Force spectroscopy

Last updated July 23, 2024

Force spectroscopy is a set of techniques for the study of the interactions and the binding forces between individual molecules.^[1]^[2] These methods can be used to measure the mechanical properties of single polymer molecules or proteins, or individual chemical bonds. The name "force spectroscopy", although widely used in the scientific community, is somewhat misleading, because there is no true matter-radiation interaction.^[3]

Force spectroscopy measures the behavior of a molecule under stretching or torsional mechanical force. In this way a great deal has been learned in recent years about the mechanochemical coupling in the enzymes responsible for muscle contraction, transport in the cell, energy generation (F1-ATPase), DNA replication and transcription (polymerases), DNA unknotting and unwinding (topoisomerases and helicases).^[8]

As a single-molecule technique, as opposed to typical ensemble spectroscopies, it allows a researcher to determine properties of the particular molecule under study. In particular, rare events such as conformational change, which are masked in an ensemble, may be observed.

Experimental techniques

There are many ways to accurately manipulate single molecules. Prominent among these are optical or magnetic tweezers, atomic-force-microscope (AFM) cantilevers and acoustic force spectroscopy. In all of these techniques, a biomolecule, such as protein or DNA, or some other biopolymer has one end bound to a surface or micrometre-sized bead and the other to a force sensor. The force sensor is usually a micrometre-sized bead or a cantilever, whose displacement can be measured to determine the force.

Atomic force microscope cantilevers

Molecules adsorbed on a surface are picked up by a microscopic tip (nanometres wide) that is located on the end of an elastic cantilever. In a more sophisticated version of this experiment (Chemical Force Microscopy) the tips are covalently functionalized with the molecules of interest.^[9] A piezoelectric controller then pulls up the cantilever. If some force is acting on the elastic cantilever (for example because some molecule is being stretched between the surface and the tip), this will deflect upward (repulsive force) or downward (attractive force). According to Hooke's law, this deflection will be proportional to the force acting on the cantilever. Deflection is measured by the position of a laser beam reflected by the cantilever. This kind of set-up can measure forces as low as 10 pN (10⁻¹¹ N), the fundamental resolution limit is given by the cantilever's thermal noise.

The so-called force curve is the graph of force (or more precisely, of cantilever deflection) versus the piezoelectric position on the Z axis. An ideal Hookean spring, for example, would display a straight diagonal force curve. Typically, the force curves observed in the force spectroscopy experiments consist of a contact (diagonal) region where the probe contacts the sample surface, and a non-contact region where the probe is off the sample surface. When the restoring force of the cantilever exceeds tip-sample adhesion force the probe jumps out of contact, and the magnitude of this jump is often used as a measure of adhesion force or rupture force. In general the rupture of a tip-surface bond is a stochastic process; therefore reliable quantification of the adhesion force requires taking multiple individual force curves. The histogram of the adhesion forces obtained in these multiple measurements provides the main data output for force spectroscopy measurement.

In biophysics, single-molecule force spectroscopy can be used to study the energy landscape underlying the interaction between two bio-molecules, like proteins. Here, one binding partner can be attached to a cantilever tip via a flexible linker molecule (PEG chain), while the other one is immobilized on a substrate surface. In a typical approach, the cantilever is repeatedly approached and retracted from the sample at a constant speed. In some cases, binding between the two partners will occur, which will become visible in the force curve, as the use of a flexible linker gives rise to a characteristic curve shape (see Worm-like chain model) distinct from adhesion. The collected rupture forces can then be analysed as a function of the bond loading rate. The resulting graph of the average rupture force as a function of the loading rate is called the force spectrum and forms the basic dataset for dynamic force spectroscopy.^[10]^[11]

In the ideal case of a single sharp energy barrier for the tip-sample interactions the dynamic force spectrum will show a linear increase of the rupture force as function of a logarithm of the loading rate, as described by a model proposed by Bell et al.^[12] Here, the slope of the rupture force spectrum is equal to the ${\frac {k_{B}T}{x_{\beta }}}$ , where $x_{\beta }$ is the distance from the energy minimum to the transition state. So far, a number of theoretical models exist describing the relationship between loading rate and rupture force, based upon different assumptions and predicting distinct curve shapes.^[11]^[13]

For example, Ma X.,Gosai A. et al., utilized dynamic force spectroscopy along with molecular dynamics simulations to find out the binding force between thrombin, a blood coagulation protein, and its DNA aptamer.^[14]

Acoustic force spectroscopy

A recently developed technique, acoustic force spectroscopy (AFS), allows the force manipulation of hundreds of single-molecules and single-cells in parallel, providing high experimental throughput.^[5] In this technique, a piezo element resonantly excites planar acoustic waves over a microfluidic chip. The generated acoustic waves are capable of exerting forces on microspheres with different density than the surrounding medium. Biomolecules, such as DNA, RNA or proteins, can be individually tethered between the microspheres and a surface and then probed by the acoustic forces exerted by the piezo sensor. With AFS devices it is possible to apply forces ranging from 0 to several hundreds of picoNewtons on hundreds of microspheres and obtain force-extension curves or histograms of rupture forces of many individual events in parallel.

This technique is mostly utilized to study DNA-bindings protein. For example, AFS was used to examine bacterial transcription with presence of antibacterial agents.^[15] Viral proteins also can be studied by AFS, for instance this technique was used to explore DNA compaction along with other single-molecule approaches.^[16]

Cells also can be manipulated by the acoustic forces directly, or by using microspheres as handles.^[17]

Optical tweezers

Another technique that has been gaining ground for single molecule experiments is the use of optical tweezers for applying mechanical forces on molecules. A strongly focused laser beam has the ability to catch and hold particles (of dielectric material) in a size range from nanometers to micrometers. The trapping action of optical tweezers results from the dipole or optical gradient force on the dielectric sphere. The technique of using a focused laser beam as an atom trap was first applied in 1984 at Bell laboratories. Until then experiments had been carried out using oppositely directed lasers as a means to trap particles. Later experiments, at the same project at Bell laboratories and others since, showed damage-free manipulation on cells using an infrared laser. Thus, the ground was made for biological experiments with optical trapping.

Each technique has its own advantages and disadvantages. For example, AFM cantilevers, can measure angstrom-scale, millisecond events and forces larger than 10 pN. While glass microfibers cannot achieve such fine spatial and temporal resolution, they can measure piconewton forces. Optical tweezers allow the measurement of piconewton forces and nanometer displacements which is an ideal range for many biological experiments. Magnetic tweezers can measure femtonewton forces, and additionally they can also be used to apply torsion. AFS devices allow the statistical analysis of the mechanical properties of biological systems by applying picoNewton forces to hundreds of individual particles in parallel, with sub-millisecond response time.

Applications

Common applications of force spectroscopy are measurements of polymer elasticity, especially biopolymers such as RNA and DNA.^[18] Another biophysical application of polymer force spectroscopy is on protein unfolding.^[19] Modular proteins can be adsorbed to a gold or (more rarely) mica surface and then stretched. The sequential unfolding of modules is observed as a very characteristic sawtooth pattern of the force vs elongation graph; every tooth corresponds to the unfolding of a single protein module (apart from the last that is generally the detachment of the protein molecule from the tip). Much information about protein elasticity and protein unfolding can be obtained by this technique. Many proteins in the living cell must face mechanical stress.

Moreover, force spectroscopy can be used to investigate the enzymatic activity of proteins involved in DNA replication, transcription, organization and repair. This is achieved by measuring the position of a bead attached to a DNA-protein complex stalled on a DNA tether that has one end attached to a surface, while keeping the force constant. This technique has been used, for example, to study transcription elongation inhibition by Klebsidin and Acinetodin.^[20]

The other main application of force spectroscopy is the study of mechanical resistance of chemical bonds. In this case, generally the tip is functionalized with a ligand that binds to another molecule bound to the surface. The tip is pushed on the surface, allowing for contact between the two molecules, and then retracted until the newly formed bond breaks up. The force at which the bond breaks up is measured. Since mechanical breaking is a kinetic, stochastic process, the breaking force is not an absolute parameter, but it is a function of both temperature and pulling speed. Low temperatures and high pulling speeds correspond to higher breaking forces. By careful analysis of the breaking force at various pulling speeds, it is possible to map the energy landscape of the chemical bond under mechanical force.^[21] This is leading to interesting results in the study of antibody-antigen, protein-protein, protein-living cell interaction and catch bonds.^[22]

Recently this technique has been used in cell biology to measure the aggregative stochastic forces created by motor proteins that influence the motion of particles within the cytoplasm. In this way, force spectrum microscopy may be used better to understand the many cellular processes that require the motion of particles within cytoplasm.^[23]

Related Research Articles

Protein folding is the physical process by which a protein, after synthesis by a ribosome as a linear chain of amino acids, changes from an unstable random coil into a more ordered three-dimensional structure. This structure permits the protein to become biologically functional.

Raman spectroscopy is a spectroscopic technique typically used to determine vibrational modes of molecules, although rotational and other low-frequency modes of systems may also be observed. Raman spectroscopy is commonly used in chemistry to provide a structural fingerprint by which molecules can be identified.

Atomic force microscopy (AFM) or scanning force microscopy (SFM) is a very-high-resolution type of scanning probe microscopy (SPM), with demonstrated resolution on the order of fractions of a nanometer, more than 1000 times better than the optical diffraction limit.

Optical tweezers are scientific instruments that use a highly focused laser beam to hold and move microscopic and sub-microscopic objects like atoms, nanoparticles and droplets, in a manner similar to tweezers. If the object is held in air or vacuum without additional support, it can be called optical levitation.

Tweezers are small hand tools used for grasping objects too small to be easily handled with the human fingers. Tweezers are thumb-driven forceps most likely derived from tongs used to grab or hold hot objects since the dawn of recorded history. In a scientific or medical context, they are normally referred to as just "forceps", a name that is used together with other grasping surgical instruments that resemble pliers, pincers and scissors-like clamps.

<span class="mw-page-title-main">Molecular motor</span> Biological molecular machines

Molecular motors are natural (biological) or artificial molecular machines that are the essential agents of movement in living organisms. In general terms, a motor is a device that consumes energy in one form and converts it into motion or mechanical work; for example, many protein-based molecular motors harness the chemical free energy released by the hydrolysis of ATP in order to perform mechanical work. In terms of energetic efficiency, this type of motor can be superior to currently available man-made motors. One important difference between molecular motors and macroscopic motors is that molecular motors operate in the thermal bath, an environment in which the fluctuations due to thermal noise are significant.

<span class="mw-page-title-main">Single-molecule experiment</span>

A single-molecule experiment is an experiment that investigates the properties of individual molecules. Single-molecule studies may be contrasted with measurements on an ensemble or bulk collection of molecules, where the individual behavior of molecules cannot be distinguished, and only average characteristics can be measured. Since many measurement techniques in biology, chemistry, and physics are not sensitive enough to observe single molecules, single-molecule fluorescence techniques caused a lot of excitement, since these supplied many new details on the measured processes that were not accessible in the past. Indeed, since the 1990s, many techniques for probing individual molecules have been developed.

Magnetic tweezers (MT) are scientific instruments for the manipulation and characterization of biomolecules or polymers. These apparatus exert forces and torques to individual molecules or groups of molecules. It can be used to measure the tensile strength or the force generated by molecules.

A nanoparticle–biomolecule conjugate is a nanoparticle with biomolecules attached to its surface. Nanoparticles are minuscule particles, typically measured in nanometers (nm), that are used in nanobiotechnology to explore the functions of biomolecules. Properties of the ultrafine particles are characterized by the components on their surfaces more so than larger structures, such as cells, due to large surface area-to-volume ratios. Large surface area-to-volume-ratios of nanoparticles optimize the potential for interactions with biomolecules.

The technique of vibrational analysis with scanning probe microscopy allows probing vibrational properties of materials at the submicrometer scale, and even of individual molecules. This is accomplished by integrating scanning probe microscopy (SPM) and vibrational spectroscopy. This combination allows for much higher spatial resolution than can be achieved with conventional Raman/FTIR instrumentation. The technique is also nondestructive, requires non-extensive sample preparation, and provides more contrast such as intensity contrast, polarization contrast and wavelength contrast, as well as providing specific chemical information and topography images simultaneously.

Adsorption is the accumulation and adhesion of molecules, atoms, ions, or larger particles to a surface, but without surface penetration occurring. The adsorption of larger biomolecules such as proteins is of high physiological relevance, and as such they adsorb with different mechanisms than their molecular or atomic analogs. Some of the major driving forces behind protein adsorption include: surface energy, intermolecular forces, hydrophobicity, and ionic or electrostatic interaction. By knowing how these factors affect protein adsorption, they can then be manipulated by machining, alloying, and other engineering techniques to select for the most optimal performance in biomedical or physiological applications.

The following outline is provided as an overview of and topical guide to biophysics:

The colloidal probe technique is commonly used to measure interaction forces acting between colloidal particles and/or planar surfaces in air or in solution. This technique relies on the use of an atomic force microscope (AFM). However, instead of a cantilever with a sharp AFM tip, one uses the colloidal probe. The colloidal probe consists of a colloidal particle of few micrometers in diameter that is attached to an AFM cantilever. The colloidal probe technique can be used in the sphere-plane or sphere-sphere geometries. One typically achieves a force resolution between 1 and 100 pN and a distance resolution between 0.5 and 2 nm.

<span class="mw-page-title-main">Single-cell analysis</span> Testbg biochemical processes and reactions in an individual cell

In the field of cellular biology, single-cell analysis and subcellular analysis is the study of genomics, transcriptomics, proteomics, metabolomics and cell–cell interactions at the single cell level. The concept of single-cell analysis originated in the 1970s. Before the discovery of heterogeneity, single-cell analysis mainly referred to the analysis or manipulation of an individual cell in a bulk population of cells at a particular condition using optical or electronic microscope. To date, due to the heterogeneity seen in both eukaryotic and prokaryotic cell populations, analyzing a single cell makes it possible to discover mechanisms not seen when studying a bulk population of cells. Technologies such as fluorescence-activated cell sorting (FACS) allow the precise isolation of selected single cells from complex samples, while high throughput single cell partitioning technologies, enable the simultaneous molecular analysis of hundreds or thousands of single unsorted cells; this is particularly useful for the analysis of transcriptome variation in genotypically identical cells, allowing the definition of otherwise undetectable cell subtypes. The development of new technologies is increasing our ability to analyze the genome and transcriptome of single cells, as well as to quantify their proteome and metabolome. Mass spectrometry techniques have become important analytical tools for proteomic and metabolomic analysis of single cells. Recent advances have enabled quantifying thousands of protein across hundreds of single cells, and thus make possible new types of analysis. In situ sequencing and fluorescence in situ hybridization (FISH) do not require that cells be isolated and are increasingly being used for analysis of tissues.

AFM-IR or infrared nanospectroscopy is one of a family of techniques that are derived from a combination of two parent instrumental techniques. AFM-IR combines the chemical analysis power of infrared spectroscopy and the high-spatial resolution of scanning probe microscopy (SPM). The term was first used to denote a method that combined a tuneable free electron laser with an atomic force microscope equipped with a sharp probe that measured the local absorption of infrared light by a sample with nanoscale spatial resolution.

Cell mechanics is a sub-field of biophysics that focuses on the mechanical properties and behavior of living cells and how it relates to cell function. It encompasses aspects of cell biophysics, biomechanics, soft matter physics and rheology, mechanobiology and cell biology.

Alireza Mashaghi is a physician-scientist and biophysicist at Leiden University. He is known for his contributions to single-molecule analysis of chaperone assisted protein folding, molecular topology and medical systems biophysics and bioengineering. He is a leading advocate for interdisciplinary research and education in medicine and pharmaceutical sciences.

A probe tip is an instrument used in scanning probe microscopes (SPMs) to scan the surface of a sample and make nano-scale images of surfaces and structures. The probe tip is mounted on the end of a cantilever and can be as sharp as a single atom. In microscopy, probe tip geometry and the composition of both the tip and the surface being probed directly affect resolution and imaging quality. Tip size and shape are extremely important in monitoring and detecting interactions between surfaces. SPMs can precisely measure electrostatic forces, magnetic forces, chemical bonding, Van der Waals forces, and capillary forces. SPMs can also reveal the morphology and topography of a surface.

Rae Marie Robertson-Anderson is an American biophysicist who is a Professor and Associate Provost at the University of San Diego. She works on soft matter physics and is particularly interested in the transport and molecular mechanics of biopolymer networks. Robertson-Anderson is a member of the Council on Undergraduate Research.

Cell biomechanics a branch of biomechanics that involves single molecules, molecular interactions, or cells as the system of interest. Cells generate and maintain mechanical forces within their environment as a part of their physiology. Cell biomechanics deals with how mRNA, protein production, and gene expression is affected by said environment and with mechanical properties of isolated molecules or interaction of proteins that make up molecular motors.

References

↑ Neuman KC, Nagy A (June 2008). "Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy". Nature Methods. 5 (6): 491–505. doi:10.1038/nmeth.1218. PMC 3397402 . PMID 18511917.
1 2 Hoffmann T, Dougan L (July 2012). "Single molecule force spectroscopy using polyproteins". Chemical Society Reviews. 41 (14): 4781–4796. doi:10.1039/c2cs35033e. PMID 22648310.
↑ Bizzarri AR, Cannistraro S (25 January 2012). Dynamic Force Spectroscopy and Biomolecular Recognition. CRC Press. pp. 1–. ISBN 978-1-4398-6237-7.
↑ Jagannathan B, Marqusee S (November 2013). "Protein folding and unfolding under force". Biopolymers. 99 (11): 860–869. doi:10.1002/bip.22321. PMC 4065244 . PMID 23784721.
1 2 Sitters G, Kamsma D, Thalhammer G, Ritsch-Marte M, Peterman EJ, Wuite GJ (January 2015). "Acoustic force spectroscopy". Nature Methods. 12 (1): 47–50. doi:10.1038/nmeth.3183. PMID 25419961. S2CID 12886472.
↑ Kishino A, Yanagida T (July 1988). "Force measurements by micromanipulation of a single actin filament by glass needles". Nature. 334 (6177): 74–76. Bibcode:1988Natur.334...74K. doi:10.1038/334074a0. PMID 3386748. S2CID 4274023.
↑ Evans E, Ritchie K, Merkel R (June 1995). "Sensitive force technique to probe molecular adhesion and structural linkages at biological interfaces". Biophysical Journal. 68 (6): 2580–2587. Bibcode:1995BpJ....68.2580E. doi:10.1016/S0006-3495(95)80441-8. PMC 1282168 . PMID 7647261.
↑ Mohapatra S, Lin CT, Feng XA, Basu A, Ha T (January 2020). "Single-Molecule Analysis and Engineering of DNA Motors". Chemical Reviews. 120 (1): 36–78. doi:10.1021/acs.chemrev.9b00361. PMID 31661246. S2CID 204974046.
↑ Ott W, Jobst MA, Schoeler C, Gaub HE, Nash MA (January 2017). "Single-molecule force spectroscopy on polyproteins and receptor-ligand complexes: The current toolbox". Journal of Structural Biology. 197 (1): 3–12. doi:10.1016/j.jsb.2016.02.011. PMID 26873782.
↑ Bhushan B (2017). Springer handbook of nanotechnology. ISBN 9783662543573. OCLC 1012104482.^{[ page needed ]}
1 2 Hinterdorfer P, Van Oijen A (2009). Handbook of single-molecule biophysics. Springer. ISBN 9780387764979. OCLC 534951120.^{[ page needed ]}
↑ Bell GI (May 1978). "Models for the specific adhesion of cells to cells". Science. 200 (4342): 618–627. Bibcode:1978Sci...200..618B. doi:10.1126/science.347575. PMID 347575.
↑ Petrosyan R (2020). "Unfolding force definition and the unified model for the mean unfolding force dependence on the loading rate". J. Stat. Mech. 2020 (33201): 033201. arXiv: 1904.03925 . Bibcode:2020JSMTE..03.3201P. doi: 10.1088/1742-5468/ab6a05 .
↑ Ma X, Gosai A, Balasubramanian G, Shrotriya P (May 2019). "Force spectroscopy of the thrombin-aptamer interaction: Comparison between AFM experiments and molecular dynamics simulations". Applied Surface Science. 475: 462–472. Bibcode:2019ApSS..475..462M. doi:10.1016/j.apsusc.2019.01.004. S2CID 104310868.
↑ Metelev M, Arseniev A, Bushin LB, Kuznedelov K, Artamonova TO, Kondratenko R, et al. (March 2017). "Acinetodin and Klebsidin, RNA Polymerase Targeting Lasso Peptides Produced by Human Isolates of Acinetobacter gyllenbergii and Klebsiella pneumoniae". ACS Chemical Biology. 12 (3): 814–824. doi:10.1021/acschembio.6b01154. PMID 28106375.
↑ Marchetti M, Kamsma D, Cazares Vargas E, Hernandez García A, van der Schoot P, de Vries R, et al. (August 2019). "Real-Time Assembly of Viruslike Nucleocapsids Elucidated at the Single-Particle Level". Nano Letters. 19 (8): 5746–5753. doi:10.1021/acs.nanolett.9b02376. PMC 6696885 . PMID 31368710.
↑ Sorkin R, Bergamaschi G, Kamsma D, Brand G, Dekel E, Ofir-Birin Y, et al. (August 2018). "Probing cellular mechanics with acoustic force spectroscopy". Molecular Biology of the Cell. 29 (16): 2005–2011. doi:10.1091/mbc.E18-03-0154. PMC 6232971 . PMID 29927358.
↑ Williams MC, Rouzina I (June 2002). "Force spectroscopy of single DNA and RNA molecules". Current Opinion in Structural Biology. 12 (3): 330–336. doi:10.1016/S0959-440X(02)00340-8. PMID 12127451.
↑ Jagannathan B, Elms PJ, Bustamante C, Marqusee S (October 2012). "Direct observation of a force-induced switch in the anisotropic mechanical unfolding pathway of a protein". Proceedings of the National Academy of Sciences of the United States of America. 109 (44): 17820–17825. Bibcode:2012PNAS..10917820J. doi: 10.1073/pnas.1201800109 . PMC 3497811 . PMID 22949695.
↑ Metelev M, Arseniev A, Bushin LB, Kuznedelov K, Artamonova TO, Kondratenko R, et al. (March 2017). "Acinetodin and Klebsidin, RNA Polymerase Targeting Lasso Peptides Produced by Human Isolates of Acinetobacter gyllenbergii and Klebsiella pneumoniae". ACS Chemical Biology. 12 (3): 814–824. doi:10.1021/acschembio.6b01154. PMID 28106375.
↑ Merkel R, Nassoy P, Leung A, Ritchie K, Evans E (January 1999). "Energy landscapes of receptor-ligand bonds explored with dynamic force spectroscopy". Nature. 397 (6714): 50–53. Bibcode:1999Natur.397...50M. doi:10.1038/16219. PMID 9892352. S2CID 4419330.
↑ Schoeler C, Malinowska KH, Bernardi RC, Milles LF, Jobst MA, Durner E, et al. (December 2014). "Ultrastable cellulosome-adhesion complex tightens under load". Nature Communications. 5 (1): 5635. Bibcode:2014NatCo...5.5635S. doi:10.1038/ncomms6635. PMC 4266597 . PMID 25482395.
↑ Guo M, Ehrlicher AJ, Jensen MH, Renz M, Moore JR, Goldman RD, et al. (August 2014). "Probing the stochastic, motor-driven properties of the cytoplasm using force spectrum microscopy". Cell. 158 (4): 822–832. doi:10.1016/j.cell.2014.06.051. PMC 4183065 . PMID 25126787.