Thermal stability and folding of type IV procollagen and effect of peptidyl-prolyl cis-trans-isomerase on the folding of the triple helix.
Intact, monomeric type IV procollagen was isolated from the medium of PF-HR9 cells. Its stability was measured by optical rotatory dispersion, differential scanning calorimetry, and trypsin susceptibility of the partially unfolded molecules. At neutral pH, a complex transition between 35 and 42 degrees C and a smaller transition at 48 degrees C are observed by optical rotatory dispersion, using a heating rate of 10 degrees C/h. Reduction of the heating rate to 1.6 degrees C/h resulted in a 1 degree C lowering of the apparent melting temperatures. A similar curve is observed in 10 mM acetic acid, with transitions about 2 degrees C lower. Differential scanning calorimetry revealed transitions at 36.0, 42.1, and 48.0 degrees C at neutral pH, with a total transition enthalpy of 17.1 kJ/mol tripeptide units. In 10 mM acetic acid, transitions at 35.6, 38.9, 41.7, and 50.0 degrees C are observed. The transition enthalpy is 16.4 kJ/mol tripeptide units. The transition enthalpy is similar to values found for interstitial collagens. Results from trypsin digestion experiments are consistent with the stability found by optical methods and calorimetry. The rate and completeness of refolding after melting were measured. In neutral buffer, the initial rate was found to be 0.041 min-1, faster than the refolding rates observed with types pN III and III collagen. Peptidyl prolyl cis-trans-isomerase increased the refolding rate to 0.083 min-1, indicating that cis-trans-isomerization is the rate-limiting step, despite the interruptions in the triple helix. Trypsin digestion experiments indicated that the refolding mechanism is similar in the presence and absence of the enzyme. Refolding was nearly complete in neutral buffer. In 10 mM acetic acid, folding was considerably slower and went to about 74% completion. In both solvents, the refolded material was only slightly less stable than the native material. Electron microscopy of partially refolded samples showed that most refolding started at the COOH terminus, but some was initiated at other sites.
Davis
J M
JM
Research Unit, Shriners Hospital for Crippled Children, Portland, Oregon 97201.
Boswell
B A
BA
Bächinger
H P
HP
D000591:Amino Acid Isomerases / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D002460:Cell Line; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D007700:Kinetics; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D010449:Peptide Mapping; D019696:Peptidylprolyl Isomerase; D011347:Procollagen / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D011487:Protein Conformation; D014357:Trypsin
D046911:Macromolecular Substances; D011347:Procollagen; D014357:Trypsin; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase