Molecular Machines: Packers and Movers, Assemblers and Shredders

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Molecular Machines Packers and Movers,
Assemblers and Shredders
Debashish Chowdhury
Physics Department, Indian Institute of
Technology, Kanpur
Home page http//home.iitk.ac.in/debch/profile_D
C.html
2nd IITK REACH Symposium, March 2008
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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).
Marcello Malpighi
(March 10, 1628 - September 30, 1694)
Founder of microscopic anatomy
http//en.wikipedia.org/wiki/Marcello_Malpighi
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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 - )
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Input
Motor
Mechanical
Output
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Natural Nano-machines within a living cell
Designs of molecular machines have been perfected
by Nature over millions or billions of years on
the principles of evolutionary biology.
Understanding mechanisms through experiments and
theoretical modeling
Design using artificial components synthesized in
the laboratory
Design using natural components extracted from
living cells
Artificial Nano-machines for practical
applications
All the design and manufacturing completed so far
have succeeded only in establishing
proof-of-principle, but still far from
commercial prototypes.
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Natural Nano-machines within a living cell
Understanding mechanisms through experiments and
theoretical modeling
In THIS TALK
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Outline of the talk
1. Introduction
2. Examples of molecular motors
I. Cytoskeletal motors
II. Nucleic acid-based motors
3. Methods of quantitative modeling to understand
mechanisms
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. Conclusion
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Examples of molecular motors I Cytoskeletal
Motors
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Cytoskeleton of a cell
Required for mechanical strength and
intra-cellular transportation.
Alberts et al., Molecular Biology of the Cell
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Cytoskeletal Motor Transport System Motor
Track Fuel
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TRACK
Track Microtubule
Track F-actin
a-b dimer
http//www.cryst.bbk.ac.uk/PPS2/course/section11/a
ctin2.gif
Diameter of a tubule 25 nm.
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Superfamilies of Cytoskeletal MOTORS
Woehlke and Schliwa (2000)
http//www.proweb.org/kinesin/CrystalStruc/Dimer-d
own-rotaxis.jpg
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Cytoskeletal Motors
Porters
Rowers
Myosin-V
Myosin-II
Kinesin-1
Animated cartoon MCRI, U.K.
Science, 27 June (2003)
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Cytoskeletal Motors
Porters
My research group works on PORTERS.
Kinesin-1 Smallest BIPED
Animated cartoon MCRI, U.K.
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SHREDDERS walk/diffuse and depolymerize
Theoretical modeling by Govindan, Gopalakrishnan
and Chowdhury (2008)
MCAK, KLP10A and KLP59C members of kinesin-13
family
Kip3p a member of kinesin-8 family
www.nature.com/.../v7/n3/thumbs/ncb1222-F7.gif
www.nature.com/.../n9/thumbs/ncb0906-903-f1.jpg
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Examples of molecular motors II Nucleic
acid-based Motors
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Central dogma of Molecular Biology and assemblers
Simultaneous Transcription and Translation
DNA
Transcription
(RNA polymerase)
RNA
Translation
(Ribosome)
Protein
Rob Phillips and Stephen R. Quake, Phys. Today,
May 2006.
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RNA polymerase a mobile workshop
RNA polymerase
DNA
RNA
A motor that moves along DNA track,
decodes genetic message,
polymerizes RNA using DNA as a template.
Roger Kornberg Nobel prize in Chemistry (2006)
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Ribosome a mobile workshop
http//www.mpasmb-hamburg.mpg.de/
Ribosome
mRNA
Protein
A motor that moves along mRNA track,
decodes genetic message,
polymerizes protein using mRNA as a template.
http//www.molgen.mpg.de/ag_ribo/ag_franceschi/
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Methods of Quantitative modeling to understand
mechanisms
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Levels of Description
Coarse-grained level Dynamical equations for
local densities of motors Too coarse to
maintain individual identities of the motors.
Molecular level Classical Newtons equations for
protein molecules of the aqueous environment
Classical Molecular Dynamics (MD) (inadequate
for length and time scales relevant for motor
protein dynamics)
Atomic level Quantum mechanical calculation of
structures numerical works based on software
packages (Quantum Chemistry)
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Level of Description adopted in our theoretical
works
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State Space
Chem. State
Position
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State Space
Chem. State
Translocation
Position
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State Space
Chem. State
Chem. reaction
Position
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State Space
Chem. State
Mechano-
Chemical transition
Position
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Mechano-chemical transitions in state-space
Translate into
Mathematical language
Master equations
Numerical protocols
Analytical
solution
Computer
simulation
Theoretical predictions
Numerical predictions
Compare
Compare
Compare
Experimental data
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Some Fundamental questions on mechanisms of
molecular motors
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Size Nano-meters Force Pico-Newtons
Question I Is the mechanism of molecular motors
identical to those of their macroscopic
counterparts (except for a difference of scale)?
NO.
(1) Far from equilibrium
(2) Made of soft matter
(3) 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. - DArcy
Thompson, On Growth and Form (1942).
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Question II What is the mechanism of energy
transduction ?
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Power Stroke
S.A. Endow, Bioessays, 25, 1212 (2003)
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Power-stroke versus Brownian ratchet
Joe Howard, Curr. Biol. 16, R517 (2006).
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Mechanisms of energy transduction by molecular
motors
Brownian ratchet
A Brownian motor operates by converting random
thermal energy of the surrounding medium into
mechanical work!!
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Smoluchowski-Feynman ratchet-and-pawl device
Feynman Lectures in Physics.
R.D.Astumian ,Scientific American, July 2001
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).
A Brownian motor does not violate 2nd law of
thermodynamics as it operates far from
equilibrium where the 2nd law is not applicable.
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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?
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Theoretical model of Single-headed kinesin
motor KIF1A
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For processivity of a molecular motor two heads
are not essential.
Single-headed kinesin KIF1A is processive because
of the electrostatic
attraction between the K-loop of the
motor and E-hook of the track.
Nishinari, Okada, Schadschneider and Chowdhury,
Phys. Rev. Lett. 95, 118101 (2005).
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Enzymatic cycle of a single KIF1A motor
K
KT
KDP
KD
K
ATP
P
ADP
Strongly Attached to MT
Weakly Attached to MT
(Diffusive)
State 1
State 2
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State-space of KIF1A and the mechano-chemical
transitions
Binding site on Microtubule
position
Chemical state
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Model of interacting KIF1A on a single
protofilament
Greulich, Garai, Nishinari, Okada,
Schadschneider, Chowdhury
wb
wb
Current occupation
Occupation at next time step
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Master eqns. for KIF1A traffic in mean-field
approximation
Si Probability of finding a motor in the
Strongly-bound state.
Wi Probability of finding a motor in the
Weakly-bound state.
dSi(t)/dt wa(1-Si-Wi) wf Wi-1(1-Si-Wi) ws
Wi wh Si wd Si
GAIN terms
LOSS terms
dWi(t)/dt wh Si wb Wi-1 (1-Si-Wi) wb Wi1
(1-Si-Wi) - wb Wi
(1-Si1-Wi1) (1-Si-1-Wi-1)
ws Wi wf Wi(1-Si1-Wi1)
i 1,2,,L
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Validation of the model of interacting KIF1A
Nishinari, Okada, Schadschneider and Chowdhury,
Phys. Rev. Lett. 95, 118101 (2005)
Low-density limit
ATP(mM)
8
0.9
0.3375
0.15
Excellent agreement with qualitative trends and
quantitative data obtained from single-molecule
experiments.
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Greulich, Garai, Nishinari, Schadschneider,
Chowdhury, Phys. Rev. E, 77, 041905 (2007)
Low-density region
High-density region
Density
Position
Co-existence of high-density and low-density
regions, separated by a fluctuating domain wall
(or, shock) Molecular motor traffic jam !!
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Lane-changing by single-headed kinesin KIF1A
motors
Chowdhury, Garai and Wang (2008)
W(x,y) ? W(x,y1) with wbl W(x,y) ? W(x,y-1)
with wbl- W(x,y) ? S(x,y1) with wfl W(x,y) ?
S(x,y-1) with wfl-
Lane-change allowed from weakly-bound state
Y
X
Lane Protofilament
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Effect of lane changing on the flux of KIF1A
motors
Chowdhury, Garai and Wang (2008)
Flux (per lane)
wfl/wf
New prediction Flux can increase or decrease
depending on the rate of fuel consumption.
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Theoretical models of RNA polymerase and
Ribosome
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Theoretical model of RNAP and RNA synthesis
T. Tripathi and D. Chowdhury, Phys. Rev. E 77,
011921 (2008)
Transcriptional bursts in noisy gene
expression, T. Tripathi and D. Chowdhury (2008),
submitted for publication
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The Ribosome
Cartoon of a ribosome E, P, A three binding
sites for tRNA
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
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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 (GDPP)
t-RNA t-RNA-EF-Tu (GDP)
t-RNA t-RNA EF-G (GTP)
t-RNA t-RNA
i
t-RNA t-RNA
t-RNA
i1
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Theoretical model of ribosomes and rates of
protein synthesis
A. Basu and D. Chowdhury, Phys. Rev. E 75, 021902
(2007)
Initiation
a
E
P
A
E
A
E
P
A
A
P
E
P
Codon (Triplet of
nucleotides on mRNA track)
ß
Termination
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Master eqn. for ribosome traffic for arbitrary l
gt 1
Position of a ribosome indicated by that of the
LEFTmost site.
P(ij) Conditional prob. that, given a ribosome
at site i, there is another ribosome at site j
1 - Q(ij)
Basu and Chowdhury, Phys. Rev. E 75, 021902 (2007)
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Effects of sequence inhomogeneity of real mRNA
Basu and Chowdhury, Phys. Rev. E 75, 021902 (2007)
Genes crr and cysK of E-coli (bacteria) K-12
strain MG1655
Rate of protein synthesis
Rate of fuel consumption
Hungry codons are the bottlenecks
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Examples of molecular motors III
Membrane-associated Rotary Motors
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Viral DNA packaging machine
The machine consists of a 10 nm diameter ring of
RNA molecule sandwiched between two protein rings.
Fuel ATP
The rotation of the rings pull the DNA just as a
rotating nut can pull in a bolt.
Pressure in a Phi-29 viral capsid 60
Atmospheric pressure
10 times the pressure in a
champagne bottle
The packaging motor can generate a force large
enough to withstand this pressure!!
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Membrane-associated Rotary Motors
Bacterial Flagellar motor
ocw.mit.edu

• Produces three ATPs per twelve protons passing
  through the it

ATP synthase
Movie
10 nm

www.biologie.uni-osnabrueck.de/biophys/Junge/pictu
res/ATPaseVideo/Synthase.Mov
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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 lifes 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).
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Thank You
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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).
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)
Funding CSIR (India), MPI-PKS (Germany).
Support IITK-TIFR MoU, IITK-NUS MoU.
Now at Stanford University