Localization of Ca(2+)-dependent conformational changes of calretinin by limited tryptic proteolysis.
Calretinin is an EF-hand Ca(2+)-binding protein expressed predominantly in some neurons. We have found that the tryptic digestion pattern of rat recombinant calretinin depends on Ca2+ concentration as determined by SDS/PAGE, amino-acid-sequence analysis and electrospray-ionization MS. Ca(2+)-saturated calretinin was cleaved between amino acids 60 and 61 to yield two fragments, which accumulated during cleavage. Small amounts of the larger fragment (amino acid residues 61-271) were further cleaved from the C-terminal end. Ca(2+)-free calretinin was also cleaved between residues 60 and 61; however, under the latter conditions the fragment 61-271 was further cleaved from the N-terminal end. Native rat calretinin was cleaved by trypsin in a similar Ca(2+)-dependent fashion. All identified fragments of recombinant calretinin bound 45Ca2+ on nitrocellulose filters, although to a different extent. The 61-271 fragment was released by EGTA from an octyl-agarose column in a manner similar to intact calretinin, while fragment 61-233 was not eluted by EGTA. These observations show that there are trypsin cleavage sites in calretinin that are available regardless of Ca2+ binding, other sites that are completely protected against trypsin on Ca(2+)-binding and sites which become partially available on Ca(2+)-binding. Together these data show that calretinin changes its conformation on Ca2+ binding and identify the regions which are exposed in apo and Ca(2+)-bound form.
Kuźnicki
J
J
Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, MD 20892, USA.
Wang
T L
TL
Martin
B M
BM
Winsky
L
L
Jacobowitz
D M
DM
D000818:Animals; D064032:Calbindin 2; D002118:Calcium / Q000737:chemistry*; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G / Q000737:chemistry / Q000648:ultrastructure*; D012995:Solubility; D014357:Trypsin / Q000494:pharmacology
C576512:Calb2 protein, rat; D064032:Calbindin 2; D010446:Peptide Fragments; D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G; D014357:Trypsin; D002118:Calcium