Thematic Minireview Series: Single-molecule Measurements in Biochemistry and Molecular Biology

For many years, one of the fundamental barriers to defining the mechanisms of molecular and cellular processes was our inability to observe individual molecules because most conventional experimental approaches reveal only the average properties of ensembles of molecules existing in different states. For example, although individual protein molecules in solution are likely to differ at any given time in their conformation and the ions and molecules with which they are interacting, most experimental approaches monitor only their average conformation and average binding properties. Defining the individual states accessible to a molecule is central to defining the mechanisms of the biological processes in which it participates.

Within the past couple of decades, a suite of extraordinarily sensitive methods has been developed that allows the dimensions and motions of individual biological molecules to be monitored and manipulated both in solution and in cells. Molecules can be visualized by attaching either fluorescent labels or microscopic beads that can be visualized with spectrophotometers or microscopes while mechanical, optical, and magnetic forces, generally applied in vitro to a molecule tethered to a surface, allow them to be manipulated. Although new methodologies are constantly being developed, the approaches most widely used at this time are optical monitoring of fluorescent labels or reporter microbeads, fluorescence resonance energy transfer, atomic force microscopy, magnetic tweezers, and optical tweezers (or traps).

These approaches differ in the molecular properties that they monitor and their temporal and spatial resolution, and, thus, different approaches are often optimal for different systems. At the same time, applying several approaches to the same problem can increase the depth of understanding that can be achieved. Observations made with single fluorescent labels or reporter microbeads typically provide insights into the movement of entire molecules, for example, the movement of myosin heads along an actin filament. Fluorescence energy resonance transfer between two fluorophores assesses the distance between them and changes in that distance over time as a result of intramolecular motion or changes in the interactions between molecules. The remaining three methods, atomic force microscopy, magnetic tweezers, and optical tweezers, all apply force to a molecule that is tethered at both ends and measure the effect of the force on the molecule. Typically, one end of the molecule is tethered to a surface, whereas the second is attached to a cantilever, in atomic force microscopy, or a microbead, with magnetic and optical tweezers. These methods are widely used to study the unfolding of RNA or proteins or changes in DNA topology. In atomic force microscopy, the cantilever to which the molecule is attached applies a mechanical force to the molecule, and the effect of the force on the molecule is evaluated by monitoring the reflection of a laser beam from the cantilever. In magnetic tweezers experiments, the microbead to which the molecule is attached is superparamagnetic and positioned below the poles of a pair of magnets so that decreasing the distance between the magnets and the surface to which the molecule is tethered will stretch the molecule, whereas rotating the magnet will exert torque on the molecule. In contrast, in an optical tweezers experiment, the bead is made of a dielectric material, and the force is generated by the electromagnetic field produced by a laser beam.

Single-molecule methods can provide novel new insights into an extraordinarily broad range of questions, including enzyme mechanisms; folding of DNA, RNA, proteins, and polysaccharides; membrane protein structure and function; motor proteins and cytoskeletal dynamics; rotary motors; processive nucleic acid enzymes; protein-nucleic acid interactions; single-molecule sequencing; vesicle trafficking; membrane fusion; and even brain imaging. The four minireviews in this series illustrate a few of the ways in which these approaches can be used.

The first minireview, by Alegre-Cebollada et al., describes how they used atomic force microscopy to compare and demonstrate differences at subatomic resolution in the transition states of disulfide bond oxidation catalyzed by various members of the thioredoxin family. By determining the relationship between the force applied to the disulfide bond and the rate of oxidation, Alegre-Cebollada et al. were able to evaluate the difference in the length of the disulfide bond in the ground and transition states for a simple S_n2 reaction. They found that, whereas the force dependence of the reaction is always positive for small reducing agents, the effect of the applied force on the rates of reactions catalyzed by thioredoxins varies with the structure and dynamics of the enzyme. This approach should be useful in studying and comparing other enzymes that cleave covalent bonds, and the minireview defines the criteria that must be met in applying this approach to other enzymes.

In the second minireview, Neuman focuses on how torque produced by magnetic and optical tweezers can be used to control and measure the supercoiling of DNA molecules and the relationship between supercoiling and the mechanical properties of DNA. Neuman reviews how these approaches have been employed to monitor supercoil relaxation by two topoisomerases, negative supercoiling by DNA gyrase, and promoter opening and transcriptional initiation by bacterial RNA polymerase. The minireview looks to future work on the topology of chromatin fibers and rotary motors such as RNA polymerase.

The third minireview, by Finzi and Dunlap, emphasizes the structures, kinetics, and thermodynamics of the DNA loops that form in transcriptional regulatory nucleoprotein complexes as a result of protein-protein interactions. The studies of the lac, gal, and λ repressors presented in this minireview utilize atomic force microscopy, magnetic tweezers, and the tethered-particle method, in which movement of a reporter bead attached to tethered DNA enables the changes in length of the DNA resulting from loop formation or release to be detected. This minireview also discusses the role of DNA-coiling and DNA-bending cofactors in both prokaryotic and eukaryotic systems, particularly Snf2 chromatin-remodeling factors.

The last minireview in this series, by Hamdan and van Oijen, describes how single-molecule flow-stretching experiments have led to new insights into how the molecular events in DNA replication in T7 phage are coordinated. In the experiments described, DNA molecules tagged at each end with microbeads were oriented by laminar flow so that their lengths could be determined. Because transitions between double- and single-stranded DNAs alter the length of the DNA, the steps in replication in which these transitions occur can be identified. Like Finzi and Dunlap, Hamdan and van Oijen were particularly interested in DNA loops created by protein-protein interactions, in this case, the loops that form in the lagging DNA strand to keep replication of the leading and lagging strands in phase. They set out to determine what triggers release of the loops. Is it the synthesis of a new primer molecule or the collision of the polymerase with the Okazaki fragment previously synthesized? In fact, they found that both mechanisms operate to ensure that Okazaki fragments of the correct length are synthesized.

This series covers only a small fraction of the topics that can be investigated with single-molecule methods. The Journal of Biological Chemistry is expanding its coverage of this important area and encourages submission of manuscripts that use single-molecule methods to provide novel and important insights into molecular and cellular processes.