title: Probing the role of the F-helix in serpin stability through a single tryptophan substitution.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, P.O. Box 13D, Victoria 3800, Australia.
authors.forename: Lisa D
authors.initials: LD
authors.lastname: Cabrita
authors.forename: James C
authors.initials: JC
authors.lastname: Whisstock
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Bottomley
mesh_terms: D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004355:Drug Stability; D019791:Guanidine; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D015843:Serpins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan; D000515:alpha 1-Antitrypsin / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D015843:Serpins; D000515:alpha 1-Antitrypsin; D014364:Tryptophan; D019791:Guanidine; D000409:Alanine