The multidomain structure of ceruloplasmin from calorimetric and limited proteolysis studies.
Differential scanning calorimetry has been used to investigate the thermal stability of three different ceruloplasmins (from sheep, chicken, and turtle) in their native state and after limited proteolysis. The three undegraded proteins showed a similar structural organization in three calorimetric domains, although their temperature of unfolding varied from 57.8 degrees C (turtle) to 71.2 degrees C (sheep) to 82.1 degrees C (chicken). The spectroscopic and the catalytic properties were totally lost at temperatures corresponding to the unfolding of the less thermostable domain in the case of sheep and chicken ceruloplasmins and to the unfolding of the most thermostable domain in the turtle protein. Trypsin, but not plasmin, digestion caused a significant decrease of the thermal stability of sheep and chicken ceruloplasmins. Turtle ceruloplasmin was insensitive to both proteases. Comparing the thermodynamic parameters of the sheep protein in its undegraded and cleaved states revealed a mismatch between the three calorimetric domains and the 3-fold internal replication of the primary structure, which is evident in the highly homologous, fully sequenced human protein. Copper removal caused the rearrangement of the molecule in only two calorimetric domains, suggesting a role of the metal atoms in organizing a new calorimetric domain, which was tentatively assigned to the less thermostable cooperative unit of the native protein.
Bonaccorsi di Patti
M C
MC
Department of Biochemical Sciences, University of Rome, La Sapienza, Italy.
Musci
G
G
Giartosio
A
A
D'Alessio
S
S
Calabrese
L
L
D000818:Animals; D002152:Calorimetry, Differential Scanning; D002570:Ceruloplasmin / Q000737:chemistry* / Q000302:isolation & purification; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D011489:Protein Denaturation; D012756:Sheep; D014357:Trypsin; D014426:Turtles
D010446:Peptide Fragments; D002570:Ceruloplasmin; D014357:Trypsin