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StructureandIntermolecularinteractionsofbio‐molecularself‐assemblies
UriRaviv,TheInstituteofChemistry,TheHebrewUniversityofJerusalem.
Understandingofsupramolecularstructuresofbiomolecules,theirdynamicsandrolesatthe
molecularlevelrequiresknowledgeofthechemistryoftheproteinsorlipidsinvolvedandtheirinteractions.ThisknowledgeisemergingfromsolutionX‐rayscatteringmethodsthatarenon‐invasiveanddonotrequirecrystallinesamples,thusofferinguniqueadvantagesforstudying
protein‐protein(PNAS,2004,101,16099)andprotein–lipidinteractions(PNAS,2005,102,11167).Thestructuralresolution,althoughlowerthanthecrystallographiclevel,issufficientfor
mostinvestigations,wherethefocusisontheassociationofproteinsand/orlipidstoformhigherordercomplexesundervarioussolutionconditions.Thisisparticularlyrelevantforunderstandingbiologicalsystemsthatareinaqueousenvironmentandarecontrolledbyvarious
parameters.Ofparticularsignificanceistheincreasingcapabilityofthosemethodstoprovidetime‐dependentstructuralinformationatthemolecularlevel‐ameansforaddressingdynamicalaspectsofself‐assembly.
Ourlabisequippedwithastate‐of‐the‐artsmallangleandwideanglex‐rayscattering
(SAXS/WAXS)set‐up(SoftMatter,2011,7,1512).Toallowdetailedanalysisofsolutionx‐rayscatteringdataauniquesoftwarewasdevelopedinourlab(Langmuir,2010,26,13110).Theprogramenablesvariouswaystosubtractbackgroundnoise,tomodelvariousgeometrical
modelsanddefineasmanylayersofthoseshapesasneeded.Thethicknessandtheelectrondensityofeachlayerarefittingparameters.Theprogramcanthenaddresstheassemblyofthoseshapesintodifferentlatticesymmetries.Thissoftwareisparticularlyusefulforanalyzing
supra‐molecularself‐assembliesandforreconstructingthesignalinit'sentirety(J.Appl.Cryst.,2010,43,1522).
Weinvestigatetheinteractionbetweenchargedmembranes,neutralmembraneswithionsandusethoseionsasaprobetoexplorethesizeoflipidrafts.Wearestudyingtheself‐assemblyof
microtubulesundervarioussolutionconditionsandwhenformingcomplexeswithmicrotubuleassociatedproteinsordrugs.Usingtime‐resolvedsolutionX‐rayscatteringat3rdgenerationsynchrotronswestudymicrotubulenucleationandtheassemblyofSV40virusesand
investigatingthestructureofbiomembranes.
Structure and Intermolecular interactions of biomolecular
self-assemblies
Uri Raviv
The Institute of Chemistry
May 6 2011
General Aims
• How biological molecules self-assemble and interact with one another in solutions?
• Focus on: Charged and Dipolar Systems
General Approach
• Similar to liquid crystals
• Focus on important length scales and study their variation
• X-ray scattering in solutions (combined with microscopy and other biophysical methods) – Non-invasive – Good statistics – Structures – Domain size – Fluctuations – Intermolecular interactions – Elastic and mechanical properties
Analysis
• Charged and dipolar membranes • Lipid-peptide complexes • Protein and peptide aggregation • Bio-membranes • Dynamic self-assembly of cytoskeleton
Proteins • Time-Dependent self-assembly of the
SV40 virus and virus-like particles
Self-Assembled Systems:
X 2θ
q = (4π/λ)sinθ
Ultra Small Angle X-ray Scattering
Samples: solutions in capillaries
The scattering amplitude F is : F(q )α
I(q) α
The intensity, I is:
The momentum transfer q is:
Area detector
Analysis of supramolecular structures
P. Szekely, A. Ginsburg, T. Ben-Nun, UR, Lamgmuir, 2010
T. Ben-Nun A. Ginsburg, P. Szekely, UR, J. Appl. Cryst., 2010
Supramolecular self-assembled structure
P. Szekely, A. Ginsburg, T. Ben-Nun, UR, Lamgmuir, 2010
T. Ben-Nun A. Ginsburg, P. Szekely, UR, J. Appl. Cryst., 2010
Charged Interfaces
Ariel Steiner Pablo Szekeley Or Sela
dw
δ
d
Electron microscopy of Golgi stack regeneration.
Charged interfaces
• Solid like-charged interfaces, such as clay minerals, swell indefinitely when diluted in water
• Very soft charged interfaces can swell only up to a maximum distance. Further dilution leads to their continuous unbinding, driven by thermal fluctuations
Self-assembled like-charged interfaces
• We used lipids with phosphatydylserine (PS) that are sufficiently rigid to swell indefinitely.
DOPS
scale bar= 50 μm
DOPS in water – Lamellar phase
Cryo TEM D. Danino, Technion
Optical microscopy
10 µm
DIC: Tom Dvir
Why ?
• Self-assembled charged interfaces have negative Gaussian modulus.
• The negative Gaussian modulus balances the elastic energy cost associated with the formation of the entropically- stabilized disordered phase.
• The disordered phase is depleted from the lamellar phase and applies an osmotic stress on it.
• The lamellar spacing is set by equating the water chemical potential and the pressures of the two phases.
Zwitterionic soft interfaces
Avanti Polar Lipids, Inc.
1,2-dipalmitoyl-sn-glycero-3-phosphocholine
1,2-dioleoyl-sn-glycero-3-phosphocholine
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
Saturated lipids adsorb multivalent ions
Or Szekely, Ariel Steiner, Pablo Szekely, Einav Amit, Roi Asor, Carmen Tamburu and UR, Langmuir, in press
Charging
Screening
Binary mixtures swell when the molar fraction of the saturated lipid decreases
DOPC: DPPC Mixtures With 5 or 10 mM CaCl2
POPC: DPPC Mixtures With 5 or 10 mM CaCl2
Form factor analysis shows that binary mixtures tend to phase separate
DOPC: DPPC
Incomplete lipid insertion
Phase separation
Ideal Mixing
The hybrid lipid (POPC) reduces the repulsive interactions between the membranes
DOPC: DPPC = 1:2 DOPC: DPPC = 1:4
Form factor analysis shows that the hybrid lipid (POPC) dissolves the lipid rafts
DOPC: DPPC = 1:1
Incomplete lipid insertion Phase separation
Ideal Mixing
The Hybrid lipid reduces the line-tension and regulates the size of lipid rafts
Or Szekely and UR, submitted
Conclusions
• Entropic attraction condenses like-charged self-assembled interfaces
• Ion- dipole interaction depends strongly on lipid tails and ion-structure.
• Hybrid lipid regulates the size of lipid rafts.
Acknowledgements
Synchrotrons: ESRF, EMBL, SOLEIL, Elettra
Funding: HFSP, ISF, BSF, JF
Grad Students: Ariel Steiner, Or Szekely, Pablo Szekely, Avi Ginsburg, Tal Ben-Nun, Roi Asor Under-grad students: Tom Dvir. Einav Amir, Roy Resh Research Staff: Carmen Tamburu, Yaelle
The deviation from ideal swelling is a bit weaker at lower membrane charge density
GC =1.4 to 16 Å
D/GC >>1
Pel = (kBT/2lB)(1/d2)
At lower membrane charge density there are less counterions their mid plane concentration is lower the repulsion in the lamellar phase is weaker
Oppositely charged interfaces (and counter-ions) also deviate from ideal-swelling but the deviation is smaller
Cl- ions are larger than Na+ ions their affinity to the Membrane is weaker their mid plane concentration is higher the repulsion in the lamellar phase is weaker