Biology of Membrane Lipids
Measure lipid binding proteins – ‘protein profiling’
- novel lipid binding proteins/modules (lipid affinity matrices)
- lipid effectors (recruitment/activation of proteins)
Measure lipid binding proteins – ‘protein profiling’ eg. lipid receptors, interfacial binding, integral interactions
- lipid composition of membranes (LC, MS)
- structure and dynamics of lipids (NMR, ESR, fluorescent probes)
– Random, coil – beta sheet transition: Alzheimer peptide
– Random, coil – alpha helix transition: Antimicrobial peptides secreted from skin of Xenopus laevis
Measure lipid modifying activities – ‘enzymatic profiling’
- identify enzymes and isoforms (cell biology and lipid enzymology)
- correlate lipid metabolism to physiology and disease
Preparation of lipid matrices
Vesicle preparation
Small unilamellar vesicles (SUVs) of DMPC were prepared by the method of Bangham et al. (1974). Briefly, MLVs (multilamellar vesicles) were prepared by hydrating a dried lipid film of DMPC with Millipore water (37°C for several hours) and subsequently sonicating with a probe sonicator (Fisher Sonic Dismembrator 300) and filtering through a 0.22-µm Millipore filter. The resulting SUVs have an average diameter of 400 Å, as found by particle sizing optical turbidity measurements (Microtrac UPA 150; Brookhaven Instruments Corp.).
-Large unilamellar vesicles (LUV)
-Small unilamellar vesicles (SUV) > 50nm
-Multilamellar vesicles (MLV)
-Giant unilamellar vesicles (GUV)
MLV -> SUV | V GUV -> LUV -> SUV
Evaporate solvent to dry lipid to a film under a stream of nitrogen
Hydrate lipid film with water saturated nitrogen (-> swelling of lipid film)
Dry lipid over dessicating phosphorous pentoxide and high vaccuum
Resuspend lipid film in aqueous buffer by vigorous shaking (-> MLV)
Resuspend lipid film in aqueous buffer by gentle shaking (-> GLV & MLV)
Freeze-thaw cycle the lipid suspension to disrupt small vesicles (-> MLV)
Extrude lipid suspension thru polycarbonate filter (-> LUV)
Sonify lipid suspension (-> SUV)
Helical wheel projections
-Division of polar and non polar regions of a phospolipid
-Peaks of scattering density at the polar and non-polar boundary
Isothermal Titration Calorimetry
Isothermal Titration Calorimetry (ITC) is a biophysical technique used to determine the thermodynamic parameters of (biochemical) interactions. It is most often used to study the binding of small molecules (such as medicinal compounds) to larger macromolecules (proteins, DNA etc.).
Measurement of this heat allows accurate determination of binding constants (K_B), reaction stoichiometry (n), enthalpy ( H) and entropy ( S), thereby providing a complete thermodynamic profile of the molecular interaction in a single experiment
How to determine binding properties of new lipid?
-Checking for isoforms
-Check gene sequence
-Mutation: what changes? At important sites, eg. for binding, for active site.
-In vitro tests: liposomes, EM, gold labelling (using chips).
-In vivo tests: test for coimmunoprecipitation, crosslinking, localization.
Impt lipids
-Glycerophospholipids: Phosphotidylinositol
-Sterol lipids: linoleic acid + cholesterol
-Sphingolipids: ceramide + sphingomyelin
Membranbe trafficking
-Donor budding off vesicle
-Taget fusion
-Components: calthrin, AP2, SV proteins, actin
-Synaptojanin
-Dual keys code
-Viral membrrane fusion (influenza)
-Lateral segregation – Lipid microdomains, lipid rafts
Phospholipid lysobiphoshatidic acid (LBPA)
-Rich in endosomes
Siganlling role
-38:4 PIP2
-PI(4, 5)P2 as a precursor for intracellular signaling molecules – PLC cascade: activating DAG and I(1,4,5)P_3
Potential roles for lipids in replication of enveloped viruses
-protection & structural integrity
-recognition & signalling
-entry replication, replication cofactors
-recruitment of viral protein during assembly
Current Opinion in Lipidology. 18(2):121-128, April 2007.
Fernandis, Aaron Z; Wenk, Markus R
-Regulation of protein function and localization: reversible phosphorylation and dephosphorylation
-Generation of messengers by hydrolysis of membrane lipid headgroups eg. Kainate (by receptor stimulation)
-Generation of lipid messengers by metabolism of hydrocarbon tails: different molecular isomers/species due to the different position of double bonds.
-Signalling lipids as biomarkers
-Lipids as ligands for protein receptors in times of stress: by facing outside instead of inside
The metabolic chain mentioned above, i.e. GPIns(4,5)P2 -> diacylglycerol -> GPA, leads to drastic alterations in the properties of the membrane at the lipid headgroup region. The net charge in this case varies from z = -2 [GPIns(4,5)P2] -> neutral (diacylglycerol ) -> z = -1 (GPA). This has important implications for protein binding to the membrane. Furthermore, the headgroups of these lipids are vastly different with respect to their occupied space and degree of hydration. Using peptide probes, such localized alterations in phagosomal membranes could be monitored. Comparison with other optical probes for lipids indicates that hydrolysis of phosphoinositides is indeed a major contributing factor in these changes [9••]. Reduced lateral pressure, by altered packing constraints in the headgroup region, affects the interaction and depth of penetration of proteins at the membrane surface. Indeed, the membrane activity of dynamin, a GTPase involved in clathrin-mediated endocytosis, may be regulated (at least in part) by such mechanisms [10,11]. Furthermore, electrostatic protein–lipid interactions are often supported by specific headgroup recognition. The phosphorylation of lipid headgroups, PI -> PIs, in particular, has pronounced effects on protein recruitment and activity at the membrane surface
Analysis
-Ubiased profiling,
-MS/MS for identification of ions in DP
-MRM quantification of ions of interest
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