Direct enthalpy measurements of the calcium-dependent interaction of annexins V and VI with phospholipid vesicles.
Annexins V and VI are two members of the annexin protein family, each of which associate with phospholipid vesicles in a calcium-dependent manner. They may be important intracellular calcium response elements. Titration calorimetry and spectroscopic techniques showed striking features of these interactions. For annexin V and VI, the total heat release from calcium-dependent binding to bilayer vesicles composed of phosphatidylserine/phosphatidylcholine was -25 and -38 kcal/mol, respectively. The enthalpy of association (delta Hassoc) for the respective protein-calcium interactions was about -11 and -5 kcal/mol. The delta Hassoc for the annexin VI-vesicle interaction was largely independent of the headgroup of the anionic phospholipid and of the pH from 7.1 to 8.4. Use of phosphatidylethanolamine as the neutral phospholipid resulted in a slightly more negative delta Hassoc. Enthalpy was either independent of vesicle size (annexin V) or showed a slightly more negative value for large vesicles (annexin VI). The delta Hassoc for annexin VI-membrane interaction was not constant during protein titration but became more exothermic with higher protein density on the membrane. This behavior was surprising because the equilibrium constant showed negative cooperativity with respect to protein density [Bazzi, M. D., & Nelsestuen, G. L. (1991) Biochemistry 30, 7970-7977]. Apparently, entropy changes occurred which were large and negative, thereby compensating for the increasingly negative enthalpy but decreasing affinity as protein density was increased. In fact, the exothermic process coincided more closely with a change in the intrinsic tryptophan fluorescence of annexin VI than with actual protein-membrane binding. Circular dichroism detected very small changes in protein secondary structure during these events. The observed delta Hassoc for annexin-membrane interaction appeared to involve contributions from the membrane as well as from the protein. Annexin-membrane binding may exert large effects on the membrane that could serve a regulatory capacity in the cell.
Plager
D A
DA
Department of Biochemistry, University of Minnesota, St. Paul 55108.
Nelsestuen
G L
GL
D017304:Annexin A5 / Q000378:metabolism*; D017317:Annexin A6 / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D008567:Membranes, Artificial; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D014364:Tryptophan / Q000737:chemistry
D017304:Annexin A5; D017317:Annexin A6; D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; D008567:Membranes, Artificial; D010743:Phospholipids; D014364:Tryptophan; D002118:Calcium