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 motifs of rat calretinin (CR) (I-II, I-III, III-VI, IV-VI), were prepared, and their Ca2+ -induced conformational changes were compared with those of full-length recombinant CR. All fragments were able to bind calcium ions as shown by 45Ca2+ overlay method on nitrocellulose and fluorescence measurements. The intrinsic tryptophan fluorescence intensity (FI) 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 calcium 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 (Kd below 0.4 microM) and with high cooperativity. An apparent Hill coefficient for Ca2+ -induced fluorescence changes in CR was about 3.7. CR bound to organomercurial-agarose Cys 101 and 266 did not form cystine. The fluorescence intensities of cysteine-linked fluorescent probes 5-iodoacetamidofluorescein and N-(1-pyreneiodoacetamide) were increased approximately 1.3-fold upon calcium binding by CR. These data indicate that CR binds Ca2+ with high affinity and cooperativity and that this binding induces a change of conformation 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 unidentified targets.
D000818:Animals; D001483:Base Sequence; D064032:Calbindin 2; D002118:Calcium / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D064030:S100 Calcium Binding Protein G / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
C576512:Calb2 protein, rat; D064032:Calbindin 2; D010446:Peptide Fragments; D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G; D002118:Calcium