Condensed phase kinetics

• The kinetic theory of gases

involves ballistic motion
• Kinetics in liquids and other
condensed phases involves
diffusion
• What are the major
characteristics?
– Many molecules interacting at
once
– Short mean free paths
– No “memory” of momenta
 Brownian motion
• Extreme case of
condensed phase
dynamics
• No memory of
momentum
• “Bath” provides:
– Random displacements
– Frictional damping
• Bath action related to
molecular collisions
 Random walks
• Markov chain: no history
dependence N! ⎛1⎞
N

• Consider a discrete time P ( n+ , N ) = ⎜ ⎟

random walk in 1D:
n+ !( N − n+ ) ! ⎝ 2 ⎠
– Equal probability of moving 2 ⎛ 2n+2 ⎞
left or right at each time step ∼ exp ⎜ − ⎟
– Position probability πN ⎝ N ⎠
distribution is binomial
⎛ ( m+ N)
2
– At a large number of steps, 1
probability becomes Gaussian ∼ exp ⎜ −
2π N ⎜ 2N
– Mean square displacement ⎝
grows linearly with time
m2 ∼ N
• These are general features
of unbiased random walks
• Can be generalized to
continuous time and space
 1D random walk
10000 trajectories 10000 trajectories
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 Diffusion coefficients
• What is the mean x, y , z t
squared m→ ,N →
l τ
displacement per ⎛ x2 ⎞
P1D ( x, t ) = ( 4π Dt )
−1 2
exp ⎜ − ⎟
unit time? ⎝ 4 Dt ⎠
• For unbiased x2 = 2 Dt
1D

random walks, it’s P2D ( x, y, t ) = ( 4π Dt )

−1 ⎛ x2 + y 2 ⎞
exp ⎜ − ⎟
pretty simple ⎝ 4 Dt ⎠
x2 + y2 = 4 Dt
• Diffusion 2D

coefficients also ⎛ x2 + y 2 + z 2 ⎞
P3D ( x, y, z , t ) = ( 4π Dt )
−3 2
exp ⎜ − ⎟
have microscopic ⎝ 4 Dt ⎠
x2 + y2 + z 2 = 6 Dt
interpretation… 2D
 Langevin equation
• Newton’s equation with
extra terms dv ( t )
• Random force m = −ζ v ( t ) + f ( t )
dt
– Energy added by bath
– Mean force is zero f (t ) = 0
• Viscous drag d v (t )
– Energy dissipated by m = −ζ v ( t )
bath dt
– Related to velocity decay v ( t ) = v ( 0 ) e −ζ t m
times
• The viscous drag is
related to the diffusion
coefficient
 Viscosity and diffusion
• Solve Langevin
d
equation for mean m ( xv ) = −ζ xv + m v 2
dt
squared position
= −ζ xv + k BT
– Multiply by x and
rearrange xv =
k BT
ζ
(1 − e −ζ t m
)
– Use the fact that the
x = B (1 − e −ζ t m )
1 d 2 kT
mean squared velocity is
2 dt ζ
kT (Maxwell dist.)
2 k BT ⎛ m −ζ t m ⎞
– Solve for <xv> with x2 =
ζ ⎝ ζ⎜ t − (1 − e ) ⎟
particle initially at origin ⎠

– Relate to <x2> and solve

 Viscosity and diffusion
• Two time scales for mean 2 k BT ⎛ m −ζ t m ⎞

squared displacement
x2 =
ζ ⎜⎝ ζ
t − (1 − e )⎟
⎠
– Related to “collisional time” τ=
m
ζ
– At short times, the motion is
ballistic x2 =
2 k BT
ζ
(t − τ (1 − e ))
−t τ

– At long times, the motion is

k BT 2
Brownian lim x 2 = t
t τ m
• Velocity doesn’t matter 12 2
= v2 t
• RMSD can be related to
diffusion coefficient 2 k BT
lim x 2 = t
t τ ζ
• The friction and diffusion = 2 Dt
coefficient are related! k BT
D=
ζ
 The Stokes-Einstein relationship
• Stokes law provides an
expression for the
ζ = 6πη a
viscous drag around
various simple shapes k BT
• This can be combined D=
with the MSD derivation ζ
above
• The final relationship is k BT
called the Stokes- =
Einstein relation 6πη a
 Mass action kinetics
• The rate of change in concentration or probability
• Describes a variety of phenomena: chemical
reactions, binding events, etc.
• Relates changes in concentrations with time to
(powers of) species concentrations
• Assumes large numbers of species
• Ignores fluctuations due to small copy numbers

aA + bB
k1
k2
P
dcP ( t )
= k1c A ( t ) cB ( t ) − k2 cP ( t )
a b

dt
 Michaelis-Menten kinetics
• Three basic reactions
– Substrate-enzyme association
– Substrate-enzyme dissociation
– Catalysis and product-enzyme dissociation
• Steady-state assumption used below

E+A EA ⎯⎯→ E + P
kon kcat
koff

koff koff + kcat

Kd = , KM =
kon kon
kcat cE ( 0 ) cS ( t )
vss =
K M + cS ( t )
 What is a diffusion-limited reaction?
• Consider a reaction where the chemical step is
instantaneous
– All reactions which form ES complex go to products
– The rate-limiting aspect of the reaction is binding to the enzyme
• Assume low substrate concentrations

E+A EA ⎯⎯→ E+P

kon kcat
koff

kcat kcat
kcat koff , cS ( t ) , KM ≈
kon kon
kcat cE ( 0 ) cS ( t )
vss ≈ ≈ kon cE ( 0 ) cS ( t )
kcat
+ cS ( t )
kon
 Diffusion-limited reactions
• Typical diffusional encounter rate is 109
to 1010 M-1 s-1
– There are lots of caveats, exceptions,
etc.: protein flexibility, electrostatics,
limited reaction surface
• Smoluchowski encounter rate:
−1
w( r ) k B T
– Assumes spherical symmetry ∞
⎡ e ⎤
– Based on solution of PDE kD ( R ) = ⎢ ∫ dr ⎥
⎣ R 4π r D ( r ) ⎦
2
– No interactions: proportional to sum of
diffusion coefficients and separation
– Interactions: related to integral of k D0 ( R ) = 4π DR
potential
• Thought to represent evolutionary
pressure
• Examples
– Superoxide dismutase
– Acetylcholinesterase
– Barstar-barnase
 Methods for diffusional encounter
simulations
• Discrete methods • Continuum methods
– Langevin dynamics – Fokker-Planck
– Brownian dynamics – Smoluchowski equation
– Monte Carlo
 Discrete simulations of binding events
• Brownian dynamics
• Use as normal dynamics methods
– Integrate stochastic equations of motion
– Average: configurations, thermodynamics, etc. (nothing
that depends on viscosities!)
• Use as encounter simulation method
 First-order BD integration
• Calculate
– Diffusion coefficient gradient
– Potential of mean force gradient
– Random displacement
• Works for large time steps provided the gradients don’t
change (much)
• Position components can be x, y, z – or separate particle
coordinates
• Coupling between particle diffusion components:
hydrodynamic interactions
∂Dij ( t ) ∂W ( t )
ri ( t + Δt ) = ri ( t ) + Δt ∑ − Δt ∑ Dij ( t ) + Ri ( Δt )
j ∂rj j ∂rj
Ri ( Δt ) = 0
Ri ( Δt ) R j ( Δt ) = 2 Dij Δt
 BD for encounter rate calculation
• Assumptions:
– Low enzyme and substrate concentrations (no enzyme-
enzyme or substrate-substrate interactions)
– Diffusion control
– Implicit solvent
• Basic idea: what is the probability that two
molecules started at distance b will encounter one
another rather than wandering off to infinity?
 BD for encounter rate calculation
• BD trajectory:
– Start two molecules at a
separation b where the
potential is centrosymmetric
– Integrate BD equation of
motion until
• Molecules satisfy reaction
criteria
• Molecules exceed separation
distance q
• A maximum number of steps
are taken
• Perform multiple BD
trajectories:
– Accumulate collision Figure from: Northrup SH, Allison SA,
frequencies McCammon JA. J Chem Phys 80 (4)
– Statistics are noisy; multiple 1517-24, 1984.
runs needed!
BD for encounter rate calculation
 BD for diffusion-limited reactions
• Collision frequencies can kD ( b ) β
be transformed into rates k=
• Think: flux through reactive
1 − (1 − β ) Ω
site! w( r ) k B T
−1
⎡ e ∞
⎤
• If all collisions result in kD ( b ) = ⎢ ∫ dr ⎥
⎣ b 4π D ( r ) r
2
reaction (diffusion-limited),
⎦
rate is related to:
∞ w( r ) k B T
– Rate of diffusion to separation e
b (can use Smoluchowski
formula)
∫ 4π D ( r ) r 2
dr
Ω=
q
– Collision frequency ∞ w( r ) k B T
e
– Probability that trajectories
leaving q returns to b ∫ 4π D ( r ) r
b
2
dr
 BD for diffusion-influenced reactions
• If only some collisions result in reaction
(probability α), rate is related to:
– All of above
– Reaction probability α
– Probability Δ that unsuccessful encounter results in later
collision
⎡ β ⎤
α kD ( b ) ⎢ ⎥
⎣ 1 − (1 − β ) Ω ⎦
k=
⎧⎪ ⎡ β ⎤ ⎫⎪
1 − (1 − α ) ⎨Δ + (1 − Δ ) ⎢ ⎥⎬
⎪⎩ ⎣1 − (1 − β ) Ω ⎦ ⎪⎭
 Interactions in BD calculations
• Forces
– Long-range influences only
– Electrostatics: approximate
charge-field calculations
Filig ≈ qilig E prot
• Poisson-Boltzmann calculation for
protein, charge model for ligand
• No desolvation
• Little “internal dielectric” screening
(some effective charge methods)
• Diffusion coefficients
αβ k BT ⎛ δ ij 1 − δ ij ⎛ rij rij ⎞⎞
≈ ⎜ I+ ⎜⎜ I + 2 ⎟
⎟⎟ ⎟
– Should include rotation, translation, Dij
and configuration changes
– No hydrodynamic interactions
cπη ⎜⎝ ai 2 Rij ⎝ rij ⎠⎠
• Probably OK for small ligands
• Stokes-Einstein isotropic diffusion ⎧ai + a j rij < ai + a j
coefficients Rij = ⎨
• Coefficients do not depend on
distance or configuration ⎩ rij rij ≥ ai + a j
– Hydrodynamic interactions
• Include water-mediated effects
• Oseen and other (approximate)
analytic forms
• Configuration- and distance-
dependent
 Application to acetylcholinesterase

• Hydrolytic enzyme in
neuromuscular junction
• Subject of extensive
computational (BD) and
experimental study
• Properties:
– Diffusion-limited catalysis
– Long, narrow active site
gorge
– Significant electrostatic
influences
 AChE/TMA binding
• Binding of neurotransmitter-
like molecule to
acetylcholinesterase
• Diffusion-controlled binding
• Significant dependence on
[NaCl]
• Sensitivity to charged
residues

Figures from: Tara S, et al. Biopolymers 46 (7)

465-74, 1998.
 DHFR-TS substrate channeling
• Bifunctional enzyme:
thymidylate synthase
produces dihydrofolate used
by dihydrofolate reductase
• Electrostatic steering between
active sites enhances
efficiency
– Reduces diffusional
broadening
– Does not “direct” between
sites

Figures from: Elcock AH, et al. J Mol

Biol 262, 370-4, 1996.
 Protein-protein encounter
• Same basic procedure as before
• Reaction criteria are harder to evaluate – often a
variable in the simulation
• Effective charge method
– Use full electrostatic grid for one molecule
– Use a smaller number of charges: termini and
charged residues
• Usually neglect:
– Desolvation terms
– Hydrodynamic interactions (with exceptions)
– Ion relaxation Figures from: Gabdoulline RR,
– Flexibility Wade RC. J Phys Chem 100
– Substrate-substrate interactions 3868-78, 1996 and ibid.
Methods 14 329-41, 1998.
 Actin polymerization
• BD simulations of actin
polymerization
• Reproduced experimental
observation of faster
polymerization at “barbed”
filament end
• Implicated electrostatics in
faster binding to barbed end
• Also observed effect of ADF-
cofilin on polymerization
Figures from: Sept D, Elcock
AH, McCammon JA. J Mol
Biol 294 (5) 1181-9, 1999.
 Lots of protein-protein association rates
• Systems studied:
– Barstar-barnase
– AChE-Fas2
– Cyt C peroxidase-Cyt C
– HyHEL antibodies and lysozyme
• Agreement with experiment is good
• Antibody/lysozyme and AChE-Fas2 rates
overestimated: not diffusion-limited?
• Electrostatics aren’t always helpful!
• Figures from: Gabdoulline RR, Wade RC.
J Mol Biol 306 1139-55, 2001.
Continuum diffusion simulation methods
• Discrete methods
– Solve stochastic ODEs
– Provide atomic problem resolution
– Facilitate integration of stochastic
phenomena
– Software: MCell, UHBD, etc.
• Continuum methods
– Solve deterministic PDEs
– Bridge larger length scales
– Facilitate integration of continuum
mechanics phenomena
– Software: SMOL
 Continuum diffusion motivation

• Demonstrate:
– Accurate description of enzyme binding kinetics (steady-
state and time-dependent)
– Simulation of synapse electrophysiology
– Extreme adaptivity of methods to bridge length scales
• Long-term goals:
– Integrate continuum and discrete methods
– More complete description of cellular-scale processes
 Smoluchowski equation
Concentration
change over time
Diffusion term Drift term
∂ρ ( x)
= ∇ ⋅ J ( x) = ∇ ⋅ D( x)[∇ρ ( x) + βρ ( x)∇W ( x)]
∂t Flux Diffusion External
coefficient potential

ρ (x) = ρ
“Bulk” boundary condition

ρ ( x) = 0
Reactive boundary condition

n( x ) ⋅ J ( x ) = 0
Reflective boundary condition

k (t ) = ∫ J ( s ) ⋅ n( s )ds
The observable: the time-dependent
rate constant
 Advances and outlook
• Receptor flexibility
• Detailed binding
mechanisms
• Imperfect reactivity;
calculate “re-entrant”
trajectories

HEL-antibody flexibility in lysozyme binding from

Gabdoulline RR, Wade RC. J Mol Biol 306 1139-
55, 2001.

Camphor release pathway from

cytochrome P450 from Guallar lab.
 Advances and outlook
• Cellular simulations
• Proteomics-scale
interactions
• Crowded
environments

Virtual Cell schematic from Slepchenko BM, Crowding and GroEL simulation from Elcock AH.
Schaff JC, Carson JH, Loew LM. Annu Rev PNAS 100 (5) 2340-4, 2003.
Biophys Biomol Struct 31 423-41, 2002.