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Cette revue sur le repliement des protéines ne fait appel à aucune expérience propre de diffusion de neutrons. Elle décrit les aspects cinétiques et thermodynamiques de la structuration des protéines sur leur chemin de repliement, en faisant référence à des techniques biophysiques variées permettant d'échantillonner des changements conformationnels sur des échelles de temps longues (ms. à s.). Les neutrons constituent une sonde évidemment complémentaire des techniques usuellement employées pour l'étude du repliement des protéines. Nous avons choisi un modèle d'étude, l'α-lactalbumine, pour donner un état de l'art des connaissances acquises à ce jour dans le domaine, et pour ainsi inciter biophysiciens et spectroscopistes à développer des techniques en temps résolu sur des échelles plus rapides, afin de pouvoir détecter les étapes précoces du repliement. Les neutrons doivent trouver une place logique dans de tels développements.

Cette revue sur le repliement des prot?ines ne fait appel ? aucune exp?rience propre de diffusion de neutrons. Elle d?crit les aspects cin?tiques et thermodynamiques de la structuration des prot?ines sur leur chemin de repliement, en faisant r?f?rence ? des techniques biophysiques vari?es permettant d'?chantillonner des changements conformationnels sur des ?chelles de temps longues (ms. ? s.). Les neutrons constituent une sonde ?videmment compl?mentaire des techniques usuellement employ?es pour l'?tude du repliement des prot?ines. Nous avons choisi un mod?le d'?tude, l'?-lactalbumine, pour donner un ?tat de l'art des connaissances acquises ? ce jour dans le domaine, et pour ainsi inciter biophysiciens et spectroscopistes ? d?velopper des techniques en temps r?solu sur des ?chelles plus rapides, afin de pouvoir d?tecter les ?tapes pr?coces du repliement. Les neutrons doivent trouver une place logique dans de tels d?veloppements.

Once two radioactive Ca2+ coming from the cytoplasm are bound to the transport sites of the nonphosphorylated ATPase, excess EGTA induces rapid dissociation of both ions, whereas excess nonradioactive Ca2+ only reaches one of the two bound Ca2+. This difference has been explained assuming that the two Ca2+ sites are in a single file channel in which the superficial Ca2+ is freely exchangeable from the cytoplasm, whereas the deeper Ca2+ is exchangeable only when the superficial site is vacant. The same experiment was done using phosphorylated ATPase to determine whether Ca2+ dissociation toward the lumen is sequential as well. Under conditions that allow ADP-sensitive phosphoenzyme to accumulate (leaky vesicles, 5 degrees C, pH 8, 300 mM KC1), we found the same two pools of Ca2+. Excess EGTA induced dissociation of both ions together with dephosphorylation. Excess nonradioactive Ca2+ induced the exchange of half the radioactive Ca2+ without any effect on the phosphoenzyme level. Our results show a close similarity between the transport sites of the nonphosphorylated and the phosphorylated enzymes, although the orientation, affinities, and dissociation rate constants are different.

Excess ATP is known to enhance Ca(2+)-ATPase activity and, among other effects, to accelerate the Ca2+ binding reaction. In previous work, we studied the pH dependence of this reaction and proposed a 3H+/2Ca2+ exchange at the transport sites, in agreement with the H+/Ca2+ counter transport. Here we studied the effect of ADP and nonhydrolyzable ATP analogues on the Ca2+ binding reaction at various pH values. At pH 6, where Ca2+ binding is monophasic and slow, ADP, adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP), or adenyl-5'-yl imidodiphosphate (AMPPNP) increased the Ca2+ binding rate constant 20-fold. At pH 7 and 8, where Ca2+ binding is biphasic, the nucleotides induce fast and monophasic Ca2+ binding. At pH 7, AMP-PCP accelerated Ca2+ binding with an apparent dissociation constant of 10 microM. At acidic pH, ADP, AMPPCP, or AMPPNP increased the equilibrium affinity of Ca2+ for ATPase, whereas at alkaline pH, these nucleotides had no effect. At pH 5.5, AMPPCP increased equilibrium Ca2+ binding with an apparent dissociation constant of 1 microM.

The experiments reported in the present paper were designed to check the model proposed for Ca2+ binding in the preceding paper (Forge, V., Mintz, E., and Guillain, F. (1993) J. Biol. Chem. 268, 10953-10960). The pH dependence of the Mg(2+)-induced variation of the intrinsic fluorescence, as well as that of the phosphorylation by Pi, confirmed that there are several species of Ca(2+)-deprived ATPase. Kinetics of Ca2+ binding as a function of pH suggested that the deprotonated form of the ATPase binds Ca2+ rapidly (k > 50 s-1), whereas the protonated forms bind Ca2+ slowly (1.3-2.7 s-1). At variance with other models which are linear, slow and rapid Ca2+ binding take two different routes, and intermediate pH values and Mg2+, which favors the deprotonated forms, result in biphasic kinetics. Mg2+ binds to all Ca(2+)-deprived species and to species having one bound Ca2+ but does not bind to ECa2. This is the reason why Mg2+ inhibits Ca2+ binding, and this inhibition is removed in the presence of adenosine-5'-O-(3-thiotriphosphate) which drives Mg2+ into the catalytic site.

H+ and Mg2+ are known to inhibit Ca2+ binding to the transport sites of sarcoplasmic reticulum-ATPase. Evaluation of the affinity for the Ca2+ binding sites requires measurement of the amount of Ca2+ bound to ATPase as a function of the free Ca2+ concentration imposed by a Ca2+ chelator. The choice of the chelator is crucial as it determines the accuracy of the free Ca2+ concentration. At pH > 7, the EGTA affinity for Ca2+ is higher than that of ATPase, inducing artifacts that alter the shape of the binding curves. Thus, we have used 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), whose affinity is unchanged at pH > or = 7. Ca2+ binding was studied at equilibrium, from pH 6 to pH 8 and from 0 to 10 mM Mg2+, using EGTA and/or BAPTA and [45Ca]Ca2+. Under all conditions, the stoichiometry was 2 Ca2+/ATPase. At variance with previous studies, the Hill coefficient was 1.1-2 and higher at pH 6 than at pH 8. In addition, it decreased in the presence of Mg2+. The Ca2+ binding curves were analyzed according to a model in which they result from a sequential binding of two Ca2+, each binding step being modified by H+ and Mg2+. The effect of H+ is described by two steps involving two H+ and one H+, with pK 7 and 7.9, respectively. At pH 6, ATPase must lose two H+ for the first Ca2+ to bind and a third H+ for the second Ca2+ to bind. At pH 9, both Ca2+ bind without any H+ exchange. Mg2+ can bind to all species, except to that saturated with Ca2+. The species having lost two H+ has a higher affinity for Mg2+ (< or = 1 mM) than the species having bound three H+ (4 mM). The above model allows us to analyze the effects of H+ and Mg2+ at each Ca2+ binding step and to explain the changes in the apparent affinity and cooperativity.