This study presents an experimental approach, based on the change of Trp fluorescence between native and denatured states of proteins, which permits to monitor unfolding equilibria and the thermodynamic stability (DeltaG degrees ) of these macromolecules in frozen aqueous solutions. The results obtained by guanidinium chloride denaturation of the azurin mutant C112S from Pseudomonas aeruginosa, in the temperature range from -8 to -16 degrees C, demonstrate that the stability of the native fold may be significantly perturbed in ice depending mainly on the size of the liquid water pool (V(L)) in equilibrium with the solid phase. The data establish a threshold, around V(L)=1.5%, below which in ice DeltaG degrees decreases progressively relative to liquid state, up to 3 kcal/mole for V(L)=0.285%. The sharp dependence of DeltaG degrees on V(L) is consistent with a mechanism based on adsorption of the protein to the ice surface. The reduction in DeltaG degrees is accompanied by a corresponding decrease in m-value indicating that protein-ice interactions increase the solvent accessible surface area of the native fold or reduce that of the denatured state, or both. The method opens the possibility for examining in a more quantitative fashion the influence of various experimental conditions on the ice perturbation and in particular to test the effectiveness of numerous additives used in formulations to preserve labile pharmaco proteins. Study holds ProTherm entries: 23590, 23591, 23592, 23593, 23594, 23595, 23596, 23597, 23598, 23599, 23600, 23601, 23602, 23603 Extra Details: Ice, VL (size of liquid water shrinks) is 0.285% liquid water pool; adsorption; protein-ice interactions; solvent accessible surface area
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:54 p.m.
|Number of data points||42|
|Proteins||Azurin ; Azurin|
|Assays/Quantities/Protocols||Experimental Assay: Cm temp:20.0 C ; Experimental Assay: m temp:20.0 C ; Experimental Assay: dG_H2O temp:20.0 C ; Experimental Assay: Cm temp:-16.0 C ; Experimental Assay: m temp:-16.0 C ; Experimental Assay: dG_H2O temp:-16.0 C ; Experimental Assay: Cm temp:-12.0 C ; Experimental Assay: m temp:-12.0 C ; Experimental Assay: dG_H2O temp:-12.0 C ; Experimental Assay: Cm temp:-10.0 C ; Experimental Assay: m temp:-10.0 C ; Experimental Assay: dG_H2O temp:-10.0 C ; Experimental Assay: Cm temp:-8.0 C ; Experimental Assay: m temp:-8.0 C ; Experimental Assay: dG_H2O temp:-8.0 C ; Experimental Assay: Cm temp:-14.0 C ; Experimental Assay: m temp:-14.0 C ; Experimental Assay: dG_H2O temp:-14.0 C|
|Libraries||Mutations for sequence AECSVDIQGNDQMQFNTNAITVDKSCKQFTVNLSHPGNLPKNVMGHNWVLSTAADMQGVVTDGMASGLDKDYLKPDDSRVIAHTKLIGSGEKDSVTFDVSKLKEGEQYMFFCTFPGHSALMKGTLTLK|