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The biochemical characterization of the PDB entry 4EZI by in silico and in vitro quantitative techniques (777.2)
Author(s) -
Hart Kaitlin,
Dodge Greg,
Horn Alison,
Craig Paul,
Bernstein Herbert
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.777.2
Subject(s) - in silico , protein data bank (rcsb pdb) , protein data bank , esterase , computational biology , chemistry , in vitro , enzyme , biochemistry , protein structure , biology , gene
This study is part of the SBEVSL project, the purpose of which is to propose and confirm the function of uncharacterized protein structures, in the Protein Data Bank, by use of in silico and in vitro quantitative techniques. For over 3000 structures in the PDB, no function has been assigned. These structures were first screened with the PyMOL molecular graphics system, along with ProMOL plugin, which compares query structures against a library of enzyme active sites. The most promising alignments were further refined by sequence and structure comparison using BLAST and Dali. A structure of considerable interest is presented here: 4EZI. When 4EZI was queried with PyMOL/ ProMOL, the best alignment was with the dipeptidyl peptidase IV, 1ORV. After analysis with BLAST and Dali, the alignments revealed 4EZI matched closely to the esterase family, specifically 3H2H. This match was confirmed experimentally. The protein was purified using methods of transformation and column chromatography. Then, the predicted molecular weight was confirmed using SDS‐PAGE. Protein concentrations were determined by a BCA assay and enzymatic activity was confirmed using p‐nitrophenol esters of short‐chain carboxylic acids. In conclusion, 4EZI has been characterized as an esterase based both on in silico and in vitro quantitative methods. Funding was provided by RIT, Dowling College, and NIGMS 2R15GM078077‐02 & 3R15GM078077‐02S1. Grant Funding Source : Supported by NIGMS 2R15GM078077‐02, 3R15GM078077‐02S1