Serpins form loop-sheet polymers through the formation of a partially folded intermediate. Through mutagenesis and biophysical analysis, we have probed the conformational stability of the F-helix, demonstrating that it is almost completely unfolded in the intermediate state. The replacement of Tyr160 on the F-helix of alpha1-antitrypsin to alanine results in the loss of a conserved hydrogen bond that dramatically reduces the stability of the protein to both heat and solvent denaturation, indicating the importance of Tyr160 in the stability of the molecule. The mutation of Tyr160 to a tryptophan residue, within a fluorescently silent variant of alpha1-antitrypsin, results in a fully active, stable serpin. Fluorescence analysis of the equilibrium unfolding behavior of this variant indicates that the F-helix is highly disrupted in the intermediate conformation. Iodide quenching experiments demonstrate that the tryptophan residue is exposed to a similar extent in both the intermediate and unfolded states. Cumulatively, these data indicate that the F-helix plays an important role in controlling the early conformational changes involved in alpha1-antitrypsin unfolding. The implications of these data on both alpha1-antitrypsin function and misfolding are discussed. Study holds ProTherm entries: 14925, 14926, 14927, 14928, 14929, 14930, 14931, 14932, 14933, 14934, 14935, 14936, 14937, 14938, 14939, 14940, 14941, 14942, 14943 Extra Details: partially folded intermediate; conformational stability; hydrogen bond; tryptophan
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:45 p.m.
|Number of data points||51|
|Proteins||Alpha-1-antitrypsin ; Alpha-1-antitrypsin|
|Assays/Quantities/Protocols||Experimental Assay: Cm ionic:NaCl: 50 mM ; Experimental Assay: m ionic:NaCl: 50 mM ; Experimental Assay: dG_H2O ionic:NaCl: 50 mM ; Experimental Assay: Cm ionic:KI,Na2S2O3: 2 M,1 mM ; Experimental Assay: m ionic:KI,Na2S2O3: 2 M,1 mM ; Experimental Assay: dG_H2O ionic:KI,Na2S2O3: 2 M,1 mM ; Experimental Assay: Tm|
|Libraries||Mutations for sequence MDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK|