Single-molecule biophysics
Single-molecule biophysics is the measurement of the behavior of individual biological molecules, as opposed to the average over the many billions in a sample. A milliliter of a one-micromolar solution holds about 6 × 1014 molecules, and an ordinary assay reports their mean. A single-molecule experiment looks at one of them and records what it does over time, how long it stays in each state, and how often it switches.
The methods share a physical constraint. At room temperature the thermal energy kBT is about 4.1 pN nm, the work of a force of a few piconewtons acting over a nanometer, which is the scale of the forces and distances a protein deals in. A molecule is therefore always being jostled by Brownian motion with about the energy it uses to do its job, and each technique is a way of seeing a signal of that size above the jostling.
Why one at a time
An average hides the sequence of events. A population of motor proteins, each stepping at its own random moment, moves smoothly in bulk, and the steps cancel. Svoboda and colleagues used optical tweezers to place silica beads carrying single molecules of the motor kinesin on microtubules, tracked the beads by interferometry, and found that kinesin moves in steps of 8 nm.[1] In 1997 Noji and colleagues glued a fluorescent actin filament to the central subunit of the enzyme F1-ATPase and watched it turn, a rotor with a radius of about 1 nm inside a barrel of about 5 nm, through more than 100 revolutions in the presence of ATP.[2]
Averages also hide differences between molecules. Two populations, one folded and one unfolded, give a mean in between that describes neither. And they hide rare states. Ha and colleagues, in the first fluorescence energy transfer measurement between a single donor and a single acceptor, pointed out that dynamic events on a molecular scale are observable in single-pair measurements "because they are not canceled out by random averaging".[3]
The idea is older than the instruments. That molecules are separate objects with their own random motion was the argument of Jean Perrin's measurements of Brownian motion; see Molecular reality (history of science). It took until the 1970s to record the current of one ion channel and until the 1980s to see one dye molecule.
Methods
| Method | What is measured | Example |
|---|---|---|
| Patch clamp | Ionic current through one channel | Currents of a few picoamperes from single channels of frog muscle, 1976[4] |
| Optical tweezers | Force and position of a bead tethered to a molecule | Double-stranded DNA stretched to a transition at about 65 pN, 1996 |
| Atomic force microscopy | Force against extension | Domains of the protein titin unfolding at 150 to 300 pN, 1997 |
| Single-molecule FRET | Distance between two dyes | A single donor and acceptor on a short DNA molecule, 1996 |
| Nanopore | Current blocked by one molecule | Single strands of DNA through a protein pore, 1996 |
Patch clamp came first. Erwin Neher and Bert Sakmann recorded single-channel currents in 1976 and shared the 1991 Nobel Prize in Physiology or Medicine for it.[5] See Erwin Neher and Bert Sakmann.
Optical tweezers hold a small bead in the focus of a laser. Ashkin and colleagues showed in 1986 that a single tightly focused beam can trap a dielectric particle in three dimensions, and Arthur Ashkin shared the 2018 Nobel Prize in Physics for the tweezers and their use on biological systems.[6][7] With a molecule tethered between the bead and a surface the trap measures force. Smith, Cui and Bustamante stretched single molecules of double-stranded DNA and found that under about 65 pN the molecule undergoes a cooperative transition to a form 70% longer than B-DNA, and that it contracts reversibly when the force is relaxed.[8] Magnetic tweezers do the same with a magnetic bead, and can also twist the molecule.
The atomic force microscope, invented in 1986, drags a sharp tip across a surface and senses the force on it.[9] Used for force spectroscopy, it pulls on a single protein. Titin, the giant protein of muscle, gave a sawtooth: the force climbed and fell with a period of 25 to 28 nm as one immunoglobulin domain after another unfolded, each at 150 to 300 pN.[10]
Fluorescence energy transfer reports distance. The efficiency with which an excited dye hands its energy to a second dye falls steeply with their separation, which Stryer and Haugland called a spectroscopic ruler in 1967.[11] Applied to single pairs, it follows conformational changes in real time. A guide of 2008 describes experiments on immobilized molecules that follow single reaction trajectories from 1 ms to many minutes.[12] The first optical detection of a single molecule, a dye in a crystal at low temperature, was reported in 1989.[13]
A nanopore needs no label. A molecule that enters the pore keeps ions from crossing, and the dip in the ionic current is its signal, the principle of the Coulter counter shrunk to the size of the molecule. In 1996 Kasianowicz and colleagues measured single strands of DNA passing through the protein pore alpha-hemolysin.[14] The signal is large enough to see without optics, but its bandwidth is limited by nanopore noise, and a molecule can cross faster than the amplifier can follow.
Limits
One molecule gives one trajectory, and a trajectory is noisy; conclusions come from thousands of events pooled into distributions, such as the dwell time of a molecule in a pore. The tether that holds a molecule to a bead or a surface changes its behavior, dyes bleach, and an experiment tends to select the molecules that happen to behave well. The throughput is low, because a trap or a pore holds one molecule at a time.
At Molecular Reality
Molecular Reality Corporation belongs to this tradition by way of the nanopore. Its live stream records the ionic current through a pore 200 µm across, far too wide to resolve a molecule, and its stated route to the small end is described in Journey to Angstromia.
See also
References
- ^ Svoboda, K.; Schmidt, C. F.; Schnapp, B. J.; Block, S. M. (1993). "Direct observation of kinesin stepping by optical trapping interferometry". Nature 365 (6448): 721-727. doi:10.1038/365721a0.
- ^ Noji, H.; Yasuda, R.; Yoshida, M.; Kinosita, K. (1997). "Direct observation of the rotation of F1-ATPase". Nature 386 (6622): 299-302. doi:10.1038/386299a0.
- ^ Ha, T.; Enderle, T.; Ogletree, D. F.; Chemla, D. S.; Selvin, P. R.; Weiss, S. (1996). "Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor". Proceedings of the National Academy of Sciences 93 (13): 6264-6268. doi:10.1073/pnas.93.13.6264.
- ^ Neher, E.; Sakmann, B. (1976). "Single-channel currents recorded from membrane of denervated frog muscle fibres". Nature 260 (5554): 799-802. doi:10.1038/260799a0.
- ^ "The Nobel Prize in Physiology or Medicine 1991". NobelPrize.org. Nobel Prize Outreach. Retrieved October 25, 2026.
- ^ Ashkin, A.; Dziedzic, J. M.; Bjorkholm, J. E.; Chu, S. (1986). "Observation of a single-beam gradient force optical trap for dielectric particles". Optics Letters 11 (5): 288-290. doi:10.1364/OL.11.000288.
- ^ "The Nobel Prize in Physics 2018". NobelPrize.org. Nobel Prize Outreach. Retrieved October 25, 2026.
- ^ Smith, S. B.; Cui, Y.; Bustamante, C. (1996). "Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules". Science 271 (5250): 795-799. doi:10.1126/science.271.5250.795.
- ^ Binnig, G.; Quate, C. F.; Gerber, C. (1986). "Atomic force microscope". Physical Review Letters 56 (9): 930-933. doi:10.1103/PhysRevLett.56.930.
- ^ Rief, M.; Gautel, M.; Oesterhelt, F.; Fernandez, J. M.; Gaub, H. E. (1997). "Reversible unfolding of individual titin immunoglobulin domains by AFM". Science 276 (5315): 1109-1112. doi:10.1126/science.276.5315.1109.
- ^ Stryer, L.; Haugland, R. P. (1967). "Energy transfer: a spectroscopic ruler". Proceedings of the National Academy of Sciences 58 (2): 719-726. doi:10.1073/pnas.58.2.719.
- ^ Roy, R.; Hohng, S.; Ha, T. (2008). "A practical guide to single-molecule FRET". Nature Methods 5 (6): 507-516. doi:10.1038/nmeth.1208.
- ^ Moerner, W. E.; Kador, L. (1989). "Optical detection and spectroscopy of single molecules in a solid". Physical Review Letters 62 (21): 2535-2538. doi:10.1103/PhysRevLett.62.2535.
- ^ Kasianowicz, J. J.; Brandin, E.; Branton, D.; Deamer, D. W. (1996). "Characterization of individual polynucleotide molecules using a membrane channel". Proceedings of the National Academy of Sciences 93 (24): 13770-13773. doi:10.1073/pnas.93.24.13770.