Biomedical Engineering Reference
In-Depth Information
and. the. sample. can. be. ascertained. by. analyzing. the. generated. force. curves. (Noy. 2011)..
Force.spectra.have.been.generated.to.study.the.binding.of.ibronectin.to.bacterial.surfaces.
(Casillas-Ituarte.et.al..2012),.the.localization.of.vascular.endothelial.growth.factor.(VEGF).
receptor.on.endothelial.cells.(Almqvist.et.al..2004),.and.the.distribution.of.β1-integrin.on.
stem.cells.(Li.et.al..2012)..Figure.16.3A.highlights.force.spectra.that.have.been.generated.
due.to.dimerization.of.the.protein.cadherin.(Kumar.and.Hoh.2001).
Hinterdorfer.and.his.colleagues.have.developed.a.single-molecule.recognition.imaging.
technique.(Dufrêne.and.Hinterdorfer.2008;.Chtcheglova.and.Hinterdorfer.2011).in.which.
a.cantilever.tip.functionalized.with.a.probe.molecule.(a.lexible.short-linker).is.oscillated.
at.a.frequency.below.resonance..The.oscillation.is.split.into.approaching.and.withdrawing.
components.. The. oscillation. signals. in. the. approaching. component. relect. the. repulsive.
tip-sample.interaction,.providing.topographs..The.signals.in.the.withdrawing.component.
relect. the. association. events. between. the. probe. molecule. and. its. counterpart,. resulting.
in. recognition. images.. This. approach. enables. one. to. correlate. the. topographical. height.
images. with. the. recognition. images. obtained. using. AFM.. Single-molecule. recognition.
imaging.has.been.used.to.study.the.distribution.of.individual.ICAM-1.molecules.on.sur-
faces.(Willemsen.et.al..1998).and.to.study.transmembrane.transporters.on.cell.surfaces.at.
the.single-molecule.level.(Puntheeranurak.et.al..2011).
Besides. molecular. recognition,. AFM. has. also. been. used. to. study. cell-adhesion. forces..
AFM-based. single-cell. force. spectroscopy. (SCFS). enables. the. quantitative. study. of. cell.
adhesion.under.physiological.conditions..The.overall.protocol.involves.(1).the.functional-
ization.of.AFM.cantilevers.to.enable.cell.attachment,.(2).the.measurement.of.adhesion.forces.
between. the. cell. and. a. functionalized. substrate,. and. (3). data. analysis. and. interpretation..
SCFS.has.been.used.to.probe.adhesive.interactions.of.single.living.mammalian.cells.with.
extracellular.matrix.(ECM).proteins.(Friedrichs.et.al..2010),.other.cells.(Hoffmann.et.al..2011),.
metal.oxide.surfaces.(Stevens.et.al..2009),.and.bacterium.with.various.surfaces.(Lower.2011).
16.4.2 Single-Molecule Mechanics
AFM.offers.new.insight.into.the.process.of.protein.folding.and.unfolding.without.the.need.
to.denature.the.proteins.with.chemicals.or.temperature..Instead,.AFM.can.probe.single,.
fully.intact.proteins.with.mechanical.forces.(Bornschlögl.and.Rief.2011)..In.these.single-
molecule.force.spectroscopy.(SMFS).experiments,.interaction.forces.are.monitored.while.
the.AFM.tip.continuously.approaches.and.retracts.from.the.biological.sample..The.obtained.
force-extension.curves.provide.key.insight.into.the.molecular.elasticity.and.localization.of.
single.molecules.either.on.isolated.systems.or.on.cellular.surfaces.(Marszalek.and.Dufrêne.
2012)..The.energy.landscape.of.the.giant.muscle.protein.titin.(Linke.and.Grützner.2008).
and. the. eukaryotic. signaling. protein. calmodulin. (Junker. and. Rief. 2009). are. paradigm.
models.in.this.ield..The.nanobiomechanics.of.several.other.biomolecules.have.also.been.
analyzed.using.SMFS..These.include.RNA.unfolding.(Heus.et.al..2011),.enzyme.mechanics.
and.catalysis.(Wang.et.al..2011),.molecular.persistence.length.and.force.ingerprint.of.pro-
teoglycans.(Harder.et.al..2010),.nuclei-acid-protein.interactions.in.intact.viruses.(Jin.et.al..
2010),.and.of.polysaccharides.and.proteins.on.live.cells.(Francius.et.al..2009).
In. recent. years,. AFM-based. single-molecule. mechanics. has. also. been. complemented.
with.other.techniques.such.as.luorescence.resonance.energy.transfer.spectroscopy.(AFM-
FRET). (He. et. al.. 2012).. This. integrated. AFM-FRET. nanoscopy. approach. can. effectively.
pinpoint.and.mechanically.manipulate.a.targeted.dye-labeled.single.protein..By.analyzing.
time-resolved.FRET.trajectories.and.correlated.AFM.force.pulling.curves.of.the.targeted.
single-molecule.enzyme,.the.protein.conformational.changes.can.be.observed.
