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


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 UniProtKB Entries

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