interactions between lipid membranes
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www.iupui.edu/~lab59. Interactions between lipid membranes. Horia I. Petrache. Department of Physics. Indiana University Purdue University Indianapolis, USA. Support:. - PowerPoint PPT PresentationTRANSCRIPT
Interactions between lipid membranes
Horia I. PetracheDepartment of Physics
Indiana University Purdue University Indianapolis, USA
www.iupui.edu/~lab59
Support:
IUPUI Biomembrane Signature Center IUPUI Integrated Nanosystems Development Institute Alpha 1 Foundation NIH Generous student volunteering
o More (better) theory
o Applications
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oily tails
Lipid molecules have two parts
dipolar head
15- 25 Å
5- 7 Å
Lipids aggregate and form bilayers (membranes)
Visible by X-ray depending on electron density.
~ 40 Å
liquid water
Zero net density contrast but...
10𝑒30 𝐴3=0.333𝑒 /Å3
lipid400𝑒
1200 𝐴3 =0.333𝑒 /Å 3
Electron densities at T = 300 K
lipid headgroup160𝑒
320 𝐴3=0.5𝑒 / 𝐴3
lipid tails9𝑒
50 𝐴3 =0.18𝑒 / 𝐴3
compared to 0.333 e/ Å3 for water
Electron densities at T = 300 K
=> can see them!
X-ray scattering from unoriented lipid membranes
X-ray scattering from oriented lipid membranes
Biophys. J. 2005, J. Lipid Research, 2006
2q1Incident beam
MLV sample
Bragg rings seen on the detector
2q2Scattered beam(s)
X-ray scattering from multilayers (1D randomly oriented lattice)
q hD sin2Bragg’s Law
q hD sin2Bragg’s Law
With D = 60 Å, = 1.54 Å, and h = 1, obtain
q = 0.74o (small angle)
=> Need a small x-ray machine
angle
x-ray source (tube)
detectorsample chamber
Wavelength = 1.54 Å (Cu source)
Sample-to-detector distances: 0.15 m, 0.6 m, and 1 m
Lattice spacings: 8 Å to 900 Å
Fixed anode Bruker Nanostar U, 40 kV x 30 mA.
Electron density of a typical lipid bilayer
0.333 e/Å3
Note: broad distributions (no sharp lipid-water interface)
Higher spatial resolution from oriented samples
J. Lipid Research 2006
(DLPC: a lipid we like)
Cryo-EM, Dganit Danino, Technion, Israel
Electron microscopy of lipids in water
Equilibrium distance means
attractive force + repulsive force = 0
F1
F2
D-spacing
=> Any measured change in distance means a change in membrane forces.
+ water => + more water =>
=> Can control spacing by hydration/dehydration (osmotic stress)
+ electrolyte =>
... or by adding ions/electrolytes
1 Molar = a pair of ions for each 55 water molecules.
100 mM = 10 times less ions or 10 times more water.
Debye screening lengths for electrostatic interactions in solution:
10 Å in 100 mM monovalent ions3 Å in 1M
q (Å-1)
Example: D-spacing increases in KBr
DLPC/water20mM KBr
40mM
60mM
80mM100mM
200mM400mM
600mM
q (Å-1)
DLPC/water20mM KBr
40mM
60mM
80mM100mM
200mM400mM
600mM
Example: D-spacing increases in KBr
Equilibrium distances depend on polarizabilities (as expected)
Numbers indicate polarizability ratios .
Szymanski, Petrache, J. Chem. Phys. 2011
KCl
KBr
Water spacing
...but need to explain a curiously large difference between the effects of KBr and KCl
screening length
D
2DKClW
KBrW DD
Looks like electrostatics but distances are large
...112 2
WDH
van der Waals
Hamaker, Parsegian, Ninham, Weiss,...
Attractive interactions between lipid bilayers
With Hamaker parameter H ~ 1-2 kBT
hydration repulsion
Repulsion #1
Empirical exponential form with two adjustable parameters: Ph ~ 1000 – 3000 atm ~ 2 – 3 A
/WDh eP
(lipids don’t want to give water away)
Rand, Parsegian, Marcelja, Ruckenstein, ...
shape fluctuation
Repulsion #2
2
2 12 C
B
KTk
KC=bending modulus = fluctuation amplitude
Helfrich, de-Gennes, Caillé
(membranes bend and undulate)
electrostatics: some analytical forms, mostly numerical calculations
Repulsion #3
Main parameters:
membrane surface charge
Debye screening length (of the electrolyte)
Poisson-Boltzmann, Debye-Huckel, Gouy-Chapman, Andelman, ...
(electric charges exist)
2
2
2/ 1
212
C
B
W
Dh K
TkD
HeP W
vdW shape fluctuationhydration
Additivity/separability model of membrane interactions
+ elec
Fitting parameters: Ph, , H, KC
Also need (DW)
Parsegian, Nagle, Petrache
Long story short: (DW) from X-ray line shape analysis
(DOPC and DOPS are two popular lipids)
Petrache et al., Phys. Rev. E 1998
Osmotic pressure
𝑃𝑜𝑠𝑚=− 𝑑𝐹𝑑𝑉𝑊
It can be measured with an osmometer.
Rand and Parsegian, 1979
Lipid
PEG
Reduce inter-membrane spacing by using osmolytes (e.g. polyethylene glycol, PEG)
Zero pressure
fluctuations
di(14:0)PC (DMPC) at 35oC
hydration
vdW
Example of interaction analysis giving Ph, , H, KC (no electrostatics)
Practical method: use well calibrated reference lipid to investigate salt/electrolyte effects on membrane interactions
Koerner et al., Biophys. J. 2011Danino et al. Biophys. J. 2009Rostovtseva et al. Biophys. J. 2008Petrache et al., PNAS 2006Kimchi et al., J. Am. Chem. Soc. 2005
Main results: Screening of vdW interactions Electrostatic charging due to affinity of polarizable ions to lipidsSome interesting complications at the water/lipid interface
KCl KBrwater
FluidDLPC at 30oC
1M salts
Water spacing (Å)
Fit with ~50% vdW reduction (no elec.)
J. Lipid Res. 2006
Detect Br- binding from data in 100 mM salt
Binding constant
Obtain vdW strength (H) vs. salt concentration
Waterspacing
ExpectDW /λD
DW )eλ/DH~( 221
Cl
Br
(according to Ninham, Parsegian)
Functional form OK but needs empirical correction
DWDDW eDH /2)/21(~
DWD /Petrache et al., PNAS 2006
Detect electrostatic charging due to zwitterions
Koerner et al., Biophys. J. 2011
Common pH buffers
Our calibrated lipid
(Koerner et al., BJ 2011)
Zwitterions (e.g. MOPS buffer) swell multilayers really well
Expect reduction of vdW attraction of membranes
weaker vdW
...and electrostatic charging
(at total 200 mM concentration)
Measure charging by competition with calibrated KBr
+¿−
neutral point: 75% MOPS, 25% KBr
% MOPS replacing KBr(at total 200 mM concentration)
Lipid multilayers are found around nerve axons
source: Public domain (Wiki)
Lipid multilayers are found around nerve axons
source: Public domain (Wiki)
Conclusions
[3] Water, mobile charges, and membrane fluctuations complicate calculations of interactions. Huge room for improvement.
[1] X-ray scattering measurements on well calibrated membrane systems provide experimental parameters for vdW and electrostatics. Experiments show larger screening length (reduced screening power of salt ions) than predicted theoretically.
[2] Can detect weak electrostatic interactions by competition measurements (e.g. MOPS vs. KBr).
Ryan Lybarger Buffers, mixtures
Jason Walsman E. coli (adaptation to ionic sol.)
Megan Koerner Zwitterions
Luis Palacio, Matt Justice X-ray
Torri Roark Lithium salts
Johnnie Wright Exclusion measurements
Visit us at www.iupui.edu/~lab59Acknowledgements
John Nagle (Carnegie Mellon Univ., USA)
Stephanie Tristram-Nagle (Carnegie Mellon Univ., USA)
Daniel Harries (Hebrew Univ., Israel)
Luc Belloni (Saclay, France)
Thomas Zemb (formerly at Saclay, France)
Adrian Parsegian (Univ. of Massachusetts, formerly at NIH)
Rudi Podgornik (University of Ljubljana, Slovenia)
Tanya Rostovtseva (NIH, USA)
Philip Gurnev (NIH, USA)
Acknowledgements (cont.)