Diplomprüfung

• is a spherical vesicle consisting of 1 or more phospholipid bilayers around an watery compartment

• amphilic phospholipids self-assemble in water with hydrophilic heads facing water & hydrophobic tails facing iwards

• closure of bilayer avoids exposed hydrophobic edges

• differ in size and lamellarity

• SUVs and LUVs are unilamellar, with LUVs generally larger.

• MLVs contain several concentric bilayers.

• GUVs are giant unilamellar vesicles, large enough for conventional optical microscopy.

• Temperature

• chain length

• saturation

• cholesterol content.

  
  

  -> Unsaturated chains disrupt close packing and increase fluidity

  -> saturated and longer chains generally increase order. -> Cholesterol buffers fluidity and reduces permeability, with effects depending on temperature and composition.

• Electrostatic interactions

• hydrogen bonds

• hydrophobic interactions

• van der Waals forces.

Their contributions depend on protein structure, lipid composition, pH and ionic strength.

• Affinity describes equilibrium binding strength and is characterised by Kd.

• Kinetics describe association and dissociation rates, kon and koff.

• For a simple 1:1 interaction, Kd = koff/kon.

• I would incubate protein with defined liposomes and use a direct or quantitative binding method such as SPR or ITC.

• also use flotation or co-sedimentation followed by SDS-PAGE/Western blot to assess membrane association

• DLS/zeta potential as complementary characterisation.

  
  

  -> Appropriate controls are essential.

• SPR measures changes in refractive index near a sensor surface and can provide real-time kon, koff and Kd.

• ITC measures heat released or absorbed during binding and provides Kd, stoichiometry and thermodynamic parameters such as ΔH.

• Energy transfers non-radiatively from an excited donor to an acceptor when they are sufficiently close and have suitable spectral overlap.

• FRET efficiency depends strongly on distance.

• It reports proximity, which is not necessarily equivalent to specific binding.

• DLS can reveal an increase in hydrodynamic diameter or PDI.

• Cryo-TEM can provide direct visual evidence of morphology and aggregation.

• DLS alone should be interpreted carefully because a small number of large particles can dominate scattering.

• Co-sedimentation detects proteins that pellet with liposomes.

• Flotation uses a density gradient in which liposomes move towards less dense fractions and carry associated proteins with them.

  -> Controls are needed to rule out protein aggregation.

• DNA has a negatively charged phosphate backbone, so it migrates towards the positively

  charged anode in an electric field.

• Use a gel retardation assay -> Free DNA migrates through agarose

• DNA–liposome complexes have altered mobility and may remain near the well.

• DLS and zeta potential provide complementary information.

• SDS-PAGE denatures proteins and gives them a broadly similar negative charge-to-mass ratio

  -> separation is mainly by molecular weight.

• Native PAGE preserves more of the protein's native structure

  -> separates according to charge, size and shape; some complexes can remain intact.

• separates molecules by hydrodynamic size using porous beads.

  -> Large molecules enter fewer pores (have shorter way) and elute first

  -> smaller molecules enter more pores and elute later.

• Ion exchange separates according to net charge using a charged stationary phase.

• Affinity chromatography relies on specific molecular recognition between the target and an immobilised binding partner.

• measures the mass-to-charge ratio, m/z, of gas-phase ions.

• The sample is ionised, ions are separated by m/z and a detector records their abundance.

• ESI produces ions from charged liquid droplets using a high electric field and often generates multiply charged ions

  -> it is commonly coupled to LC.

• MALDI uses a laser-absorbing matrix to facilitate desorption and ionisation and often produces predominantly singly charged ions.

• elected precursor peptide ion is fragmented, and the fragment ions are analysed.

• Fragment patterns can be matched to theoretical peptide spectra in databases to identify peptides and infer proteins.

• No. DLS may detect protein binding, liposome aggregation, protein aggregation or other changes.

• I would use an orthogonal binding assay such as SPR or ITC and a separation-based assay such as flotation to investigate the interaction.

The 10 nM interaction has stronger affinity because lower Kd means stronger binding.

• Methods provide complementary information.

• DLS measures hydrodynamic size

• zeta potential measures electrokinetic surface potential

• SPR measures binding kinetics

• ITC measures binding thermodynamics.

  
  

  -> Combining independent methods helps distinguish binding from indirect effects.

• Run a gel retardation assay across increasing lipid-to-DNA ratios and quantify residual free DNA.

• Complement it with DLS and zeta-potential measurements.

• Complete retardation under the assay conditions suggests little detectable free DNA, but does not alone prove complete complexation.

• Dissolved ions screen charges and reduce electrostatic attraction between oppositely charged groups.

• The effect depends on ionic strength and whether other interactions also contribute.

• Detecting binding shows that an interaction occurs under the tested conditions.

• Measuring affinity requires quantitative data, ideally across a concentration range, and fitting an appropriate binding model to estimate Kd.

I would compare:

• size and PDI by DLS

• morphology by cryo-TEM

• zeta potential under matched buffer conditions.

  
  

  -> Also assess stability over time and, if binding is the objective, use a suitable binding assay such as SPR or ITC.

  -> Lipid composition, preparation method and concentration must be controlled.

• Protein aggregation or precipitation can cause pelleting without membrane binding.

• Run protein-only controls and consider a flotation assay or an orthogonal binding method

• Possible reasons include immobilisation artefacts, different buffer or surface conditions, masstransport effects, weak heat signal, low binding enthalpy, sample concentration errors or different molecular states

• The methods should be checked for comparable conditions and sample quality.

• concentration series reveals how the response depends on analyte concentration and helps estimate affinity or distinguish saturable binding from non-specific effects.

• A single concentration cannot normally provide a reliable Kd.

• Include protein alone, liposomes alone, buffer blank and a non-binding or altered-lipid control where possible.

• Match salt, pH, temperature and solvent conditions. Confirm liposome integrity and protein quality, and use independent replicates.

• Prepare liposomes with a defined lipid composition and confirm their initial size distribution by DLS.

  Incubate liposomes with a concentration series of the protein under controlled buffer, temperature and time

  conditions.

• Use SPR or ITC if quantitative affinity/kinetic or thermodynamic information is required.

• Use co-sedimentation or flotation followed by SDS-PAGE/Western blot as an orthogonal test for membrane

  association.

• Measure DLS and zeta potential before and after incubation to detect changes in size distribution and electrokinetic surface properties.

• Include controls: protein alone, liposomes alone, buffer blank and, where possible, a non-binding protein or

  lipid-composition control.

-> Interpretation: no single change in DLS, zeta potential or pellet signal proves specific binding. Agreement between

independent methods makes the conclusion more robust.

• Prepare cationic liposomes and DNA at a range of lipid-to-DNA charge ratios.

• Incubate to allow lipoplex formation.

• Run an agarose gel retardation assay to assess the amount of free DNA.

• Use DLS to assess complex size and PDI.

• Use zeta potential to assess changes in surface potential as the ratio changes.

• Use cryo-TEM if morphology is important.

-> Interpretation: a loss of free-DNA migration supports complex formation, but should be interpreted alongside

particle-size and surface-potential data