THE ROLE OF BETA-SHEET INTERACTIONS IN DOMAIN STABILITY, FOLDING, ANDTARGET RECOGNITION REACTIONS OF CALMODULIN

Single-residue mutations have been made of the hydrophobic Ile or Val residue in position 8 of each of the four calcium-binding loop sequenc es (sites I-IV) of Drosophila calmodulin, These highly conserved resid ues are part of the hydrophobic core of either calmodulin domain and a re involved in the structural link of two calcium-binding sites via a shea antiparallel beta-sheet. In the ape-form, the replacement of ne ( Or Val) by Cry causes a significant destabilization, shown by the unfo lding of the secondary structure of the domain carrying tile mutation. In the presence of calcium, the deficiency in alpha-helical structure at 20 degrees C is restored for the mutants at site I, II, or III but not at site IV, which requires the further binding of a high-affinity target peptide to re-establish the native conformation. The extent of the destabilization is seen in the depression of the melting temperat ure of individual domains, which can be as large as 80 degrees C in th e case of Ca-4-CaM(V136G). However, because of low values of the unfol ding enthalpy for calmodulin domains, only relatively low values of <2 kcal/mol are implied for Delta Delta G, the free energy of destabiliz ation due to mutation, Consistent with this, the secondary structure o f any unfolded mutant domain is highly sensitive to solvent compositio n and is largely refolded in the presence of 12.5% (v/v) aqueous trifl uoroethanol. Compared to wild-type calmodulin, the affinities of the m utants for calcium and target peptides from sk-MLCK at 20 degrees C ar e significantly reduced but the effects are relatively small. These re sults indicate that the conformation of calmodulin can be dramatically altered by mutation of a single highly conserved residue but that cha nges in solvent or the binding of a target sequence can readily compen sate for this, restoring the wild-type properties. The results also su ggest that the integrity of both the apo- and hole-forms of calmodulin is important for the maintenance of its biological function and confi rm the importance of conserving the structural function of the residue s involved in the beta-sheet interactions.