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 in solution, re-producing the characteristic structure of native protein domains [Shaw, G.S., Hodges, R.S., & Sykes, B. D. (1990) Science 249, 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 proteins. A synthetic analogue of calcium binding loop III of calmodulin, AcDKDGDGYISAAE-NH2, has been studied by 1H NMR spectroscopy. For the La(3+)-saturated peptide, concentration dependent broadenings and shifts of certain signals have been observed indicating dimerization process 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 constants of the process for various temperatures within the range of 10-80 degrees C. The dimerization constant changes according to van't Hoff relationship with delta S = 233 J/mol.K and delta H = 62 kJ/mol. A distance of 11.4 +/- 0.4 A between the ions coordinated by dimer molecules has been determined by measurements of Tb(3+)-->Ho3+ luminescence 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 binding to the peptide dimers must be highly cooperative. Therefore, cooperativity of ion binding to domains of EF-hand proteins is, at least partly, due to local interactions between binding loops.