proteins for their ligands, are all examples in which stresses and strains develop in molecules as they move along a reaction coordinate.

to be continued

reference: Bustamante, C.Of torques, forces, and protein machines(2004) Protein Science, 13 (11), pp. 3061-3065.doi: 10.1110/ps.041064504