Single amino acid substitutions of alpha 1-antitrypsin that confer enhancement in thermal stability.


Abstract

A recombinant alpha 1-antitrypsin variant which increased thermal stability was obtained from random mutagenesis followed by screening. The clone was identified as having a single mutation of Phe51-->Cys. Heat deactivation of purified recombinant alpha 1-antitrypsin produced in Escherichia coli revealed that the mutation slowed down the deactivation rate 10-fold at 57 degrees C, increasing thermal stability of recombinant protein to almost that of natural glycosylated plasma form. The mutant protein also exhibited increased stability against denaturant. The urea-induced unfolding monitored by the changes in fluorescence intensity at 360 nm showed that the mutation shifted midpoint of the transition from 1.9 M to 2.8 M. The mutation site is particularly interesting in that some genetic variants mapped at adjacent positions were shown previously to cause aggregation of the polypeptides, while the Phe51-->Cys mutation decreased aggregation rate significantly during heat deactivation. The association rate constant with porcine pancreatic elastase revealed that the mutation did not affect inhibitory activity significantly. The site identified may be critical for regulating stability of alpha 1-antitrypsin. Characterization of various single amino acid substitutions at position 51 suggests that volume and flexibility of hydrophobic side chain at the site are critical factors for enhancing the stability of alpha 1-antitrypsin. Study holds ProTherm entries: 5142, 5143 Extra Details: additive : EDTA(1 mM),

Submission Details

ID: 3wHRdYsW4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:28 p.m.

Version: 1

Publication Details
Kwon KS;Kim J;Shin HS;Yu MH,J. Biol. Chem. (1994) Single amino acid substitutions of alpha 1-antitrypsin that confer enhancement in thermal stability. PMID:8144550
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
1QMB 2000-02-06 2.6 Cleaved alpha-1-antitrypsin polymer
1EZX 2000-10-25 2.6 CRYSTAL STRUCTURE OF A SERPIN:PROTEASE COMPLEX
1OO8 2003-08-05 2.65 CRYSTAL STRUCTURE OF A1PI-PITTSBURGH IN THE NATIVE CONFORMATION
1ATU 1997-08-20 2.7 UNCLEAVED ALPHA-1-ANTITRYPSIN
3NDF 2010-07-28 2.7 Cleaved antitrypsin with P8-P6 Asp
1PSI 1996-12-07 2.92 Intact recombined alpha1-antitrypsin mutant PHE 51 to LEU
1D5S 2000-04-02 3.0 CRYSTAL STRUCTURE OF CLEAVED ANTITRYPSIN POLYMER
9API 1990-10-15 3.0 THE S VARIANT OF HUMAN ALPHA1-ANTITRYPSIN, STRUCTURE AND IMPLICATIONS FOR FUNCTION AND METABOLISM
7API 1990-10-15 3.0 THE S VARIANT OF HUMAN ALPHA1-ANTITRYPSIN, STRUCTURE AND IMPLICATIONS FOR FUNCTION AND METABOLISM
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