Chapter 6 – Forces  237

6.5.4  SINGLE-​MOLECULE AFM FORCE SPECTROSCOPY

AFM can also be used to investigate the mechanical elasticity of single biomolecules

(Figure 6.7a) in a technique called AFM force spectroscopy. In the simplest form, AFM force

spectroscopy experiments involve nonspecific binding of the biomolecule in question to a

gold-​ or platinum-​coated coverslip followed by dipping an AFM tip into the surface solu­

tion. Upon retracting the AFM tip back, there is a probability that a section of a molecule

is nonspecifically tethered between the tip, for example, by hydrophobic binding, and the

coverslip. In having tethered a section of a single molecule, the tip–​cantilever system can

then be used to investigate how the molecular restoring force varies with its end-​to-​end

extension, similar to optical and magnetic tweezers discussed previously in this chapter.

Simple AFM force spectroscopy devices can be limited to just one axis of controllable

movement for the vertical axis controlled by a piezo actuator to move the AFM tip rela­

tive to the sample (these one-​axis instruments in effect relying on lateral sample drift to

move to a different region of the sample, so there is a paradoxical benefit in having a mar­

ginally unstable system). Single-​molecule AFM force spectroscopy experiments are often

performed on modular proteins, either purified from the native source or using smaller syn­

thetic molecules that allow shorter sections of the native molecules to be probed in a more

controllable way than the whole native molecule. The large muscle protein titin, discussed

previously in the context of optical tweezers, has proved to be an invaluable model system in

AFM force spectroscopy studies. In one of the best examples of such pioneering experiments,

single molecule constructs consisting of up to eight repeats of the same protein “Ig” domain

(Rief et al., 1997).

The properties of the molecule titin are worth discussing in greater detail due to its import­

ance in force spectroscopy experiments and our subsequent understanding of molecular

mechanical properties. Titin is an enormous molecule whose molecular weight lies in the

MDa range, consisting of ~30,000 individual amino acid residues and is part of a filamentous

system in muscle, which act as springs to align the functional subunits of muscle tissue called

sarcomeres. Most of the molecule is composed of repeating units of β-​barrel modules of

~100 amino acid residues each, which either belong to a class called “fibronectin” (Fn) or

“immunoglobulin” (Ig), with a combined total in excess of 370 combined Fn and Ig domains.

The increased likelihood of unfolding of the β-​barrel structure of Fn or Ig domains as force

is increased on the titin possibly confers a shock-​absorber effect, which ensures that the myo­

fibril, the smallest fully functional filamentous subunit of muscle tissue compared to multiple

repeating sarcomeres, can maintain structural integrity even in the presence of anomalously

high forces, which could damage the muscle. Titin is made in a variety of different forms with

different molecular weights depending on the specific type of muscle tissue and its location

in the body, and there is good evidence to indicate that this allows the titin molecular stiffness

to be catered to the range of force experienced in a given muscle type.

In fishing for surface-​bound titin constructs, a variable number of Ig modules in the range

1–​8 can be tethered between the gold surface and the AFM tip depending on the essen­

tially random position of the nonspecific binding to both. These domains unfold in the same

manner as those described for mechanical stretch experiments on titin using optical twee­

zers, with a consequent sudden drop in entropic force from the molecule and increase in

molecular extension of ~30 nm due to an Ig domain making a transition from a folded to

an unfolded conformation. Thus, the resultant force-​extension relation has a characteristic

sawtooth pattern, with the number of “teeth” corresponding to the number of Ig domains

unfolded in the stretch, and therefore varying in the range 1–​8 in this case (Figure 6.7b).

These sawtooth patterns are important since they indicate the presence of a single-​molecule

tether, as opposed to multiple tethers, which might be anticipated if the surface density of

molecules is sufficiently high. The sawtooth pattern thus denotes a molecular signature.

AFM force spectroscopy can also be used with greater binding specificity by chemically

functionalizing both the AFM tip and the gold or platinum surface. Many AFM force spec­

troscopy devices are also used in conjunction with an xy nanostage that allows lateral con­

trol of the sample to allow reproducible movements to different sample regions as well as