Probing the equilibrium denaturation of the serpin alpha(1)-antitrypsin with single tryptophan mutants; evidence for structure in the urea unfolded state.


Abstract

The native conformation of proteins in the serpin superfamily is metastable. In order to understand why serpins attain the native state instead of more stable conformations we have begun investigations into the equilibrium-unfolding of alpha(1)-antitrypsin. alpha(1)-Antitrypsin contains two tryptophan residues, Trp194 and Trp238, situated on the A and B beta-sheets, respectively. Site-directed mutagenesis was used to construct two single-tryptophan variants. Both variants were fully active and had similar secondary structure and stabilities to alpha(1)-antitrypsin. The denaturation of alpha(1)-antitrypsin and its variants was extremely similar when followed by far-UV CD, indicating the presence of a single intermediate. Fluorescence analysis of the unfolding behavior of each single tryptophan variant indicated that the sole tryptophan residue reported the structural changes within its immediate environment. These data suggest that the A beta-sheet is expanded in the intermediate state whilst no structural change around the B beta-sheet has occurred. In the urea-induced unfolded state, Trp238 does not become fully solvated, suggesting the persistence of structure around this residue. The implications of these data on the folding, misfolding and function of the serpin superfamily are discussed. Study holds ProTherm entries: 12708, 12709, 12710, 12711, 12712, 12713, 12714, 12715, 12716, 12717, 12718, 12719, 12720, 12721, 12722, 12723, 12724 Extra Details: proteinase inhibitor; protein folding; conformational disease;,residual structure; fluorescence spectroscopy

Submission Details

ID: t3FWtuqB4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:44 p.m.

Version: 1

Publication Details
Tew DJ;Bottomley SP,J. Mol. Biol. (2001) Probing the equilibrium denaturation of the serpin alpha(1)-antitrypsin with single tryptophan mutants; evidence for structure in the urea unfolded state. PMID:11700071
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
3NDD 2010-07-28 1.5 Cleaved antitrypsin with P10 Pro, and P9-P6 Asp
5NBU 2018-03-21 1.67 Crystal structure of native alpha-1-antitrypsin with seven stabilising mutations
5NBV 2018-03-21 1.73 Crystal structure of native alpha-1-antitrypsin with seven stabilising mutations
3NE4 2011-12-14 1.81 1.8 Angstrom structure of intact native wild-type alpha-1-antitrypsin
4PYW 2015-06-10 1.91 1.92 angstrom crystal structure of A1AT:TTAI ternary complex
2QUG 2008-08-12 2.0 Crystal structure of alpha-1-antitrypsin, crystal form A
1QLP 1999-09-27 2.0 2.0 ANGSTROM STRUCTURE OF INTACT ALPHA-1-ANTITRYPSIN: A CANONICAL TEMPLATE FOR ACTIVE SERPINS
1HP7 2001-03-14 2.1 A 2.1 ANGSTROM STRUCTURE OF AN UNCLEAVED ALPHA-1-ANTITRYPSIN SHOWS VARIABILITY OF THE REACTIVE CENTER AND OTHER LOOPS
1IZ2 2003-02-11 2.2 Interactions causing the kinetic trap in serpin protein folding
3DRM 2009-03-31 2.2 2.2 Angstrom Crystal Structure of Thr114Phe Alpha1-Antitrypsin
1OPH 2003-08-05 2.3 NON-COVALENT COMPLEX BETWEEN ALPHA-1-PI-PITTSBURGH AND S195A TRYPSIN
3CWL 2008-09-23 2.44 Crystal structure of alpha-1-antitrypsin, crystal form B
3CWM 2008-09-23 2.51 Crystal structure of alpha-1-antitrypsin complexed with citrate
1EZX 2000-10-25 2.6 CRYSTAL STRUCTURE OF A SERPIN:PROTEASE COMPLEX
1QMB 2000-02-06 2.6 Cleaved alpha-1-antitrypsin polymer
1OO8 2003-08-05 2.65 CRYSTAL STRUCTURE OF A1PI-PITTSBURGH IN THE NATIVE CONFORMATION
3NDF 2010-07-28 2.7 Cleaved antitrypsin with P8-P6 Asp
1ATU 1997-08-20 2.7 UNCLEAVED ALPHA-1-ANTITRYPSIN
1PSI 1996-12-07 2.92 Intact recombined alpha1-antitrypsin mutant PHE 51 to LEU
7API 1990-10-15 3.0 THE S VARIANT OF HUMAN ALPHA1-ANTITRYPSIN, STRUCTURE AND IMPLICATIONS FOR FUNCTION AND METABOLISM
9API 1990-10-15 3.0 THE S VARIANT OF HUMAN ALPHA1-ANTITRYPSIN, STRUCTURE AND IMPLICATIONS FOR FUNCTION AND METABOLISM
1D5S 2000-04-02 3.0 CRYSTAL STRUCTURE OF CLEAVED ANTITRYPSIN POLYMER
8API 1990-10-15 3.1 THE S VARIANT OF HUMAN ALPHA1-ANTITRYPSIN, STRUCTURE AND IMPLICATIONS FOR FUNCTION AND METABOLISM
3DRU 2009-03-31 3.2 Crystal Structure of Gly117Phe Alpha1-Antitrypsin
2D26 2005-11-29 3.3 Active site distortion is sufficient for proteinase inhibit second crystal structure of covalent serpin-proteinase complex
5IO1 2016-06-08 3.34 CRYSTAL STRUCTURE OF RECOMBINANT HUMAN Z ALPHA-1-ANTITRYPSIN
1KCT 1997-01-11 3.46 ALPHA1-ANTITRYPSIN
3T1P 2011-08-17 3.9 Crystal structure of an alpha-1-antitrypsin trimer

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