Biomolecular Motors

Presented By:

Arushe Tickoo

DTU, Delhi

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The typical biomolecular motor is a protein that uses free energy from ATP (Adenosine Tri-

phosphate) or other nucleotide triphosphate (NTP) hydrolysis as its fuel to produce mechanical

force.

ATP is the universal currency of energy in biological systems and, is generated from glucose by

a sequence of reactions called glycolysis.

The hydrolysis of ATP to ADP and inorganic phosphate is energetically favorable and the pool of

ATP in a cell is far higher than that of its hydrolysis products, hence serving as a remarkable

store of chemical energy that is used to perform the various cellular functions.

Motor proteins are involved in several critical cellular processes and have functions ranging

from ATP synthesis and muscle contraction.

Biomolecular motor

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Flagellated bacteria, such as Escherichia coli, swim by rotating thin helical filaments, each driven at its

base by a reversible rotary motor, powered by an ion flux. A motor is about 45 nm in diameter and is

assembled from about 20 different kinds of parts. It develops maximum torque at stall but can spin several

hundred Hz.

Its direction of rotation is controlled by a sensory system that enables cells to accumulate in regions

deemed more favorable.

No more than 50 nm in Diameter It spins clockwise (CW) orcounterclockwise (CCW) at speeds on the order of

100 Hz, driving long thin helical filaments that enable cells to swim Receptors near the surface of the cell count

molecules of interest (sugars, amino acids, dipeptides) and control the direction of flagellar rotation. If a leg of

the search is deemed favorable, it is extended,

Flagellar motor

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Fig; A schematic diagram

of the flagellar motor

CheY-P

It is the chemotaxis

signaling molecule that

binds to FliM, and FlgM

is the anti-sigma factor

pumped out of the cell by

the transport apparatus;

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A cell swims steadily in a direction roughly parallel to its long axis for about a second—it is said to

“run”—and then moves erratically in place for a small fraction of a second—it is said to “tumble”—

and then swims steadily again in a new direction. When a cell runs at top speed, all of its flagellar

filaments spin CCW, the filaments form a bundle that pushes the cell steadily forward. When a cell

tumbles, one or more filaments spin CW; these filaments leave the bundle, and the cell changes

course

Motors switch from CCW to CW and back again approximately at random. The likelihood of

spinning CW is enhanced by a chemotactic signaling protein, CheY.

When phosphorylated, CheY binds to the cytoplasmic face of the flagellar motor. The

phosphorylation of CheY is catalyzed by a kinase, the activity of which is controlled by

chemoreceptors.

The different components of the motor are named after the genes that encode them. genes for which

mutant cells lacked flagellar filaments were called fla (for flagellum), but after more than 26 had been

found.

fla genes are now called flg, flh, fli, or flj, depending upon their location on the genetic map. Genes for

which mutant cells produce paralyzed flagella are called mot (for motility). Four of the all gene

products that are involved in gene regulation (FlgM, FlhC, FlhD, FliA);

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The M-ring (for membrane) has affinity for inner-

membrane fractions,

The S-ring is seen just above the inner membrane,

The P-ring (for peptidoglycan) is at the right place to be

embedded in the peptidoglycan,

and the L-ring (for lipopolysaccharide) has affinity for

outer-membrane fractions.

It was found that both the M- and S-rings (now called the

MS-ring) comprise different domains of the same protein,

FliF. Therefore, they function as a unit.

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FliG, FliM, and FliN are also referred to as the “switch complex,” since many mutations of fliG, fliM,

and fliN lead to defects in switching (in control of the direction of rotation)

Operons encoding the proteins of the chemotaxis system of E.

colia

Class 1 Class 2 Class 3

flhDC flgAMN fliC

flgBCDEFGHIJKL motABcheAW

flhBAE tar tap cheRBYZ

fliAZY aer

fliDST trg

Class 1 contains the master operon, flhDC, the expression of which is required for transcription of class 2 and class 3

operons.

Class 2 contains eight operons that encode components required for construction of the hook-basal body complex,

and class 3 contains six more that encode components required for filament assembly and motor function

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ATP Synthase

ATP synthase is one of the wonders of the molecular world. ATP synthase is an enzyme, a molecular motor, an

ion pump, and another molecular motor all wrapped together in one amazing nanoscale machine. It plays an

indispensable role in our cells, building most of the ATP that powers our cellular processes.

Human mitochondrial (mt) ATP synthase, or complex V consists of two functional domains: F1, situated in the

mitochondrial matrix, and Fo, located in the inner mitochondrial membrane. Complex V uses the energy created

by the proton electrochemical gradient to phosphorylate ADP to ATP.

ATP synthase consists of two well defined protein entities: the F1 sector, a soluble portion situated in the

mitochondrial matrix, and the Fo sector, bound to the inner mitochondrial membrane. F1 is composed of three

copies of each of subunits α and β, and one each of subunits γ, δ and ε. F1 subunits γ, δ and ε constitute the

central stalk of complex V. Fo consists of a subunit c-ring (probably comprising eight copies, as shown in

bovine mitochondria and one copy each of subunits a, b, d, F6 and the oligomycin sensitivity-conferring

protein (OSCP). Subunits b, d, F6 and OSCP form the peripheral stalk which lies to one side of the complex.

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Human mitochondrial ATP synthase, or

complex V, consists of two functional domains,

F1 and Fo. F1 comprises 5 different subunits

(three α, three β, and one γ, δ and ε) and is

situated in the mitochondrial matrix. Fo contains

subunits c, a, b, d, F6, OSCP and the accessory

subunits e, f, g and A6L. F1 subunits γ, δ and ε

constitute the central stalk of complex V.

Subunits b, d, F6 and OSCP form the peripheral

stalk. Protons pass from the intermembrane

space to the matrix through Fo, which transfers

the energy created by the proton

electrochemical gradient to F1, where ADP is

phosphorylated to ATP.

Structure of ATP Synthase

Jonckheere I, Smeitink J A M, and Rodenburg R J; Mitochondrial ATP synthase: architecture, function and

pathology (2011), J Inherit Metab Dis

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Fo Region F1 Region

The F1 portion is soluble and consists of a

hexamer, denoted a3b3. This hexamer is arranged

in an annulus about a central shaft consisting of the

coiled-coil γ subunit.

The Fo portion consists of three transmembrane

subunits: a, b2 and c10-14. The remainder of Fo

consists of the transmembrane subunits a, and

b2; the latter is attached by the d subunit to the

a3b3 hexamer so that it anchors the a subunit

to F1. Thus there are two ‘stalks’ connecting Fo

to F1: ge and b2d.

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The binding change mechanism. Notation for site occupancies: T = ATP bound, DP = ADP • Pi bound, D =ADP bound. b subunits are numbered clockwise. The length of the arrows indicates the relative bindingaffinities (a) The system starts with either (β 1, β 2, β 3) = (E, T D•P, D) (b) Clockwise rotation of gincreases the binding affinity of ADP in b1, traps ATP in b2, and promotes Pi binding on b3. (c) Furtherrotation of g traps ADP and allows Pi binding in b1, releases the tightly bound ATP and allows ADPbinding in b2, and traps Pi in b3.

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In steps 1 and 2 a site binds ADP and phosphate (not necessarily in that order). While trapped in the catalytic

site in step 3, reactants (ADP and Pi ) and product (ATP) are in chemical equilibrium. Step 4 requires the

input of mechanical torque from Fo on g to trap the reactants in the ATP state and to pry open the site

releasing the tightly bound ATP. The way in which this works is found in the shape of the a3b3 hexamer and

the g shaft

At the top of the a3b3 hexamer is a hydrophobic ‘sleeve’ in which the g shaft rotates. Further down, however,

the annulus is offset from the center, so that as g rotates clockwise, it sequentially pushes outwards on each

catalytic site. In addition, the e subunit is located eccentrically and attached to the g and c subunits so that, as

g rotates, it comes into contact sequentially with each b subunit in a conserved region called the DELSEED

sequence (named for the single letter abbreviation of its constituent amino acids). Together, this asymmetric

rotation exerts stress on the catalytic site loosening its grip on ATP so that thermal fluctuations can free it into

solution.

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A single molecule of F1-ATPase acts as a rotary motor, the smallest known, by direct observation

of its motion. A central rotor of radius approximately 1 nm, formed by its gamma-subunit, turns

in a stator barrel of radius approximately 5nm formed by three alpha- and three beta-subunits. F1-

ATPase, together with the membrane-embedded proton-conducting unit F0, forms the H+-ATP

synthase that reversibly couples transmembrane proton flow to ATP synthesis/hydrolysis in

respiring and photosynthetic cells. In the presence of ATP, the filament rotated for more than 100

revolutions in an anticlockwise direction when viewed from the 'membrane' side. The rotary

torque produced reached more than 40 pN nm(-1) under high load.

Direct observation of the rotation of F1-ATPase.

Noji H, Yasuda R, Yoshida M, Kinosita K Jr.

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In the realm of ATP-driven linear biomotors, the most prominent examples are dynein, kinesin and

myosin, which is involved in muscle contraction. Muscle contraction is a sophisticated process involving

actin and myosin and is a result of actin filaments sliding on myosin heads. Actin filaments are formed of

375 amino acid long subunits that are associated with ATP. Myosin transport along actin is not limited to

muscle contraction, and is involved in several cellular transport processes.

It is thought that the two heads of the myosin move along in a hand-over-hand mechanism based on the

~74nm displacements observed by fluorescent labeling methods.

Interaction of an actin filament

with myosin-coated surface

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1. Itoh, H., et al., (2004) Mechanically driven ATP synthesis by F1-ATPase. Nature,. 427(6973): p. 465-8.

2. Noji, H., et al., (1997) Direct observation of the rotation of F1-ATPase. Nature,. 386(6622): p. 299-302.

3. Oster G, Wang H; ATP Synthase: Two rotary molecular motors working together; University of

California, Berkeley

4. Antoniel M etal., (2014 May) The Oligomycin-Sensitivity Conferring Protein of Mitochondrial ATP

Synthase ; Int J Mol Sci.; 15(5): 7513–7536.

5. Oster G and Wang H; (April 1999) ATP synthase: two motors, two fuels, Elsevier Science

6. Manuela Antoniel, (2014 May) The Oligomycin-Sensitivity Conferring Protein of Mitochondrial ATP

Synthase: Emerging New Roles in Mitochondrial Pathophysiology, International Journal of Molecular

Sciences

References