Role of Trp-187 in the annexin V-membrane interaction: a molecular mechanics analysis.

The minimized energy mapping of annexin V Trp-187 chi1 x chi2 isomerization supports the existence of two preferential rotameric orientations of the Trp side chain upon annexin V binding to membranes, in agreement with the time-resolved fluorescence results. They correspond to the perpendicular trans (-173 degrees, 73 degrees) and g- (-71 degrees, 83 degrees) rotamers and represent 59 and 28% of the population, respectively. The analysis of their local environment makes it possible to assign the trans rotamer to the long component and the g- rotamer to the short component of the biexponential fluorescence decay. The orientation of these rotamers relative to the protein core suggests a dual role for Trp-187, which might be involved both in the interaction with the phospholipid bilayer and in the formation of the annexin V 2-D array at the surface of the membrane.

Conformational adaptation of annexin V upon binding to liposomes: a time-resolved fluorescence study.

The fluorescence intensity decay of the single tryptophan residue, Trp-187, of free annexin V is described by the sum of three lifetime components (5.4, 1.3, and 0.4 ns), which may be correlated to three ground-state classes of Trp conformers. The two major classes (44 and 48%) are embedded in the protein matrix. When annexin V binds to calcium and liposomes made of dioleoylphosphatidylcholine and dioleoylphosphatidylserine, similar results are obtained whatever the (10-200) lipid ratio. The Trp fluorescence decay is fitted with only two components (6.9-7.2 and 2.0-2.2 ns). Decay-associated spectra reveal that the longest lifetime of bound annexin V can be related to Trp residues (60%) located in a partially polar environment, which could correspond to the protein-membrane interface. The shortest lifetime is attributed to Trp residues (40%) which reside in a hydrophobic surrounding: these Trp residues would penetrate into the phospholipid membrane and contribute to the stabilization of the 2D-array of annexin V molecules.

Formation of two-dimensional arrays of annexin V on phosphatidylserine-containing liposomes.

Annexins are intracellular proteins which bind to membranes in a Ca(2+)-dependent manner and which have been proposed to play regulatory roles in different membrane processes. In the present study, the stoichiometry of the Ca(2+)-dependent binding of annexin V to phosphatidylserine molecules incorporated into liposomes was studied by fluorescence spectroscopy. The Ca(2+)-dependence of the binding was determined using liposomes made of dioleoylphosphatidylserine (PS) and dioleoylphosphatidylcholine (PC), with a PC/PS molar ratio ranging from 1 to 800. These liposomes were shown to be mostly unilamellar by cryoelectron microscopy. [Ca2+]1/2 concentrations required for half-maximal binding of annexin V range from 57 microM at PC/PS = 1 up to 96 mM at PC/PS = 800. Titration of accessible PS molecules showed that annexin V molecules bind equally well to liposomes of PC/PS ratio ranging from 1 to 400. The stoichiometry of the binding between annexin V and PS, determined at low PS content, is eight annexin V molecules per one PS molecule. We propose a novel model of the Ca(2+)-dependent interaction between annexin V and lipid membranes, based on the formation of two-dimensional arrays of annexin V molecules, stabilized by both protein-lipid and protein-protein interactions.

Ca(2+)- and Mg(2+)-dependent binding of annexin II and its complex to phospholipid vesicles; a comparative spectroscopic study.

The binding of annexin II (p36) and of its complex ((p36)2 (p11)2) to model phospholipid vesicles led to conformational changes of the proteins which were studied by fluorescence spectroscopy. Titrations with liposomes of different kinds showed that the presence of the p11 dimer in the complex enhances the conformational change of the annexin II. Comparative liposome titrations were carried out in the presence of Mg2+: the binding of the protein to liposome induced a much smaller protein conformational change in this case. Moreover, Ca2+ and phospholipid binding order experiments showed that the protein conformational change only occurred when both are bound.

A fluorescence spectroscopy study of the calpactin I complex and its subunits p11 and p36: calcium-dependent conformation changes.

A fluorescence study of the calpactin I complex, a heterotetramer composed of two molecules of p36 and two molecules of p11, and its subunits, was performed to clarify their conformation. The analysis of the fluorescence characteristics of the single Trp of p36, in the absence of Ca(2+), shows that: (i) in the complex, Trp is buried within the protein matrix and subjected to static quenching from nearby groups; (ii) for p36 the results are similar, but Trp seems even more shielded than in the complex. Adding Ca(2+) to the calpactin I complex, or to p36, shifts the Trp emission maximum wavelengths, and increases the quantum yields which reflect a conformational change, burying the Trp in a more hydrophobic environment. In the presence and even in the absence of Ca(2+), the binding of phosphatidylserine liposomes induces a conformational change, detected by fluorescence measurements. The Ca(2+) dissociation constants, as determined by fluorescence titrations, are similar for the complex and p36 (KD approximately 0.5 x 10(-3) M). The affinity is enhanced a 1000-times in the presence of negatively charged phospholipids. In p11, both Try residues are located in a hydrophobic environment and the protein fluorescence does not change upon Ca(2+) addition.