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3 Nanomechanics of DNA, Proteins, and Cells

3 Nanomechanics of DNA, Proteins, and Cells

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11



Biology on the Nanoscale



Fig. 11.29 (a) A DNA

molecule is stretched between

beads held in a micropipette

and a force-measuring optical

trap. The measured extension

is the sum of contributions

from the single-stranded

DNA (ssDNA) and

double-stranded DNA

(dsDNA). (b) Force versus

extension for ssDNA and

dsDNA obtained with the

instrumentation in (a). Arrows

show changes in extension

observed at constant tension

during polymerization (Poly)

or force-induced exonuclease

(removal of nucleotide

sections from the DNA)

activity (Exo). (Reprinted

with permission from [11.74].

© 2003 Nature Publishing

Group.)



replicate the stretched molecule at given tension. As the ssDNA was converted into

dsDNA by the polymerase, the replication process could be followed by monitoring the extension (below 6 pN) or contraction (above 6 pN) of the molecule (see

Fig. 11.29).

The force-extension behavior of single supercoiled DNA molecules can further

elucidate enzyme efficiency by performing elasticity measurements. By the rotation

of a bead attached to a stretched DNA molecule, a torsional strain can be applied to

this molecule (Fig. 11.30a) which finally gives rise to buckling and the formation

of plectonemes (DNA units projecting out of the molecular axis) and reduces the

apparent extension of the molecule. By the activity of the enzyme topoisomerase II,

which is known to relax supercoils in eukaryotic cells [11.74], the extension of a

single supercoiled DNA molecule is step-wise increased (Fig. 11.30b). The size of

these steps corresponds to the removal of two DNA turns by cutting the molecule

(Fig. 11.30c) so that the helix is passed through itself and finally resealed. It has been

additionally shown in single-molecule studies that the topoisomerase IV enzyme has

a chiral substrate specificity: it relaxes overwound DNA more efficiently than underwound DNA. This makes the enzyme relax the supercoils forming during replication

while avoiding counterproductive relaxation in non-replicating DNA [11.74].



11.3



Nanomechanics of DNA, Proteins, and Cells



559



Fig. 11.30 The elastic behavior of supercoiled DNA molecules for single-molecule topoisomerase

enzyme assays. (a) Molecules are stretched and twisted in magnetic tweezers until the DNA

molecule buckles and forms plectonemes, shortening the apparent extension. (b) Under addition of topoisomerase II to plectonemed DNA molecules, 90 nm steps appear in the extension,

corresponding to the removal of two turns as discussed by the model in (c). (c) Model of the topoisomerase II action. The enzyme cleaves both strands of the supercoiled DNA and passes the double

helix through itself, leading to the removal of two turns. (Reprinted with permission from [11.74].

© 2003 Nature Publishing Group)



11.3.3 Unzipping of DNA

During replication the enzyme DNA helicase must generate force to unzip the

parental DNA strands. In single-molecule studies the forces for mechanical separation were measured (see Fig. 11.31) for poly (dG-dC) DNA to FG−C = (20 ± 3)

pN and for poly (dA–dT) DNA to FA−T = (9 ± 3) pN [11.75]. These forces can

be ascribed to the difference in the stabilities of the triple hydrogen bonds of the

guanine–cytosine base pair or the double hydrogen bond of the adenine–thymine

base pair, respectively. By this type of experiments the helicase function might be

studied directly [11.74].



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Fig. 11.31 (a) Measurement of the force between the base pairs of DNA. (a) Measuring principle in the atomic force microscope (AFM). (b) The steps in the stress–strain curve of a poly

(dG–dC) DNA molecule with triple hydrogen bonds yield the binding force FG−C = 20 pN (bottom curve). (c) For poly (dA–dT) DNA with double hydrogen bonds the binding force FA−T = 9

pN is determined. (Reprinted with permission from [11.75]. © 1999 Nature Publishing Group)



11.3.4 Protein Mechanics

Proteins perform an array of tasks in living cells, from signal transduction to

metabolic and catalytic functions, and mechanical support (see [11.6]). Proteins

consist of 20 different amino acids (Fig. 11.32a) arranged in a specific polypeptide sequence (Fig. 11.32b). Hydrogen bonding leads to the formation of secondary

structures, such as α-helices and β-sheets (Fig. 11.32c, d) which fold to globular domains due to short-range forces such as hydrophobic, electrostatic, van der



11.3



Nanomechanics of DNA, Proteins, and Cells



561



Fig. 11.32 Basic structural features, characteristic length, and timescales of proteins. (a) Protein

structures are constructed from 20 different amino acids. A monomer of an amino acid with a

side-chain labeled R. (b) A short peptide comprising two different amino acids yielding 202 , or

400 possible sequences. (c) α-Helices and (d) β-sheets form proteins’ secondary structure. (e) The

3D protein structures consist out of globular structures formed by the secondary structures. (f) The

characteristic timescales for protein motion and deformation span from 1 s to 10−10 s. (Reprinted

with permission from [11.6]. © 2003 Nature Publishing Group)



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Biology on the Nanoscale



Waals, and hydrogen bonding (Fig. 11.32e) with size of 1–100 nm. Proteins in a cell

undergo continuous motion and structural changes on 5 nm (domains) to 0.05 nm

(single atom) scales over a wide range of timescales (10−15 –100 s; Fig. 11.32f).

Protein molecules can form domain hinge motion in cells with significant biological implications. The molecular motor myosin “moves” on an actin filament

by generating hinge motion of its head, where as the rotation of the γ-submit of

the F1 -ATPase might be driven by the hinge motion of the β-subunit (see [11.6]).

Such as proteins can transform from a biologically active state to a denatured of



Fig. 11.33 Deformation and unfolding of a multidomain protein when stretched by an AFM. The

sawtooth pattern corresponds to a sequential deformation and unfolding of individual domains.

When the distance between the substrate and the cantilever increases (1–2) the force increases

and the protein elongates. When a domain unfolds (3) the elongation of the protein reduces the

force almost to zero. Further extension again stresses the cantilever (4). The consecutive domain

deformation of the molecule (recombinant human tenascin C) obeys the worm-like chain (WLC)

model with the persistence length fixed to 0.56 nm and the contour length L0 for each peak adjusted

as shown. (Reprinted with permission from [11.6]. © 2003 Nature Publishing Group)



11.4



Molecular Motors and Machines



563



inactive state in response to small changes in temperature or pH of the surroundings,

the application of mechanical forces can lead to protein domain deformation and

unfolding (Fig. 11.33). By these mechanical studies the force for, e.g., unfolding a

protein domain can be measured specifically [11.76, 11.77].



11.4 Molecular Motors and Machines

Life implies movement. Motion in biology is a cascade of mechanical transduction

processes, from the molecular scales of time and length upwards, which are powered by tiny protein machines known as molecular motors. Among the best known

are motors that use sophisticated intramolecular amplification mechanisms to take

nanometer steps along protein tracks in the cytoplasm. Three types of cytoplasmic

motors are known (see [11.15, 11.78, 11.79]): myosins, which move on actin filaments, and kinesins, and dyneins, which use microtubules as tracks (see Fig. 11.34).

In all three motor classes, ATP hydrolysis causes a conformational change in a globular motor domain that is amplified and translated into movement with the help of

accessory structural motifs. These motors transport a wide variety of cargo, power

cell locomotion, drive cell division and, when combined in large ensembles, allow

organisms to move. Motor defects may lead to severe diseases or may even be lethal.

The basic principles of these motors and of the rotational ATPase nanomachine

synthesizing ATP will be presented below.



Fig. 11.34 Cytoskeletal motors. (a) Myosin II; (b) conventional kinesin; (c) ciliary dynein. Top

panel: cryoelectron micrographs of rotary-shadowed single molecules. Bottom panel: schematic

overviews. The diameter of the myosin II motor domains (yellow) is about 15 nm with the associated proteins (brown). Coiled-coil domains are represented by parallel black lines. (Reprinted with

permission from [11.79]. © 2003 Nature Publishing Group)



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11.4.1 Myosin

The myosins are a large family of molecular motors, where the human genome

includes about 40 myosin genes.

Myosin V is an actin-based molecular motor that has a key function in organelle

and messenger RNA (mRNA) transport, as well as in membrane trafficking. This

motor was the first member of the myosin superfamily shown to be processive,

meaning that a single motor protein can “walk” hand-over-hand along an actin

filament for many steps before detaching. The active state is extended whereas

the inactive state is compact (see [11.80]). Each myosin V molecule consists of

two heads (see Fig. 11.35) that contain an amino-terminal motor domain followed

by a lever-arm, a coiled-coil dimerization domain (S2), and a carboxy-terminal

globular cargo-binding domain. After ATP hydrolysis (see [11.15]), the loss of

the phosphate group from ATP leaves a space of approximately 0.5 nm, which is

thought to cause a rearrangement of structural elements flanking the ATP-binding

site. The rearrangement in this first level of amplification is coordinated with

structural changes in the actin-binding site. The next level of amplification involves

the communication of the initial conformational change in the active ATP site



Fig. 11.35 Orientation of the myosin V inhibited structure on actin from Spodoptera frugiperda

(Sf9) cells. (a) Only one myosin V head can be docked to the actin strand, rendered light-blue

and gray. The unbound head extends up to the viewer. (b) Cargo-binding domain (yellow) on the

motor domain (magenta) are rendered in a space-filling representation. The ATP-binding region

and Loop1 are colored green. A decrease in the flexibility of Loop1 may decrease ADP release rates

and the direct binding of the cargo binding domain to Loop 1 could stabilize its motion and decrease

ADP release rates, thus accounting for the decreased ATPase activity of the folded monomer.

(c) High-magnification electron micrographs of actin-bound myosin V molecules. (Reprinted with

permission from [11.80]. © 2006 Nature Publishing Group)



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