Protease digestion studies of an equilibrium intermediate in the unfolding of creatine kinase.
Protease digestion experiments have been used to characterize the structure of an equilibrium intermediate in the unfolding of creatine kinase (CK) by low concentrations (0.625 M) of guanidine hydrochloride (GdnHCl). Eighteen of the major products of digestion by trypsin, chymotrypsin and endoproteinase Glu-C have been identified by microsequencing after separation by SDS/PAGE and electroblotting on poly(vinylidene difluoride) membranes. The C-terminal portion (Gly215 to Lys380) was much more resistant to digestion than the N-terminal portion (Pro1 to Gly133), although the area most sensitive to proteolysis was in the middle of the CK sequence (Arg134 to Arg214). These experiments are consistent with the two-domain model for the CK monomer. The structure of the intermediate is proposed to consist of a folded C-terminal domain and a partly folded N-terminal domain separated by an unfolded central linker. Protease susceptibility is clustered within two N-terminal regions and one central region. These regions are evidently exposed as a result of the partial unfolding and/or separation of the N-terminal domain. Further evidence for the structure of this intermediate comes from gel filtration studies. Treatment of CK with 0.625 M GdnHCl resulted in slow aggregation at 37 degrees C, but not at 12 degrees C, a phenomenon previously reported for phosphoglycerate kinase. The aggregation did not occur at higher GdnHCl concentrations and was unaffected by a reducing agent. It is proposed that aggregation is a consequence of non-specific interactions between hydrophobic regions, possibly domain/domain interfaces, which become exposed in the intermediate.
Webb
T
T
MRIC Biotechnology Group, N.E. Wales Institute, Plas Coch, Wrexham, U.K.
Jackson
P J
PJ
Morris
G E
GE
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D002918:Chymotrypsin / Q000378:metabolism; D003402:Creatine Kinase / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D007527:Isoenzymes; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011487:Protein Conformation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary
D007527:Isoenzymes; D003402:Creatine Kinase; D010450:Endopeptidases; D002918:Chymotrypsin