ISOLATED CALCIUM-BINDING LOOPS OF EF-HAND PROTEINS CAN DIMERIZE TO FORM A NATIVE-LIKE STRUCTURE

Helix-loop-helix fragments of EF-hand proteins are known to dimerize i n solution, re-producing the characteristic structure of native protei n domains [Shaw, G. S., Hedges, R. S., & Sykes, B. D. (1990) Science 2 49, 280-283]. In this paper we present evidence that isolated calcium- binding loops can also dimerize, when saturated with lanthanide ions, interacting with each other in a similar way as do loops in intact pro teins. A synthetic analogue of calcium binding loop III of calmodulin, AcDKDGDGYISAAE-NH2, has been studied by H-1 NMR spectroscopy. For the La3+-saturated peptide, concentration dependent broadenings and shift s of certain signals have been observed indicating dimerization proces s of intermediate rate on the NMR time scale. Analysis of signal shape and position of the Tyr7 ring protons as a function of concentration makes it possible to determine the association and dissociation rate c onstants of the process for various temperatures within the range of 1 0-80 degrees C. The dimerization constant changes according to van't H off relationship with Delta S = 233 J/mol . K and Delta H = 62 kJ/mol. A distance of 11.4 +/- 0.4 Angstrom between the ions coordinated by d imer molecules has been determined by measurements of Tb3+-->Ho3+ lumi nescence energy transfer. This value suggests that the dimer structure is similar to that of two-loop structural elements in native EF-hand proteins. From a thermodynamic cycle it can be shown that La3+ ion bin ding to the peptide dimers must be highly cooperative. Therefore, coop erativity of ion binding to domains of EF-hand proteins is, at least p artly, due to local interactions between binding loops.