Thermal stability of the pore-forming domain of colicin A was studied by high sensitivity differential scanning calorimetry and circular dichroism spectroscopy. In the pH range between 8 and 5, the thermal denaturation of the protein in solution occurs at 66-69 degrees C and is characterized by the calorimetric enthalpy of approximately 90 kcal/M. At pH below 5, there is a rapid pH-dependent destabilization of the pore-forming domain resulting in the lowering of the midpoint denaturation temperature and a decrease in the calorimetric enthalpy of denaturation. Circular dichroism spectra in the near and far ultraviolet show that the thermotropic transition is associated with collapse of the native tertiary structure of the pore-forming domain, although a large proportion of the helical secondary structure remains preserved. The present data indicate some similarity also between acid-induced and temperature-induced denaturation of the pore-forming domain of colicin A. Association of the pore-forming domain with phospholipid vesicles of dioleoylphosphatidylglycerol results in total disappearance of the calorimetric transition, even at pH values as high as 7. Since lipid binding also induces collapse of the near ultraviolet circular dichroism spectrum, these data indicate that interaction with the membrane facilitates a conformational change within the pore-forming domain to a looser (denaturated-like) state. These findings are discussed in relation to the recent model (van der Goot, F. G., Gonzalez-Manas, J. M., Lakey, J. H., Pattus, F. (1991) Nature 354, 408-410) which postulates that a flexible "molten globule" state is an intermediate on the pathway to membrane insertion of colicin A. Study holds ProTherm entries: 5210, 5211, 5212, 5213, 5214, 5215 Extra Details:
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:29 p.m.
|Number of data points||6|
|Proteins||Colicin-A ; Colicin-A|
|Assays/Quantities/Protocols||Experimental Assay: Tm pH:8.0 ; Experimental Assay: Tm pH:7.0 ; Experimental Assay: Tm pH:5.0 ; Experimental Assay: Tm pH:4.0 ; Experimental Assay: Tm pH:3.5 ; Experimental Assay: Tm pH:3.0|
|Libraries||Mutations for sequence VAEKAKDERELLEKTSELIAGMGDKIGEHLGDKYKAIAKDIADNIKNFQGKTIRSFDDAMASLNKITANPAMKINKADRDALVNAWKHVDAQDMANKLGNLSKAFKVADVVMKVEKVREKSIEGYETGNWGPLMLEVESWVLSGIASSVALGIFSATLGAYALSLGVPAIAVGIAGILLAAVVGALIDDKFADALNNEIIRPAH|