Calreticulin (CRT) is a soluble chaperone involved in the conformational maturation of glycoproteins in the endoplasmic reticulum. Using biochemical and biophysical techniques including circular dichroism, proteolysis, and analytical ultracentrifugation, we have determined the effects of calcium and zinc ions on the structural properties of human CRT. Circular dichroism analysis has shown that the binding of calcium and zinc ions to CRT induces no significant changes in the secondary structure of the protein but affects in very distinct ways the local tertiary packing of these elements. More specifically, these studies have revealed that CRT adopts a more rigid and thermally stable structure upon binding calcium ions and a more loosely packed and thermally destabilized structure upon binding zinc ions. Consistent with these results, proteolysis experiments demonstrated that the intrinsic conformational flexibility of CRT can be modulated toward either a decrease or an increase in susceptibility to cleavage by chymotrypsin upon binding calcium or zinc ions, respectively. Results from sedimentation analysis indicated that the global three-dimensional structure of CRT is essentially unchanged upon binding calcium ions. In marked contrast, CRT self-associates reversibly to form dimers upon binding zinc ions. Collectively, our results provide evidence that calcium and zinc ions induce strikingly different changes in the biochemical and structural properties of CRT. Study holds ProTherm entries: 11764 Extra Details: additive : EDTA(2 mM), chaperone; structural properties; secondary structure; zinc ions
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:43 p.m.
|Number of data points||1|
|Proteins||Calreticulin ; Calnexin|
|Assays/Quantities/Protocols||Experimental Assay: Tm|
|Libraries||Mutations for sequence SKSKPDTSAPTSPKVTYKAPVPSGEVYFADSFDRGTLSGWILSKAKKDDTDDEIAKYDGKWEVDEMKETKLPGDKGLVLMSRAKHHAISAKLNKPFLFDTKPLIVQYEVNFQNGIECGGAYVKLLSKTPELNLDQFHDKTPYTIMFGPDKCGEDYKLHFIFRHKNPKTGVYEEKHAKRPDADLKTYFTDKKTHLYTLILNPDNSFEILVDQSIVNSGNLLNDMTPPVNPSREIEDPEDQKPEDWDERPKIPDPDAVKPDDWNEDAPAKIPDEEATKPDGWLDDEPEYVPDPDAEKPEDWDEDMDGEWEAPQIANPKCESAPGCGVWQRPMIDNPNYKGKWKPPMIDNPNYQGIWKPRKIPNPDFFEDLEPFKMTPFSAIGLELWSMTSDIFFDNFIVCGDRRVVDDWANDGWGLKKAADGAAEP|