CONFORMATIONAL-CHANGES AND CALCIUM-BINDING BY CALRETININ AND ITS RECOMBINANT FRAGMENTS CONTAINING DIFFERENT SETS OF EF HAND MOTIFS

Four recombinant fragments, representing different sets of EF-hand mot ifs of rat calretinin (CR) (I-II, I-III, III-VI, IV-VI), were prepared , and their Ca2+-induced conformational changes were compared with tho se of full-length recombinant CR. All fragments were able to bind calc ium ions as shown by Ca-45(2+) overlay method on nitrocellulose and fl uorescence measurements. The intrinsic tryptophan fluorescence intensi ty (F-I) of apo-CR reversibly increased about 3-fold upon addition of calcium, indicating a change of conformation. The FI of fragments I-II (Trp 25) and I-III (Trp 25 and 116) increased about 1.4-fold on calci um binding, but that of fragment III-VI (Trp 116) increased 3.5-fold. Calcium titration of CR monitored by Trp fluorescence intensity showed that recombinant CR and some fragments bound Ca2+ with high affinity (K-d below 0.4 mu M) and with high cooperativity. An apparent Kill coe fficient for Ca2+-induced fluorescence changes in CR was about 3.7. CR bound to organomercurial-agarose independent of Ca2+ concentrations a nd could be eluted with 2-mercaptoethanol or DTT, indicating that Cys 101 and 266 did not form cystine. The fluorescence intensities of cyst eine-linked fluorescent probes 5-iodoacetamidofluorescein and N-(1-pyr eneiodoacetamide) were increased approximately 1.3-fold upon calcium b inding by CR. These data indicate that CR binds Ca2+ with high affinit y and cooperativity and that this binding induces a change of conforma tion that involves the interaction of different parts of the molecule. Taken together, our results suggest that CR works as an on/off switch within a narrow range of free Ca2+ by interacting with as yet unident ified targets.