The activation domain of human procarboxypeptidase A2 (ADA2h), a globular open-sandwich alpha + beta domain with 80 residues and no disulfide bridges, has been studied by thermodynamic and kinetic analysis. Equilibrium denaturation by urea or temperature is fully reversible at pH 7.0 and fits to a two-state transition. The Gibbs energy of unfolding extrapolated to null concentration of chemical denaturant, delta GH2O, at pH 7.0 and 298 K, is calculated to be 17.0 +/- 1 kJ mol-1, which is within experimental error of the value determined by differential scanning calorimetry, 15.1 +/- 2 kJ mol-1. Kinetics of unfolding and refolding followed by fluorescence do not show the presence of any kinetic intermediate accumulating in the folding reaction. A value for delta GH2O of 17.9 +/- 0.7 kJ mol-1 can be extrapolated from the kinetic data. All these data indicate that the folding pathway of this domain is consistent with a two-state model (with the exception of the cis-Pro intermediates). More importantly, the analysis of this and several other small domains or proteins supports the hypothesis that stable kinetic folding intermediates are not necessary for a protein to fold. There seems to be a relationship between the size of a protein and the presence of stable kinetic intermediates. Globular proteins with less than 80 residues and no disulfide bonds follow a two-state transition, while proteins larger than 100 residues present stable kinetic folding intermediates. Study holds ProTherm entries: 4928 Extra Details: open-sandwich alpha + beta domain; kinetic intermediate;,folding reaction; two-state model
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:27 p.m.
|Number of data points||2|
|Proteins||Carboxypeptidase A2 ; Carboxypeptidase A2|
|Assays/Quantities/Protocols||Experimental Assay: m ; Experimental Assay: dG_H2O|
|Libraries||Mutations for sequence LETFVGDQVLEIVPSNEEQIKNLLQLEAQEHLQLDFWKSPTTPGETAHVRVPFVNVQAVKVFLESQGIAYSIMIEDVQVLLDKENEEMLFNRRRERSGNFNFGAYHTLEEISQEMDNLVAEHPGLVSKVNIGSSFENRPMNVLKFSTGGDKPAIWLDAGIHAREWVTQATALWTANKIVSDYGKDPSITSILDALDIFLLPVTNPDGYVFSQTKNRMWRKTRSKVSGSLCVGVDPNRNWDAGFGGPGASSNPCSDSYHGPSANSEVEVKSIVDFIKSHGKVKAFIILHSYSQLLMFPYGYKCTKLDDFDELSEVAQKAAQSLRSLHGTKYKVGPICSVIYQASGGSIDWSYDYGIKYSFAFELRDTGRYGFLLPARQILPTAEETWLGLKAIMEHVRDHPY|