the influence of trehalose on melting and dynamics in dehydrated
TRANSCRIPT
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The influence of trehalose on melting and dynamics in dehydrated phospholipidbilayers
Janna K. Maranas
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User friendly packages are available for simulations in biology
NanoscaleMolecular Dynamics
NAMD GROMACSGroningen Machine for Chemical Simulationswww.ks.uiuc.edu/Research
/namd/ www.igc.ethz.ch/GROMOS
AMBERAssisted Model Building with Energy Refinement
http://amber.scripps.edu/
PACKAGES
FORCE FIELDS
AMBER
all-atom
ff94 ff96 ff99 ff99SB ff02 ff03
CHARMM
all-atom
CHARMM22 CHARMM27
united atomCHARMM19
GROMOS
united atom
GROMOS87 GROMOS96
AMBER
Nucleotides
Membranes
Proteins
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Accessible properties are similar to polymers; presence of water and initial configuration are different
Initial protein structures
www.rcsb.org
Computational methods for structure prediction
Experimentally determined NMR structures
http://boinc.bakerlab.org/rosetta/
Adapted from Ding et al., Trends Biotechnol, 2005. 23,9:450-455
Complete dynamics are difficult to access
102
103
107
Simulation size of biological systems
19751970 1980 1985 1990 1995 2000 2005 2010
104
105
106
Sanbonmatsu K et al. J.Struc.Biol. 2007. 15,470-480
Num
ber o
f ato
ms
Modeling solvent effects
water models: TIP3P, TIP4P, SPC/E,F3C
Tim
e sc
ale
(s)
10-15
10-14
10-13
10-12
10-11
10-10
10-9
10-8
10-7
10-6
10-5
10-4
10-3
10-2
10-1
110102
103
Backbone librations
Large conformational
changesProtein Folding
Protein aggregation
All-atom molecular dynamics
Side chain motions
10-11 10-10 10-9 10-8 10-7 10-6
Length scale (m)
Small moleculesProtein complexes
Proteins
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Conformational dynamics of trialanine in waterMu et al., J.Phys.Chem B 2003, 107:5064-5073
Possible conformations for trialanine from simulation
Ion permeation through a narrow membrane channelAllen et al., Biophys. J. 2006, 90:3447-3468
Conductance prediction across membrane (pS)
0.300.816.921
GROMOS87CHARMM27AMBER94Experimental
Educate yourself when choosing a force field
CHARMM22AMBER ff96
Ramachandran Plot (φ, ψ angles)OPLS96
GROMOS96
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Models for watersimulation of ubiquitin
Showalter et al., J. Chem. Theory Comput. 2007, 3(3):961-975
AMBER force fields variantssimulation of alanine tetrapeptide
Hornak et al., Proteins: Struct. Funct. Bioinf. 2006,65: 712-725
Ramachandran plots
AMBER99NPT
AMBER99NVT
AMBER99SBNPT
AMBER99SBNVT
0
0.02
0.04
0.06
0.08
0.1SPC TIP3P
Variations within force field families and with water are less important
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Resurrection PlantResurrection Plant((SelaginellaSelaginella))
Trehalose and sucrose: preservation of biologicals•Proteins: enzymes, antibodies•Cells: stem cells, platelets, bacteria, sperm, seed embryos
Nature uses sugars to survive dessication
PhospholipidPhospholipid
Head GroupHead Group
Alkyl Alkyl TailsTails
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De-hydration Re-hydration
hydrated bilayer
bilayer dried with trehalose
hydrated bilayer
bilayer dried without trehalose
What drives melting of freeze-dried liposomes: tails & heads?
What is the nature of the transition and “liquid crystalline” state with trehalose?
How does trehalose affect the dynamics of lipid heads & tails?
Trehalose stabilizes membranes during re-hydration
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•hTrehalose•dhDPPC•hdDPPC•hhDPPC+dTrehalose•ddDPPC+hTrehalose•dhDPPC+dTrehalose•hdDPPC+dTrehalose
hhDPPC
dTrehalose
dhDPPC
This study combines simulation with QENS
dhDPPC
QENS follows proton motion
“hide” specific parts with deuteration
Systems investigated•128 DPPC molecules•100 water molecules•256 trehalosemolecules
•384 DPPC molecules•300 water molecules
QENS
Atomistic MD
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Layer thickness
The GROMOS force field is accurate for this system
Area per lipid, 320 K
Expt: 64 Å2
[ave. value over xray, NMR, neutron]
Sim: 60 Å2
Hydrated bilayers
Dry bilayers
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We combined HFBS and DCS dynamic runs and used HFBS for elastic scans
Δt ≅ 0.5-40 ps Δt ≅ 240-5000 ps
NG4 Disk-chopper time-of-flight spectrometer
NG2 Backscattering spectrometer
Dynamic run
Elastic scan
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dhDPPC hdDPPC
Q=0.62-1.75 Å-1
melting transition: lipid tailsSignificant mobility before Tm
Elastic Scans: melting ofTails, Heads andWhole Lipid
Melting in dry bilayers without trehalose is driven by lipid tails
0
0.5
1
1.5
2
2.5
3
3.5
4
200 250 300 350 400 450 500Temperature (K)
Inte
nsity
(arb
. uni
ts)
hPMMA
hPEO
amorphousSemi-crystalline
Melting: lipid tails
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Three regimes in tail dynamics:1. Vibrational motion2. “Localized” motion3. Translational motion
Dry Bilayer395K
Dry Bilayer310K
Molecular Simulations
Previous QENS hydrated bilayers: equivalence of tail protons
(König et al.,J. Phys II, 1992)
Head and Tail mean displacement
Mobility varies between heads and tails and with tail position
( )2 2exp / 3I Q u∝ −No translational motion:
u = r – re
re
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Trehalose restricts headgroupmobilityTrehalose and phospholipidheadgroups: similar mobility
Q=0.62-1.75 Å-1
TrehaloseHeadgroups
Trehalose+LipidHeadgroups+Sugar
Trehalose restricts headgroup mobility
QENS: elastic scans
QENS T=395K HFBS
0
0.2
0.4
0.6
0.8
1
100 1000 10000time (ps)
I (q,
t)
d-DPPC + h-trehalose at q=1.60
hd-DPPC + d-trehalose at q=1.60
QENS: dynamics at 395 K
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PMMA
“poly methyl methacrylate”
This behavior is encountered in polymer mixtures
Without hydrogen bonds: ΔTg = 180K
-4
-2
0
2
4
6
1.5 2 2.5 3 3.5
1000/T (K-1)
log
tau
(ns)
CH2 C
CH3
CH3
C
O
O
CH2 OCH2
PEO “poly ethylene oxide”
Tg ~ 220 K, more mobile
Tg ~ 400 K, less mobile
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Significant sugar-headgroup hydrogen bonding is likely
* Zhang, Runt, et al., Macromolecules (2004, 2003)
0
0.2
0.4
-2 -1 0 1 2 3 4 5 6log10[Frequency (Hz)]
95908580757065605552
40% PVPh/PVEE
One dynamic response: 30 - 70% PVPh
Hydrogen bonding = similar dynamics
Trehalose and lipid headgroups have significant hydrogen bonding
With hydrogen bonds: ΔTg = 185 K
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Q=0.62-1.75 Å-1
Trehalose lowers melting transition and restricts mobilitySome differences below TmLarge difference above Tm
Tails with and without, sugar
Trehalose also restricts mobility of lipid tails
Dry Bilayer395K
Dry Bilayerwith trehalose395K
??
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No translational motion:
Trehalose decreases the mean displacements and restrains the lipid mobility
( )2 2exp / 3I Q u∝ −Tails mean displacements withand without trehalose
Molecular Simulations
Dry Bilayer/Trehalose395K
Dry Bilayer/Trehalose310K
Molecular nature of mobility is different when trehalose is present
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The melting transition differs with trehalose
310 K
395 K
Tm“melting” = bottom of tails
Without trehaloseWith trehalose
Tm“melting” = top of tails395 K
310 K
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Dynamic runs were made on tail labeled samples
0.0001
0.001
0.01
0.1
1
-6 -4 -2 0 2 4Energy transfer (meV)
Inte
nsity
(arb
. uni
ts.)
dhDPPC 390KResolution DCS
HFBS
Δt ≅ 0.5-40 ps Δt ≅ 240-5000 ps
NG4 Disk-chopper time-of-flight spectrometer
NG2 Backscattering spectrometer
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τ: characteristic timeβ: distribution of times
E: motion in restricted geometry
DCS HFBS
Tail motion in QENS window has two processes
( )( ) ( )[ ]i
iiii tEEKWW
KWWKWWAtqIβτ/exp1
, 21
−−+=
=
τ: typical time scale for trans/gauche isomerization
If varying mobilities, β < 1
KWW Limiting cases
β= 1: same τ for all protons
E = 0: no spatial restriction
Fit lines
Fast process: trans/gauche isomerization 7-45 ps
Slow process: lipid rotation 2-12 ns
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fast processQ=0.98
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1 10 100 1000
time (ps)
I(Q
,t)
T=310 dhDPPC T=330 dhDPPCT=350 dhDPPC T=370 dhDPPCT=395 dhDPPC T=370 dhDPPC+dtreh
Isolating the fast process reveals conformational transitions
( ) ( )[ ]11111 /exp1 βτtEEKWW −−+=
trehalose decreases conformational transitionseffect is similar to decreasing T to between 310 & 330larger spatial restriction of protons
below Tm
fast process Q=1.58
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1 10 100 100time (ps)
I(q,
t)
T=310 dhDPPCT=330 dhDPPCT=350 dhDPPCT=370 dhDPPCT=395 dhDPPCT=370 dhDPPC+dtreh
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0
1
10
100
-0.20 -0.10 0.00 0.10 0.20 0.30 0.40
log Q (angstrom-1)
τ1
T=310 dhDPPC T=330 dhDPPCT=350 dhDPPC T=370 dhDPPCT=395 dhDPPC T=370 dhDPPC+dtreh
y = 7.1081x-1.9801
y = 3.0589x-2.1704
0
2
4
6
8
10
12
14
16
18
0.00 0.50 1.00 1.50 2.00 2.50
Q (angstrom-1)
τ1
T=310 dhDPPCT=330 dhDPPCT=350 dhDPPCT=370 dhDPPCT=395 dhDPPCT=370 dhDPPC+dtreh
Trehalose slows conformational transitions and increases spatial localization
Characteristic times: Slower with decreasing T
Little change with trehalose
Hydrated bilayers, T=333 K: τconf = 7-45 psVenable, et al, Science, 262, 223 (1993)
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.50 1.00 1.50 2.00 2.50Q (angstrom-1)
E1
T=310 dhDPPCT=330 dhDPPCT=350 dhDPPCT=370 dhDPPCT=395 dhDPPCT=370 dhDPPC+dtreh
Spatial localizationLarger with decreasing
temperature AND with trehalose
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Modeling of spatial localization quantifies the variation in mobility along lipid tails
Q dependence of EISF
( ) ( ) 2
13⎥⎦
⎤⎢⎣
⎡=
QrQrjQE
( ) ( )∑=
⎥⎦
⎤⎢⎣
⎡=
N
n n
n
QrQrj
NQE
2
2
131
Single sphere size
varying sphere sizes
E1
E1
Pure lipid tails
Diffusion in impenetrable spheres
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Simulation directly probes difference in moblity with carbon position
Simulation: neat lipid tails
E1
QENS with simulation input
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Five things to rememberlipid headgroups and trehalose:
significant hydrogen bonding
Spatial extent of mobility varies with tail postion
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.50 1.00 1.50 2.00 2.50Q (angstrom-1)
E1
T=310 dhDPPCT=330 dhDPPCT=350 dhDPPCT=370 dhDPPCT=395 dhDPPCT=370 dhDPPC+dtreh
Trehalose decreases spatial extent of mobility
Melting: lipid tails
nature of main transition may be different with trehalose