Holo- and apocytochrome P450cam were studied by differential scanning calorimetry (DSC), limited proteolysis, second-derivative spectroscopy, circular dichroism, and size-exclusion chromatography. The holoprotein shows three folding units (domains) in DSC. The prosthetic group is related to the most unstable domain, which has a thermal transition at 41.9 degrees C. Compared with the holoprotein, apocytochrome P450cam has a reduced helix content. The protein is compact as judged by the Stokes radius and is still able to undergo a two-state transition. However, the enthalpy change at thermal melting is reduced from 980 kJ/mol for the holoprotein to 135 kJ/mol for the apo form. Parts of the molecule have a destabilized tertiary structure. This is indicated by second-derivative spectroscopy, circular dichroism in the near-ultraviolet region, and a high susceptibility to proteolytic digestion. Apocytochrome P450cam is considered a native protein with the extremely low stability of delta G = 7.5 kJ/mol, thus showing at the same time intermediate-like properties. The importance of the properties for in vivo folding are discussed. Study holds ProTherm entries: 4549 Extra Details: KCN(1 mM) was added in the experiment folding units; helix content; Stokes radius; susceptibility;,tertiary structure
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:26 p.m.
|Number of data points||1|
|Proteins||Camphor 5-monooxygenase ; Camphor 5-monooxygenase|
|Assays/Quantities/Protocols||Experimental Assay: dG|
|Libraries||Mutations for sequence TTETIQSNANLAPLPPHVPEHLVFDFDMYNPSNLSAGVQEAWAVLQESNVPDLVWTRCNGGHWIATRGQLIREAYEDYRHFSSECPFIPREAGEAYDFIPTSMDPPEQRQFRALANQVVGMPVVDKLENRIQELACSLIESLRPQGQCNFTEDYAEPFPIRIFMLLAGLPEEDIPHLKYLTDQMTRPDGSMTFAEAKEALYDYLIPIIEQRRQKPGTDAISIVANGQVNGRPITSDEAKRMCGLLLVGGLDTVVNFLSFSMEFLAKSPEHRQELIERPERIPAACEELLRRFSLVADGRILTSDYEFHGVQLKKGDQILLPQMLSGLDERENACPMHVDFSRQKVSHTTFGHGSHLCLGQHLARREIIVTLKEWLTRIPDFSIAPGAQIQHKSGIVSGVQALPLVWDPATTKAV|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|