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Molecular Machines: Packers and Movers, Assemblers and Shredders Debashish Chowdhury Physics Department, Indian Institute of Technology, Kanpur Home page:

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Presentation on theme: "Molecular Machines: Packers and Movers, Assemblers and Shredders Debashish Chowdhury Physics Department, Indian Institute of Technology, Kanpur Home page:"— Presentation transcript:

1 Molecular Machines: Packers and Movers, Assemblers and Shredders Debashish Chowdhury Physics Department, Indian Institute of Technology, Kanpur Home page: 2 nd IITK REACH Symposium, March 2008

2 “Nature, in order to carry out the marvelous operations in animals and plants, has been pleased to construct their organized bodies with a very large number of machines, which are of necessity made up of extremely minute parts so shaped and situated such as to form a marvelous organ, the composition of which are usually invisible to the naked eye, without the aid of microscope”- Marcello Malpighi (seventeenth century); As quoted by Marco Piccolino, Nature Rev. Mol. Cell Biology 1, 149-152 (2000). (March 10, 1628 - September 30, 1694)March 101628 September 301694 Founder of microscopic anatomy Marcello Malpighi

3 “The entire cell can be viewed as a factory that contains an elaborate network of interlocking assembly lines, each of which is composed of a set of large protein machines…. Why do we call the large protein assemblies that underline cell function protein machines? Precisely because, like machines invented by humans to deal efficiently with the macroscopic world, these protein assemblies contain highly coordinated moving parts” - Bruce Alberts, Cell 92, 291 (1998). President of the National Academy of Sciences USA (1993-2005) Editor-in-chief, SCIENCE (March, 2008 - )

4 Machine InputOutput Motor Input Output Mechanical

5 “Natural” Nano-machines within a living cell “Artificial” Nano-machines for practical applications Understanding mechanisms through experiments and theoretical modeling Design using natural components extracted from living cells Design using artificial components synthesized in the laboratory All the design and manufacturing completed so far have succeeded only in establishing “proof-of-principle”, but still far from commercial prototypes. Designs of molecular machines have been perfected by Nature over millions or billions of years on the principles of evolutionary biology.

6 “Natural” Nano-machines within a living cell Understanding mechanisms through experiments and theoretical modeling In THIS TALK

7 Outline of the talk  Introduction 2. Examples of molecular motors I. Cytoskeletal motors II. Nucleic acid-based motors 3. Methods of quantitative modeling to understand mechanisms 8. Conclusion 4. Some fundamental questions on mechanisms of molecular motors 5. Theoretical model of single-headed kinesin motor KIF1A 6. Theoretical models of RNA polymerase and Ribosome 7. Examples of molecular motors III: Membrane-associated rotary motors

8 Examples of molecular motors I: Cytoskeletal Motors

9 Cytoskeleton of a cell Alberts et al., Molecular Biology of the Cell Required for mechanical strength and intra-cellular transportation.

10 Cytoskeletal Motor Transport System = Motor + Track + Fuel

11  -  dimer Protofilament Diameter of a tubule: ~ 25 nm. Track: MicrotubuleTrack: F-actin TRACK

12 Woehlke and Schliwa (2000) Superfamilies of Cytoskeletal MOTORS

13 Cytoskeletal Motors PortersRowers Animated cartoon: MCRI, U.K. Kinesin-1 Myosin-V Myosin-II Science, 27 June (2003)

14 Cytoskeletal Motors Porters Animated cartoon: MCRI, U.K. Kinesin-1: Smallest BIPED My research group works on “PORTERS”.

15 MCAK, KLP10A and KLP59C : members of kinesin-13 family Kip3p: a member of kinesin-8 family SHREDDERS: walk/diffuse and depolymerize Theoretical modeling by Govindan, Gopalakrishnan and Chowdhury (2008)

16 Examples of molecular motors II: Nucleic acid-based Motors

17 (RNA polymerase) Translation (Ribosome) DNA RNA Protein Transcription Central dogma of Molecular Biology and assemblers Simultaneous Transcription and Translation Rob Phillips and Stephen R. Quake, Phys. Today, May 2006.

18 RNA polymerase: a mobile workshop DNARNA decodes genetic message, RNA polymerase polymerizes RNA using DNA as a template. A motor that moves along DNA track, Roger Kornberg Nobel prize in Chemistry (2006)

19 Ribosome: a mobile workshop mRNAProtein decodes genetic message, Ribosome polymerizes protein using mRNA as a template. A motor that moves along mRNA track,

20 Methods of Quantitative modeling to understand mechanisms

21 Atomic level: Quantum mechanical calculation of structures; numerical works based on software packages (Quantum Chemistry) Molecular level: Classical Newton’s equations for protein + molecules of the aqueous environment; Classical Molecular Dynamics (MD) (inadequate for length and time scales relevant for motor protein dynamics) Brownian level: Langevin eqn. for the individual proteins (equivalent: Fokker-Planck or Master equations) Levels of Description Coarse-grained level: Dynamical equations for local densities of motors; Too coarse to maintain individual identities of the motors.

22 Brownian level: Master eqn./Fokker-Planck eqn. for the individual proteins Level of Description adopted in our theoretical works

23 Chem. State Position State Space

24 Translocation State Space Chem. State Position

25 Chem. reaction Chem. State Position State Space

26 Mechano- Chemical transition Chem. State Position State Space

27 Translate intoMathematical language Master equationsNumerical protocols Analyticalsolution Computersimulation Theoretical predictionsNumerical predictions Experimental data Compare Mechano-chemical transitions in “state-space” Compare

28 Some Fundamental questions on mechanisms of molecular motors

29 Question I: Is the mechanism of molecular motors identical to those of their macroscopic counterparts (except for a difference of scale)? Size: Nano-meters; Force: Pico-Newtons NO.  Far from equilibrium  Made of soft matter  Dominant forces are non-inertial “…gravitation is forgotten, and the viscosity of the liquid,…,the molecular shocks of the Brownian movement, …. Make up the physical environment….The predominant factor are no longer those of our scale; we have come to the edge of a world of which we have no experience, and where all our preconceptions must be recast”. - D’Arcy Thompson, “On Growth and Form” (1942).

30 FORCES on molecular motors Random thermal forces ; bombardment by water molecules (“Brownian”-type motion) Viscous forces; inertial forces are negligibly small (Low-Reynold’s number).

31 Question II: What is the mechanism of energy transduction ?

32 Power Stroke S.A. Endow, Bioessays, 25, 1212 (2003)

33 Power-stroke versus Brownian ratchet Joe Howard, Curr. Biol. 16, R517 (2006).

34 Brownian ratchet Power Stroke Input energy drives the motor forward Random Brownian force tends to move motor both forward and backward. Input energy merely rectifies backward movements. Mechanisms of energy transduction by molecular motors A Brownian motor operates by converting random thermal energy of the surrounding medium into mechanical work!!

35 R.D.Astumian,Scientific American, July 2001 Smoluchowski-Feynman ratchet-and-pawl device Using the ratchet-and-pawl device, Feynman showed that it is impossible to extract mechanical work spontaneously from thermal energy of the surrounding medium if the device is in equilibrium (consequence of the 2 nd law of thermodynamics). Feynman Lectures in Physics. A Brownian motor does not violate 2 nd law of thermodynamics as it operates far from equilibrium where the 2 nd law is not applicable.

36 Question III: Why are the porters processive? (i.e., how does a porter cover a long distance without getting detached from the track?) Answer: The “fuel burning” (ATP hydrolysis) by the two heads of a 2-headed kinesin are coordinated in such a way that at least one remains attached when the other steps ahead. Then, why is a single-headed kinesin processive?

37 Theoretical model of Single-headed kinesin motor KIF1A

38 For processivity of a molecular motor two heads are not essential. Nishinari, Okada, Schadschneider and Chowdhury, Phys. Rev. Lett. 95, 118101 (2005). Single-headed kinesin KIF1A is processive because of the electrostatic attraction between the “K-loop” of the motor and “E-hook” of the track.

39 KKTKDPKDK ATPPADP State 1State 2 Strongly Attached to MT (Diffusive) Weakly Attached to MT Enzymatic cycle of a single KIF1A motor

40 Binding site on Microtubule ii-1i+1 hh ss 111 222 bb bb ff aa dd 1,2 Two “chemical” states “State-space” of KIF1A and the mechano-chemical transitions position Chemical state

41 Model of interacting KIF1A on a single protofilament bb bb Current occupation Occupation at next time step ff dd aa 1 2 2 21 2 21 Greulich, Garai, Nishinari, Okada, Schadschneider, Chowdhury

42 Master eqns. for KIF1A traffic in mean-field approximation dS i (t)/dt =  a (1-S i -W i ) +  f W i-1 (1-S i -W i ) +  s W i –  h S i –  d S i dW i (t)/dt =  h S i +  b W i-1 (1-S i -W i ) +  b W i+1 (1-S i -W i ) -  b W i {(1-S i+1 -W i+1 ) + (1-S i-1 -W i-1 )} –  s W i –  f W i (1-S i+1 -W i+1 ) i = 1,2,…,L S i = Probability of finding a motor in the Strongly-bound state. W i = Probability of finding a motor in the Weakly-bound state. GAIN termsLOSS terms

43 Validation of the model of interacting KIF1A Excellent agreement with qualitative trends and quantitative data obtained from single-molecule experiments. Low-density limit Nishinari, Okada, Schadschneider and Chowdhury, Phys. Rev. Lett. 95, 118101 (2005)ATP(mM)∞ 0.9 0.3375 0.15

44 Position Density Greulich, Garai, Nishinari, Schadschneider, Chowdhury, Phys. Rev. E, 77, 041905 (2007) Co-existence of high-density and low-density regions, separated by a fluctuating domain wall (or, shock): Molecular motor traffic jam !! Low-density regionHigh-density region

45 X Y W(x,y) → W(x,y+1) with  bl+ W(x,y) → W(x,y-1) with  bl- W(x,y) → S(x,y+1) with  fl+ W(x,y) → S(x,y-1) with  fl- Lane-changing by single-headed kinesin KIF1A motors Chowdhury, Garai and Wang (2008) Lane = Protofilament Lane-change allowed from weakly-bound state

46 Chowdhury, Garai and Wang (2008)  fl  f Flux (per lane) New prediction: Flux can increase or decrease depending on the rate of fuel consumption. Effect of lane changing on the flux of KIF1A motors

47 Theoretical models of RNA polymerase and Ribosome

48 T. Tripathi and D. Chowdhury, Phys. Rev. E 77, 011921 (2008) Theoretical model of RNAP and RNA synthesis “Transcriptional bursts in noisy gene expression”, T. Tripathi and D. Chowdhury (2008), submitted for publication

49 The Ribosome The ribosome has two subunits: large and small The small subunit binds with the mRNA track The synthesis of protein takes place in the larger subunit Processes in the two subunit are well coordinated by tRNA Cartoon of a ribosome; E, P, A: three binding sites for tRNA

50 Biochemical cycle of ribosome during polypeptide elongation Basu and Chowdhury (2007) E P A t-RNA t-RNA t-RNA-EF-Tu (GTP) t-RNA t-RNA-EF-Tu (GDP+P) t-RNA t-RNA-EF-Tu (GDP) t-RNA t-RNA EF-G (GTP)t-RNA i i+1 t-RNA

51 α β EPA E P AEPA EP A Theoretical model of ribosomes and rates of protein synthesis A. Basu and D. Chowdhury, Phys. Rev. E 75, 021902 (2007) Initiation Termination Codon (Triplet of nucleotides on mRNA track)

52 dP 1 (i;t)/dt =  h2 P 5 (i-1;t) Q(i-1|i-1+l) +  p P 2 (i;t) –  a P 1 (i;t) dP 2 (i;t)/dt =  a P 1 (i;t) – [  p +  h1 ] P 2 (i;t) dP 3 (i;t)/dt =  h1 P 2 (i;t) – k 2 P 3 (i;t) dP 4 (i;t)/dt = k 2 P 3 (i;t) –  g P 4 (i;t) dP 5 (i;t)/dt =  g P 4 (i;t) –  h2 Q(i|i+ l ) P 5 (i;t) Master eqn. for ribosome traffic for arbitrary l > 1 Position of a ribosome indicated by that of the LEFTmost site. P(i|j) = Conditional prob. that, given a ribosome at site i, there is another ribosome at site j = 1 - Q(i|j) Basu and Chowdhury, Phys. Rev. E 75, 021902 (2007)

53 Effects of sequence inhomogeneity of real mRNA Genes crr and cysK of E-coli (bacteria) K-12 strain MG1655 “Hungry codons” are the bottlenecks Basu and Chowdhury, Phys. Rev. E 75, 021902 (2007) Rate of protein synthesis Rate of fuel consumption

54 Examples of molecular motors III: Membrane-associated Rotary Motors

55 Viral DNA packaging machine Pressure in a Phi-29 viral capsid ~ 60 Atmospheric pressure ~ 10 times the pressure in a champagne bottle The machine consists of a 10 nm diameter ring of RNA molecule sandwiched between two protein rings. The rotation of the rings pull the DNA just as a rotating nut can pull in a bolt. Fuel: ATP The packaging motor can generate a force large enough to withstand this pressure!!

56 Movie Produces three ATPs per twelve protons passing through the it ATP synthase Bacterial Flagellar motor Membrane-associated Rotary Motors 10 nm

57 Conclusion Combination of powerful techniques from several disciplines has already provided some insight into the mechanisms of natural nano-machines. “Does life provide us with a model for nanotechnology that we should try and emulate- are life’s soft machines simply the most effective way of engineering in the unfamiliar environment of the very small?”- R.A.L. Jones, Soft Machines (OUP, 2007). Molecular Machines Chemistry Molecular Cell Biology Physics Nano-technology

58 Thank You

59 Acknowledgements Collaborators (Last 4 years): On Ribosome: Aakash Basu*, Ashok Garai, T.V. Ramakrishnan (IITK/IISc/BHU). On RNA Polymerase: Tripti Tripathi, Prasanjit Prakash. On Helicase: Ashok Garai, Meredith D. Betterton (Phys., Colorado). On Chromatin-remodeling enzymes: Ashok Garai, Jesrael Mani. On KIF1A: Ashok Garai, Philip Greulich (Th. Phys., Univ. of Koln), Andreas Schadschneider (Th. Phys., Univ. of Koln), Katsuhiro Nishinari (Engg, Univ. of Tokyo), Yasushi Okada (Med., Univ. of Tokyo), Jian-Sheng Wang (Phys., NUS). On MCAK & Kip3p: Manoj Gopalakrishnan (HRI), Bindu Govindan (HRI). On MT-Motor tug-of-war: Dipanjan Mukherjee, Debasish Chaudhuri (MPI-PKS Dresden). Funding: CSIR (India), MPI-PKS (Germany). Discussions: Roop Mallik (TIFR) Krishanu Ray (TIFR) Stephan Grill (MPI-PKS and MPI-CBG, Dresden) Joe Howard (MPI-CBG, Dresden) Frank Julicher (MPI-PKS, Dresden) Gunter Schuetz (FZ, Juelich)  Now at Stanford University Support: IITK-TIFR MoU, IITK-NUS MoU.

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