Transcript Document 7420429

Protein Structure, Dynamics and Function using
Molecular Dynamics and Scattering Techniques
Vandana Kurkal-Siebert
IWR, University of Heidelberg
Contents
•Enzyme Hydration Activity  Dynamics
(Pig Liver Esterase)
•Enzyme Immobilization  Activity  Dynamics
(Dihydrofolate Reductase)
•Glass-like property of proteins : The Boson peak
(Myoglobin)
•Protein:Protein interactions
(Myoglobin)
•Structural basis for functional efficiency
(Cellulosomes)
Is hydration water
essential for enzyme activity ?
A commonly discussed threshold value is 0.2g/g of protein
Is it necessary for catalytic function ? or
Is it for diffusion of substrate and product ?
Pig liver esterase
Activity at < 3% hydration
Lind et al ; Biochim. Biophys. Acta, 1702, 103 (2004)
Is there a correlation between low
hydration enzyme activity and
picosecond dynamics ?
Neutron scattering
experiments
Hydration dependence of dynamical transition
Hydration dependence of quasielastic intensity
Dynamical transition and
quasielastic intensity
MSD
is due to
(for proteins: 180-220K)
Dynamical Transition
Temp
Simplistic view of protein dynamics
Below dynamical transition temperature
• molecule is believed to be vibrating harmonically
• No quasielastic intensity
Neutron Scattering Spectrum
Hydration dependent Dynamical transition
Kurkal et al ; Biophys. J., 89, 1282 (2005)

Elastic Intensity  S (q ,0) 
1
N atom

j
2
2
2 ( q / 6 ) u j
bj e
Neglecting non - Gaussian contributi on :
1
N atom
N atom
j 1
b
2
j
exp ( u 2j q 2 / 6)  b 2j exp ( u 2 q 2 / 6)
Hydration dependence on
quasielastic intensity
Kurkal et al ; Biophys. J., 89, 1282 (2005)
E
Quasi
S INT
(qmean , E, T )   [ S sample(qmean , E, T )  Svan (qmean , E, T )]dE
0
What did we learn ?
Kurkal et al ; Biophys. J., 89, 1282 (2005)
Linearity below DT temp does not mean Harmonicity
Water facilitates activity, but is not an absolute requirement
Enzyme Immobilization and Dynamics:
A Quasielastic Neutron Scattering Study
Tehei et al ; Biophys. J., 90, 1090 (2006)
Stability, Activity and Dynamics
Immobilization
1g of dry activated support mixed with 200ml of 5mg/ml enzyme
in 0.1M sodium phosphate buffer 25mg per 100mg of support
Immobilization of 25mg of enzyme /100mg of support achieved
Activity reduced by 87%
Questions asked
Does immobilization modulate internal dynamics ?
If yes, how ?
Can we correlate the reduction in activity to modulation in internal dynamics ?
Neutron Scattering spectrum
MSD of high freq. vibration
S (Q,  )  e
 u 2 Q2
Sum of Lorentians
n


 A0 (Q) ( )   Ai (Q) Lint ernal (i ,  )
i 1


EISF
Quasielastic Component
Comparative Dynamics : native versus
immobilized DHFR
T=285K, Q = 0.74A-1
u
2
T=285K, Q = 1.73A-1
0.19+/- 0.02 A2 (Immobilized)
0.23+/- 0.04 A2 (Native)
HWHM of the Lorentzian : A measure of
Quasielastic scattering
Continuous Diffusion =>   DQ 2
Diffusion in a sphere
 sin( Qr )  Qr cos(Qr ) 
A0 (Q)  p  (1  p)  3

3
(
Qr
)


mobile protons
immobile protons
EISF as a function of Q
Diffusion in a sphere
 sin( Qr )  Qr cos(Qr ) 
A0 (Q)  p  (1  p)  3

3
(
Qr
)


mobile protons immobile protons
Radius
Immobilized
2.59+/-0.20A
Native
2.47+/-0.20A
No difference between Native and Immobilized enzyme
Diffusion coefficients, Residence times
and energy barriers
At high Q
The line widths follow
Behaviour at high Q varies
DQ 2
(Q) 
1  DQ 2
Diffusion coefficient
Residence time of a hydrogen
0  4.333  D / r 2
D
τ
Immobilized
0.34+/-0.07 * 10-5cm2/s
20.36+/-1.80ps
Energy barriers :
  1 / 
Native
0.47+/-0.09 * 10-5cm2/s No difference
7.95+/-1.02ps
   0e( E
a
/ k BT )
Ea = 0.54+/-0.12 kcal/mol
What did we learn ?
Tehei et al ; Biophys. J., 90, 1090 (2006)
Immobilization reduces activity
Internal dynamics is indeed modulated by immobilization
Immobilization does not introduce diffusion limitation.
Immobilization does not alter the local picosecond timescale motions
Immobilization increases the energy barrier for proton jumps.
Reduction in activity is due to increase in the local energy barrier
and not due to the diffusion limitation introduced by
immobilization
Inelastic Scattering : Boson peak
Kurkal et al ; Chem. Phys., 317, 267-273(2005)
Boson peak
Low-frequency modes : why
are they interesting ?
•Ligand Binding results in softening of low frequency modes.
•Conformational change via low-frequency modes.
Boson peak : A long standing enigma in
Biological physics
What is a Boson peak ?
Observed in Raman and neutron scattering spectra
Experimentally known
Boson peak position in proteins do not show
systemic dependence.
Boson peak
Dehydration and increase of temperature : boson
peak shifts to lower frequencies
Experimental Incoherent neutron scattering spectra
Experimentally unknown :
Kurkal et al ; Chem. Phys., 317, 267-273(2005)
Which motions give rise to Boson peak ?
Questions that we ask are
Can we reproduce the experimental Boson peak ?
Can we identify the motions responsible for Boson peak ?
Is there any correlation between dynamical transition phenomena and the Boson peak
Classical dynamics and Dynamical
structure factor
d 2 xi
Fi  mi ai  mi 2
dt
Fi  iV (R)
CHARMM Energy Function:
 k (b  b )
Ebonded 
b
2
0

bonds
 k (  
angles
Enonbonded
0
)2 

 k (  
k (1  cos[ n   ]) 
dihedrals
0
)2
impropers
  12   6 
1 qi q j
  4 ij  ij    ij    
 r   i , j 4 r
 rij 
i, j
ij
 ij  

Classical dynamics and Dynamical structure factor
1
Finc (q, t ) 
N

j
e
iqrj ( 0 ) iqrj ( t )
e
FFT
1
Sinc (q, E ) 
2

F
inc

(q, t )e it dt
Single molecule simulations
Hydrated system : MbCO + ~350 water molecules.
Dry system : MbCO + no water
Temperatures : 150K and 300K
Effect of hydration and temperature
reproduced, but not the position of the
Boson peak
Position shifted to lower frequency.
What is the reason ?
Electrostatics description ? Or Environment ?
Environment ? Would crystal simulation help ?
System : Monoclinic unit cell of
MBCO
Space group : P21
Two molecules per unit cell
Electrostatics : Particle mesh Ewald
Boson peak reproduced 
Temperature and hydration effects reproduced 
Additional peak observed : Is it an artifact of periodic boundaries ?
Is it an artifact of periodic boundary
condition ?
Crystal : Monoclinic ; P21 space group
4 unit-cells put together = 8 molecules in total
Additional peak still present, not one but two
Search for the origin of additional peaks
Possible reasons
Interaction between
unit-cells between unit cells
Interaction
Interaction between
proteins in a unit cell
Intermolecular interaction
between proteins in a unit-cell
Search for the origin of additional peaks
Interaction between unit cells
Interaction between
proteins in a unit cell
Intermolecular interaction : Evidences
V. Kurkal-Siebert & J. C. Smith ; J. Am. Chem. Soc., 128, 2356 (2006)
Major contribution from translation
Intermolecular interaction absent in 1molecule
crystal simulation
Position of Boson peak shifts to lower
frequency with 1molecule crystal simulation
Ultra low-frequency peak arises from intermolecular interaction
Intermolecular interaction is essential for reproducing experimental Boson peak
position
Intermolecular Interaction : Evidence
V. Kurkal-Siebert & J. C. Smith ; J. Am. Chem. Soc., 128, 2356 (2006)
Ultra low-frequency peak arises from intermolecular interaction
Intermolecular Interaction :
Temperature and Hydration dependence
Intermolecular Interaction
Temperature dependence
V. Kurkal-Siebert, R. Agarwal & J. C. Smith ; (Phys. Rev. Lett. sub)
Environmental force constant
95.7 N/m
ke 
0.38 N/m
2k B
 d u2

 dT





Measure of resilience
Center-of-mass MSD display transition at ~240K
Autocorrelation function of the
distances of COM motion




1  exp( 1t )
1  exp( 2t )
d (t ) d (t    k1 exp( t v )(cost vt  t v sin t vt )  k2
 (1  k1  k2 )
2
2t v
 1t
 2t


Vibrational and diffusional contribution
to the COM motion:
Temperature dependence
Intermolecular Interaction
Potential of Mean Force
PMF   (dt )  kBT ln( P(dt ))
Force constants and frequencies of
intermolecular vibrations :
Temperature dependence
Px
2k BT ln
P0
k
2
( x  x0 )


 t v
 t v
k1 exp( 
)(cos t vt 
sin t vt )
2
2t v


Elucidation of Boson peak : Principal
component analysis
Aij  mi m j  (ri (t )  ri )( rj (t )  r )
m
m
j
Contributions of set of modes to Boson peak
 S (Q, E ) all  S (Q, E ) Nmodes
R  1 

S (Q, E ) all

Interaction between
proteins in a unit cell



E
Potential Mean Force of
Principal Component Modes
V. Kurkal-Siebert & J. C. Smith ; J. Am. Chem. Soc., 128, 2356 (2006)
PMF   k (qk )  k BT ln( Pk (qk ))
S  
N
p ln
i 1 i
pi
Boson peak arises from low frequency, collective, harmonic modes
What did we learn ?
Environment plays an important role in investigating vibrational dynamics
Boson peak arises from low-frequency, collective harmonic motions.
Neutron scattering spectroscopy can be used as a tool for investigating
protein:protein interaction.
Intermolecular interaction in hydrated protein systems exhibit dynamical
transition at ~240K ; hydration causes two orders of mode-softening above
dynamical transition temp.
Major contribution to the mode-softening comes from the diffusive process.
The intermolecular interaction peak intensity and width may provide valuable
information regarding the strength of intermolecular interaction.
Structural Basis of Cellulosome efficiency:
Investigation via SAXS and MD
What is a cellulosome ?
Multienzymatic complexes that efficiently degrade crystalline cellulose
Detailed Structural analysis desired
hurdles
Diversity and Complexity
Specific control of Cohesin-Dockerin interactions :
Designer Cellulosomes
highly ordered hybrid minicellulosomes containing
Cellulose-binding module
Scaffoldin
Two cohesins of divergent specificity
Two Cellulases bearing dockerin
complementary to Cohesins
induce
drastic increase in activity towards recalcitrant celluloses
Mechanism ?
Solution Structure of Designer Cellulosomes : SANS
and MD studies
Hammel et al , J. Biol. Chem., 280, 38562 (2005)
S4
Fc-S4
Fc-S4-Ft
Fc-S4-At
SAXS Scattering Profiles and RG
I (q )  I (0) exp( q 2 RG2 / 3)
(in Å)
39.8
Fc-S4
45.1
Fc-S4-Ft
61.4
Fc-S4-At
64.9
• RG values correlate with molecular masses
• Fc-S4-At is more anisotropic
Increasing
S4
Pair-distance distribution function
Extended profiles for higher distances => different modules
adopt an extended conformation with respect to each other
Low Resolution shapes from ab initio modelling
(using program GASBOR and CREDO :Biophys J., Vol. 80, 2001)
Model clearly suggest high flexibility of the Scaffoldin linker
Models derived from Rigid-Body Modelling
Model for synergistic activity of cellulosomes
Acknowledgements
Our group@
Prof. Jeremy Smith
Prof. John Finney (Univ. College, London)
Prof. Roy Daniel (Waikato Univ. New Zealand)
Dr. Moeava Tehei (ILL,Grenoble)
Murielle Lopez (Waikato Univ. New Zealand)
Dr. Ruediger Siebert (Academy of Sciences, Heidelberg)
Dr. Veronique Receveur (CNRS, Marseille)
Dr. Michael Hammel (CNRS, Marseille)
DFG
University of Heidelberg
Thank you all for your kind attention