Molecular Machines: Packers and Movers, Assemblers and Shredders Debashish Chowdhury Physics...
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Transcript of Molecular Machines: Packers and Movers, Assemblers and Shredders Debashish Chowdhury Physics...
Molecular Machines: Molecular Machines: Packers and Movers, Assemblers and Packers and Movers, Assemblers and
ShreddersShredders Debashish Chowdhury
Physics Department,
Indian Institute of Technology,
Kanpur
Home page: http://home.iitk.ac.in/~debch/profile_DC.html
2nd IITK REACH Symposium, March 2008
“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)
http://en.wikipedia.org/wiki/Marcello_Malpighi
Founder of
microscopic anatomy
Marcello Malpighi
“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 - )
MachineInput Output
MotorInput
Output
Mechanical
“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.
“Natural” Nano-machines within a living cell
Understanding mechanisms through experiments and theoretical modeling
In THIS TALK
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
Examples of molecular motors I:
Cytoskeletal Motors
Cytoskeleton of a cell
Alberts et al., Molecular Biology of the Cell
Required for mechanical strength
and intra-cellular transportation.
Cytoskeletal Motor Transport System = Motor + Track + Fuel
- dimer
Protofilament
Diameter of a tubule: ~ 25 nm.
Track: Microtubule Track: F-actin
http://www.cryst.bbk.ac.uk/PPS2/course/section11/actin2.gif
TRACK
Woehlke and Schliwa (2000)
Superfamilies of Cytoskeletal MOTORS
http://www.proweb.org/kinesin/CrystalStruc/Dimer-down-rotaxis.jpg
Cytoskeletal Motors
Porters Rowers
Animated cartoon: MCRI, U.K.
Kinesin-1
Myosin-V Myosin-
II
Science, 27 June (2003)
Cytoskeletal Motors
Porters
Animated cartoon: MCRI, U.K.
Kinesin-1: Smallest BIPED
My research group works on “PORTERS”.
MCAK, KLP10A and KLP59C :
members of kinesin-13 family
Kip3p:
a member of kinesin-8 family
SHREDDERS: walk/diffuse and depolymerizeTheoretical modeling by Govindan, Gopalakrishnan and Chowdhury (2008)
www.nature.com/.../v7/n3/thumbs/ncb1222-F7.gifwww.nature.com/.../n9/thumbs/ncb0906-903-f1.jpg
Examples of molecular motors II:
Nucleic acid-based Motors
(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.
RNA polymerase: a mobile workshop
DNA RNA
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)
Ribosome: a mobile workshop
http://www.molgen.mpg.de/~ag_ribo/ag_franceschi/
mRNA Protein
decodes genetic message,
Ribosome
polymerizes protein using mRNA as a template.
A motor that moves along mRNA track,
http://www.mpasmb-hamburg.mpg.de/
Methods of Quantitative modeling
to
understand mechanisms
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.
Brownian level:
Master eqn./Fokker-Planck eqn. for the individual proteins
Level of Description adopted in our theoretical works
Chem. State
Position
State Space
Translocation
State Space
Chem. State
Position
Chem. reaction
Chem. State
Position
State Space
Mechano-
Chemical transition
Chem. State
Position
State Space
Translate into
Mathematical language
Master equations Numerical protocols
Analytical
solution Computer
simulation
Theoretical predictions Numerical predictions
Experimental data
CompareCompa
re
Mechano-chemical transitions in
“state-space”
Compare
Some
Fundamental questions
on
mechanisms
of
molecular motors
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).
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).
Question II: Question II:
What is the mechanism of energy What is the mechanism of energy transduction ?transduction ?
Power Stroke
S.A. Endow, Bioessays, 25, 1212 (2003)
Power-stroke versus Brownian ratchet
Joe Howard, Curr. Biol. 16, R517 (2006).
Brownian ratchetPower 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!!
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 2nd law of thermodynamics).
Feynman Lectures in Physics.
A Brownian motor does not violate 2nd law of thermodynamics as it operates far from equilibrium where the 2nd law is not applicable.
Question III: Question III: Why are the porters processive? Why are the porters processive? (i.e., how does a porter cover a (i.e., how does a porter cover a long distance without getting long distance without getting
detached from the track?)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?
Theoretical model
of
Single-headed kinesin motor KIF1A
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.
K KT KDP KD K
ATP P ADP
State 1 State 2
Strongly Attached to MT
(Diffusive)
Weakly Attached to MT
Enzymatic cycle of a single KIF1A motor
Binding site on Microtubule
ii-1 i+1
h
s
1 11
2 2 2bb
f
a
d
1,2 Two “chemical” states
“State-space” of KIF1A and the mechano-chemical transitions
position
Chemical state
Model of interacting KIF1A on a single protofilament
b b
Current occupation
Occupation at next time step
fd a
1 2 2 21 2 21
Greulich, Garai, Nishinari, Okada, Schadschneider, Chowdhury
Master eqns. for KIF1A traffic in mean-field approximation
dSi(t)/dt = a(1-Si-Wi) + f Wi-1(1-Si-Wi) + s Wi – h Si – d Si
dWi(t)/dt = h Si + b Wi-1 (1-Si-Wi) + b Wi+1 (1-Si-Wi)
- b Wi {(1-Si+1-Wi+1) + (1-Si-1-Wi-1)}
– s Wi – f Wi(1-Si+1-Wi+1)
i = 1,2,…,L
Si = Probability of finding a motor in the Strongly-bound state.
Wi = Probability of finding a motor in the Weakly-bound state.
GAIN terms LOSS terms
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)ATP(mM)
∞∞0.90.9
0.33750.3375
0.150.15
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 region High-density region
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 motorsChowdhury, Garai and Wang (2008)
Lane = Protofilament
Lane-change allowed from weakly-bound state
Chowdhury, Garai and Wang (2008)
flf
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
Theoretical models
of
RNA polymerase
and
Ribosome
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
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
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 t-RNA
t-RNA t-RNA
i
i+1
t-RNA
α
β
E P A E P A E P A E P A
Theoretical model of ribosomes and rates of protein synthesisA. Basu and D. Chowdhury, Phys. Rev. E 75, 021902 (2007)
Initiation
Termination
Codon
(Triplet of nucleotides on mRNA track)
dP1(i;t)/dt = h2 P5(i-1;t) Q(i-1|i-1+l) + p P2(i;t) – a P1(i;t)
dP2(i;t)/dt = a P1(i;t) – [ p + h1] P2(i;t)
dP3(i;t)/dt = h1 P2(i;t) – k2 P3(i;t)
dP4(i;t)/dt = k2 P3(i;t) – g P4(i;t)
dP5(i;t)/dt = g P4(i;t) – h2 Q(i|i+l) P5(i;t)
Master eqn. for ribosome traffic for arbitrary l > 1Position 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)
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
Examples of molecular motors III:
Membrane-associated Rotary Motors
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!!
www.biologie.uni-osnabrueck.de/biophys/Junge/pictures/ATPaseVideo/Synthase.Mov
Movie
•Produces three ATPs per twelve protons passing through the it
ATP synthase
Bacterial
Flagellar
motor
Membrane-associated Rotary Motors
10 nm
ocw.mit.edu
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
Thank You
AcknowledgementsCollaborators (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.