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
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