Human platelet P-235, a talin-like actin binding protein, binds selectively to mixed lipid bilayers.
The interaction of platelet talin (P-235) with mixtures of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylserine (DMPS) as well as with pure lipids was studied in reconstituted lipid bilayers. Incorporation of platelet talin into vesicles was achieved by self-assembly during cycles of freeze-thawing of co-dispersions containing vesicles and the purified protein. The yield of protein incorporation as a function of lipid composition was determined by measuring the protein/lipid ratio using protein assay, phosphate determination and gel electrophoresis in parallel. Protein-lipid interactions are monitored by high sensitive differential scanning calorimetry (DSC) measuring (i) the shifts of transition states delta Ts* and delta Tl*, where Ts represents the solidus line, the onset of lipid chain melting, and Tl the liquidus line, the endpoint of chain melting, and (ii) the heats of transition. Cytoplasmic talin differs from a membrane bound form by its ability and mode of lipid interaction. The latter partially penetrates into the hydrophobic region of the bilayer, which renders a low incorporation rate even into neutral lipids. This interaction is greatly enhanced in the presence of charged lipids: a marked shift of Tl occurs due to a selective electrostatic interaction of the protein with the membrane surface. Evidence for a selective binding is also provided by Fourier transform infrared spectroscopy (FTIR). Right-side-out oriented platelet talin can be cleaved by proteinases, which truncate the extrinsic electrostatic binding domain but not the hydrophobic. In addition, reconstituted platelet talin, like in vivo, can be cleaved by thrombin. The interaction of cytoplasmic platelet talin with lipid bilayers is purely electrostatic. Our data suggest that protein reconstitution by freeze-thawing is an equilibrium process and that the protein distribution between the membrane and water is determined by the Nernst distribution law. Consequently, the work of protein transfer from water into the bilayer can be measured as a function of charged lipids.
Heise
H
H
Biophysic Department, Technical University of Munich, Garching, F.R.G.
Bayerl
T
T
Isenberg
G
G
Sackmann
E
E
D001798:Blood Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D003593:Cytoplasm / Q000187:drug effects / Q000648:ultrastructure; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D010450:Endopeptidases / Q000494:pharmacology; D005614:Freeze Fracturing; D006358:Hot Temperature; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D010710:Phosphates / Q000737:chemistry; D010743:Phospholipids / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013055:Spectrophotometry, Infrared; D016608:Talin; D013917:Thrombin / Q000494:pharmacology
D001798:Blood Proteins; D003598:Cytoskeletal Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010710:Phosphates; D010743:Phospholipids; D016608:Talin; D012965:Sodium Chloride; D010450:Endopeptidases; D013917:Thrombin