Premium
Studying B.Stearothermophilis DHFR Y127A Mutant and Investigating its Effect on Catalytic Activity
Author(s) -
Cho Sarah,
Okondo Marian,
Goodey Nina
Publication year - 2015
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.29.1_supplement.891.13
Subject(s) - chemistry , ligand (biochemistry) , mutant , conformational change , dihydrofolate reductase , fluorophore , cysteine , alanine , stereochemistry , residue (chemistry) , active site , amino acid , biochemistry , enzyme , fluorescence , receptor , physics , quantum mechanics , gene
Dihydrofolate reductase from B. stearothermophilus (BsDHFR) catalyzes the formation of tetrahydrofolate (THF), which is used for the synthesis of purines and thymidylates and is necessary for cell growth. The goal of this project is to examine the role of amino acid residues away from the active site that have been found to alter ligand specificity. Specifically, the replacement of distal residue Tyr127 of BsDHFR by an Alanine was found to result in a significant change in ligand specificity. This was an especially interesting finding because Tyr127 is located far away from the active site. Previously, a fluorescently labeled construct was developed and used to study the conformational equilibrium, the rates of ligand binding, and the associated conformational changes in BsDHFR. The construct used for this study had two mutations (C73A/S131C) to create a single reactive cysteine handle at position 131 to study conformational changes away from the active site. The studies were conducted using a covalently‐bound, environmentally sensitive fluorophore and stopped‐flow methods. We introduced the mutation Y127A into the C73A/S131C BsDHFR contruct to make C73A/ Y127A /S131C BsDHFR. This gave us a construct that can be labeled for conformational analysis with the Y127A mutation previously found to alter ligand specificity allowing us to compare the conformational equilibrium and motions associated with ligand binding between the wildtype and Y127A contruct. After the mutation was successfully introduced, the mutant BsDHFR was expressed in E. coli BL21 cells. The protein was purified by anion exchange chromatography. The purified protein was labeled with conformationally sensitive fluorophore PyMPO. We plan to use this construct to determine whether or not the Y127A mutation affects the conformational equilibrium of DHFR and this may be the origin of the altered ligand specificity.