Bio-Molecular motors

Flageller motors and working of atpase motor
Video explanation

1. Biomolecular Motors Presented By: Arushe Tickoo 2K16/IBT/03 M. Tech (1 st yr )

2. 28/04/2017 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

3. 28/04/2017 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. N o 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

4. 28/04/2017 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;

5. 28/04/2017 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 );

6. 28/04/2017 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.

7. 28/04/2017 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. coli a 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

8. 28/04/2017 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: F 1 , situated in the mitochondrial matrix, and F o , 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 F 1  sector, a soluble portion situated in the mitochondrial matrix, and the F o  sector, bound to the inner mitochondrial membrane. F 1  is composed of three copies of each of subunits α and β, and one each of subunits γ, δ and ε. F 1  subunits γ, δ and ε constitute the central stalk of complex V. F o  consists of a subunit c-ring (probably comprising eight copies, as shown in bovine mitochondria and one copy each of subunits a, b, d, F 6  and the oligomycin sensitivity-conferring protein (OSCP). Subunits b, d, F 6  and OSCP form the peripheral stalk which lies to one side of the complex.

9. 28/04/2017

10. 28/04/2017 Human mitochondrial ATP synthase, or complex V, consists of two functional domains, F 1  and F o . F 1  comprises 5 different subunits (three α, three β, and one γ, δ and ε) and is situated in the mitochondrial matrix. F o  contains subunits c, a, b, d, F 6 , OSCP and the accessory subunits e, f, g and A6L. F 1  subunits γ, δ and ε constitute the central stalk of complex V. Subunits b, d, F 6  and OSCP form the peripheral stalk. Protons pass from the intermembrane space to the matrix through F o , which transfers the energy created by the proton electrochemical gradient to F 1 , 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

11. 28/04/2017 Fo Region F1  Region The F1 portion is soluble and consists of a hexamer , denoted a 3 b 3 . 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 a 3 b 3 hexamer so that it anchors the a subunit to F1. Thus there are two ‘stalks’ connecting Fo to F1: ge and b2 d .

12. 28/04/2017

13. 28/04/2017

14. 28/04/2017 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 binding affinities (a) The system starts with either ( β 1 , β 2 , β 3 ) = (E, T D•P , D) ( b) Clockwise rotation of g increases the binding affinity of ADP in b1, traps ATP in b2, and promotes Pi binding on b3. (c) Further rotation of g traps ADP and allows Pi binding in b1, releases the tightly bound ATP and allows ADP binding in b2, and traps Pi in b3.

15. 28/04/2017 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.

16. 28/04/2017

17. 28/04/2017 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.

18. 28/04/2017 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

19. 28/04/2017 Itoh , H., et al., (2004 ) Mechanically driven ATP synthesis by F1-ATPase . Nature,. 427(6973): p. 465-8. Noji , H., et al., (1997 ) Direct observation of the rotation of F1-ATPase . Nature,. 386(6622): p. 299-302. Oster G, Wang H; ATP Synthase: Two rotary molecular motors working together; University of California, Berkeley Antoniel M etal., ( 2014 May) The Oligomycin -Sensitivity Conferring Protein of Mitochondrial ATP Synthase ; Int J Mol Sci .; 15(5): 7513–7536 . Oster G and Wang H; ( April 1999) ATP synthase: two motors, two fuels , Elsevier Science 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