Probing actin and liposome interaction of talin and talin-vinculin complexes: a kinetic, thermodynamic and lipid labeling study.
Talin purified from human platelets and chicken gizzard smooth muscle is an actin and lipid binding protein. Here, we have investigated the effect of vinculin on (a) talin-nucleated actin polymerization and (b) insertion of talin into lipid bilayers. Calorimetric data show ternary complex formation between talin, vinculin, and actin. Actin-talin, actin-vinculin and actin-(talin-vinculin) binding and rate constants as well as actin polymerization rates for all three protein species have been determined by steady state titration, stopped-flow, and fluorescence assay. In contrast to an increase of the polymerization rate by a factor of less than 2 for actin-talin and actin-(talin-vinculin) when lowering the temperature, we measured a decrease in rates for actin alone and actin-vinculin. The overall equilibrium constants (Keq) in the van't Hoff plot proved linear and were of one-step reactions. Thermodynamic data exhibited signs of van der Waal's binding forces. Using the photoactivatable lipid analogue [3H]PTPC/11, which selectively labels membrane-embedded hydrophobic domains of proteins, we also show that talin partially inserts into the hydrophobic bilayer of liposomes. This insertion occurs in a similar manner irrespective of preincubation with vinculin.
Goldmann
W H
WH
Biophysics Department, Technical University of Munich, Garching, FRG.
Niggli
V
V
Kaufmann
S
S
Isenberg
G
G
D000199:Actins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry*; D046911:Macromolecular Substances; D010718:Phosphatidylserines / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D016608:Talin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013816:Thermodynamics; D016596:Vinculin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D000199:Actins; D008081:Liposomes; D046911:Macromolecular Substances; D010718:Phosphatidylserines; D016608:Talin; D016596:Vinculin