Probing the role of the F-helix in serpin stability through a single tryptophan substitution

Biochemistry
Lisa D CabritaStephen P Bottomley

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 functio...Continue Reading

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Citations

Dec 4, 2002·Journal of Molecular Biology·Glyn L DevlinStephen P Bottomley
Dec 25, 2002·Journal of Molecular Biology·Dimitri GilisStephen P Bottomley
Jul 25, 2009·Biochemistry·Tanusree SenguptaPatrick L Wintrode
Jan 25, 2011·Nature Structural & Molecular Biology·Beena Krishnan, Lila M Gierasch
Sep 29, 2011·Journal of Molecular Biology·Anja S Knaupp, Stephen P Bottomley
Mar 31, 2004·Photochemical & Photobiological Sciences : Official Journal of the European Photochemistry Association and the European Society for Photobiology·Kirsten LotteAndreas Brockhinke
Nov 3, 2006·Current Opinion in Structural Biology·James C Whisstock, Stephen P Bottomley
Jul 31, 2007·Protein Science : a Publication of the Protein Society·Je-Hyun BaekMyeong-Hee Yu
Oct 12, 2004·Biochemical and Biophysical Research Communications·Mary C PearceStephen P Bottomley
Apr 7, 2005·Journal of Molecular Biology·Nobuyuki TakahashiMasaaki Hirose
Jul 19, 2011·International Journal of Mass Spectrometry·Anindya Sarkar, Patrick L Wintrode
Aug 10, 2010·Journal of Molecular Biology·Sayaka TakeharaMaki Onda
Feb 9, 2012·Journal of Amino Acids·Mohammad Sazzad KhanMohamad Aman Jairajpuri
Feb 3, 2018·Scientific Reports·Li YangStephen P Bottomley
Sep 27, 2016·Scientific Reports·Benjamin T PorebskiAshley M Buckle
Jul 20, 2007·The Journal of Biological Chemistry·Lisa D CabritaStephen P Bottomley
Apr 16, 2003·European Journal of Biochemistry·Troels WindPeter A Andreasen
Mar 9, 2019·Scientific Reports·Emilia M MarijanovicAshley M Buckle
Apr 9, 2008·The Journal of Biological Chemistry·Maki OndaMasaaki Hirose
Jul 30, 2016·The Biochemical Journal·Peter G W Gettins, Steven T Olson

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