The structure and stability of apocytochrome b562 were explored using absorption and circular dichroism spectroscopic methods. The polypeptide chain retains a well-defined structure when the prosthetic heme group is removed from cytochrome b562. Circular dichroism measurements estimate 60% helicity for apocytochrome b562, compared with 80% helicity found in holocytochrome b562. At low pH, apocytochrome b562 displays a midpoint pH of 2.9, while ferricytochrome b562 displays a midpoint pH of 2.3. The unfolding of the apoprotein by urea and heat can be well approximated by the two-state transition model. The stability of apocytochrome b562 is significantly reduced from that of the holoprotein. The free energy of stabilization (delta G degrees) and the midpoint transition temperature (Tm) for apocytochrome b562 are found to be 3.2 +/- 0.5 kcal/mol and 52.3 +/- 0.9 degrees C, respectively, compared with 6.6 +/- 0.5 kcal/mol and 67.2 +/- 0.5 degrees C for ferricytochrome b562. The smaller heat capacity change upon unfolding of apocytochrome b562 than that of ferricytochrome b562, estimated from the thermodynamic parameters, indicates that apocytochrome b562 possesses a smaller hydrophobic core than holocytochrome b562. Size-exclusion chromatography studies indicate that the apoprotein is slightly more extended in molecular dimension than ferricytochrome b562. The data suggest that apocytochrome b562 resembles a "molten globule" or a "collapsed form" of the holoprotein, in which secondary structure formation is largely complete while the global folding is either only partially complete or dynamically expanded. Study holds ProTherm entries: 3958, 3959, 3960, 3961 Extra Details: hydrophobic core; molten globule;collapsed form;,partially complete
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:23 p.m.
|Number of data points||8|
|Proteins||Soluble cytochrome b562 ; Soluble cytochrome b562|
|Assays/Quantities/Protocols||Experimental Assay: dG_H2O ; Experimental Assay: dCp ; Experimental Assay: Tm ; Experimental Assay: dHvH|
|Libraries||Mutations for sequence ADLEDNMETLNDNLKVIEKADNAAQVKDALTKMRAAALDAQKATPPKLEDKSPDSPEMKDFRHGFDILVGQIDDALKLANEGKVKEAQAAAEQLKTTRNAYHQKYR|