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. 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

Submission Details

ID: 3jfJvPFC4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:45 p.m.

Version: 1

Publication Details
Cabrita LD;Whisstock JC;Bottomley SP,Biochemistry (2002) Probing the role of the F-helix in serpin stability through a single tryptophan substitution. PMID:11926819
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1ATU 1997-05-11T00:00:00+0000 2.7 UNCLEAVED ALPHA-1-ANTITRYPSIN
1D5S 1999-10-11T00:00:00+0000 3.0 CRYSTAL STRUCTURE OF CLEAVED ANTITRYPSIN POLYMER
1D5S 1999-10-11T00:00:00+0000 3.0 CRYSTAL STRUCTURE OF CLEAVED ANTITRYPSIN POLYMER
1EZX 2000-05-12T00:00:00+0000 2.6 CRYSTAL STRUCTURE OF A SERPIN:PROTEASE COMPLEX
1EZX 2000-05-12T00:00:00+0000 2.6 CRYSTAL STRUCTURE OF A SERPIN:PROTEASE COMPLEX
1HP7 2000-12-12T00:00:00+0000 2.1 A 2.1 ANGSTROM STRUCTURE OF AN UNCLEAVED ALPHA-1-ANTITRYPSIN SHOWS VARIABILITY OF THE REACTIVE CENTER AND OTHER LOOPS
1IZ2 2002-09-19T00:00:00+0000 2.2 Interactions causing the kinetic trap in serpin protein folding
1KCT 1996-08-06T00:00:00+0000 3.46 ALPHA1-ANTITRYPSIN
1OO8 2003-03-03T00:00:00+0000 2.65 CRYSTAL STRUCTURE OF A1PI-PITTSBURGH IN THE NATIVE CONFORMATION
1OPH 2003-03-05T00:00:00+0000 2.3 NON-COVALENT COMPLEX BETWEEN ALPHA-1-PI-PITTSBURGH AND S195A TRYPSIN

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
93.1 Alpha-1-antitrypsin O00394 A1AT_CHLAE
92.4 Alpha-1-antitrypsin P01010 A1AT_PAPAN
96.4 Alpha-1-antitrypsin Q5RCW5 A1AT_PONAB
100.0 Alpha-1-antitrypsin P01009 A1AT_HUMAN