Folding energetics of ligand binding proteins II. Cooperative binding of Ca2+ to annexin I.

The calcium binding properties of annexin I as observed by thermodynamic DSC studies have been compared to the structural information obtained from X-ray investigation. The calorimetric experiment permitted to evaluate both the reaction scheme - including binding of ligand and conformational changes - and the energetics of each reaction step. According to published X-ray data Annexin I has six calcium binding sites, three medium-affinity type II and three low-affinity type III sites. The present study shows that at 37 degrees C annexin I binds in a Hill type fashion simultaneously two calcium ions in a first step with medium affinity at a concentration of 0.6 mM and another three Ca(2+) ions again cooperatively at 30 mM with low affinity. Therefore it can be concluded that only two medium-affinity type II binding sites are available. The third site, that should be accessible in principle appears to be masked presumably due to the presence of the N terminus. In view of the large calcium concentration needed for saturation of the binding sites, annexin I may be expected to be Ca(2+) free in vivo unless other processes such as membrane interaction occur simultaneously. This assumption is consistent with the finding, that the affinity of annexins to calcium is usually markedly increased by the presence of lipids.

X-ray structure of full-length annexin 1 and implications for membrane aggregation.

Annexins comprise a multigene family of Ca2+ and phospholipid- binding proteins. They consist of a conserved C-terminal or core domain that confers Ca2+-dependent phospholipid binding and an N-terminal domain that is variable in sequence and length and responsible for the specific properties of each annexin. Crystal structures of various annexin core domains have revealed a high degree of similarity. From these and other studies it is evident that the core domain harbors the calcium-binding sites that interact with the phospholipid headgroups. However, no structure has been reported of an annexin with a complete N-terminal domain. We have now solved the crystal structure of such a full-length annexin, annexin 1. Annexin 1 is active in membrane aggregation and its refined 1.8 A structure shows an alpha-helical N-terminal domain connected to the core domain by a flexible linker. It is surprising that the two alpha-helices present in the N-terminal domain of 41 residues interact intimately with the core domain, with the amphipathic helix 2-12 of the N-terminal domain replacing helix D of repeat III of the core. In turn, helix D is unwound into a flap now partially covering the N-terminal helix. Implications for membrane aggregation will be discussed and a model of aggregation based on the structure will be presented.

Crystallization and preliminary X-ray analysis of full-length annexin I comprising the core and N-terminal domain.

Annexin I, a member of the annexin family of Ca(2+)- and phospholipid-binding proteins, has been crystallized with the complete N-terminus. Annexins are structurally divided into a conserved protein core and an N-terminal domain that is variable in sequence and length. Three-dimensional structures of annexins comprising the protein core and a short N-terminal domain (annexins III, IV, V, VI, XII) or a truncated form almost completely lacking the N-terminal domain (annexins I and II) have been published so far. Here, the crystallization of annexin I comprising not only the core but also the complete N-terminal domain is reported. The crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 63.6, b = 96.3, c = 127.4 A, and diffract to better than 2 A. Assuming a molecular weight of 38.7 kDa for annexin I and an average value of 2.5 A(3) Da(-1) for V(M), two molecules per asymmetric unit are present.

Coupling photoisomerization of retinal to directional transport in bacteriorhodopsin.

In order to understand how isomerization of the retinal drives unidirectional transmembrane ion transport in bacteriorhodopsin, we determined the atomic structures of the BR state and M photointermediate of the E204Q mutant, to 1.7 and 1.8 A resolution, respectively. Comparison of this M, in which proton release to the extracellular surface is blocked, with the previously determined M in the D96N mutant indicates that the changes in the extracellular region are initiated by changes in the electrostatic interactions of the retinal Schiff base with Asp85 and Asp212, but those on the cytoplasmic side originate from steric conflict of the 13-methyl retinal group with Trp182 and distortion of the pi-bulge of helix G. The structural changes suggest that protonation of Asp85 initiates a cascade of atomic displacements in the extracellular region that cause release of a proton to the surface. The progressive relaxation of the strained 13-cis retinal chain with deprotonated Schiff base, in turn, initiates atomic displacements in the cytoplasmic region that cause the intercalation of a hydrogen-bonded water molecule between Thr46 and Asp96. This accounts for the lowering of the pK(a) of Asp96, which then reprotonates the Schiff base via a newly formed chain of water molecules that is extending toward the Schiff base.

Slow unfolding and refolding kinetics of the mesophilic Rop wild-type protein in the transition range.

We describe the guanidinium hydrochloride induced folding kinetics of the four-helix-bundle protein Rop wild-type (wt) under equilibrium conditions at three temperatures. The choice of appropriate denaturant conditions inside the transition range permitted, in combination with equilibrium transition curves, the determination of both unfolding and refolding rate constants. The ratio of the rate constants at zero denaturant concentration provided equilibrium constants and standard free energy changes that are in good agreement with values obtained in previous differential scanning calorimetry studies. The DeltaG0D values for 19, 25 and 40 degrees C calculated from the present kinetic studies are, respectively, 66.8, 70.8 and 57.2 kJ.mol-1. The unfolding reactions are extremely slow under these conditions. Equilibrium was reached only after 18, 12 and 6 days at 19, 25 and 40 degrees C. These results demonstrate that for Rop wt high stability correlates with slow folding kinetics.

A comparison of the energetics of annexin I and annexin V.

The annexins comprise a family of soluble Ca2+- and phospholipid-binding proteins. Although highly similar in three-dimensional structure, different annexins are likely to exhibit different biochemical and functional properties and to play different roles in various membrane related events. Since it must be expected that these functional differences arise from differences in the characteristic thermodynamic parameters of these proteins, we performed high-sensitivity differential scanning microcalorimetry (DSC) and isothermal guanidinium hydrochloride (GdnHCl)-induced unfolding studies on annexin I and compared its thermodynamic parameters with those of annexin V published previously. The DSC data were analyzed using a model that permits quantitative treatment of the irreversible reaction. It turned out, however, that provided a heating rate of 2 K min-1 is used, unfolding of annexin I can be described satisfactorily in terms of a simple two-state reaction. At pH 6.0 annexin I is characterized by the following thermodynamic parameters: t1/2=61.8 degrees C, DeltaHcal=824 kJ mol-1 and DeltaCp=19 kJ mol-1 K-1. These parameters result in a stability value of DeltaG0D (20 degrees C)=51 kJ mol-1. The GdnHCl induced isothermal unfolding of annexin I in Mes buffer (pH 6.0), yielded DeltaG0D (buffer) values of 48, 60 and 36 kJ mol-1 at 20, 12 and 5 degrees C, respectively. These DeltaG0D values are in reasonable agreement with the values obtained from the DSC studies. The comparison of annexin I and annexin V under identical conditions (pH 8.0 or pH 6.0) shows that despite the pronounced structural homology of these two members of the annexin familiy, the stability parameters are remarkably different. This difference in stability is consistent with and provides a thermodynamic basis for the potential different in vivo functions proposed for these two annexins.

Ca2+-independent interaction of annexin I with phospholipid monolayers.

At pH 6.0, the interaction of annexin I, a proteolytic fragment of annexin I and annexin V, was studied with monolayers composed of dipalmitoylphosphatidylserine (DPPS), dipalmitoylphosphatidylcholine (DPPC) or DPPS/DPPC mixtures (molar ratio 1:4). The measurements reveal that only annexin I shows a significant increase in the surface pressure at constant surface area in the absence of Ca2+ ions. We interpret these pressure changes as reflecting penetration of the protein. Kinetic analyses of the annexin I/monolayer interaction at pH 6.0 in the presence and absence of Ca2+ ions show differences between the interaction mechanisms that support the occurrence of a pH-regulated process. At pH 7.4, Ca2+ ions are required for the interaction.