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The Role of Subunit Interfaces of Malate Dehydrogenase in Protein Folding & Unfolding
Author(s) -
Abano John,
Bell Ellis,
Bell Jessica
Publication year - 2016
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.30.1_supplement.600.18
Subject(s) - malate dehydrogenase , protein subunit , biochemistry , proteolysis , dimer , protein folding , folding (dsp implementation) , chemistry , protein quaternary structure , enzyme , citrate synthase , mutant , biophysics , biology , gene , organic chemistry , electrical engineering , engineering
Malate dehydrogenase (MDH) is an essential enzyme for several metabolic pathways including the production of NADH as a precursor for ATP synthesis in the mitochondrion and glyoxysome. In the cytosol MDH is involved in the reduction of oxaloacetate as part of the aspartate‐malate shuttle.. The enzyme is a dimer of chemically identical subunits and while much is known about the enzymatic steps, little is known about the folding mechanism of MDH and the link between folding and regulation or catalysis. The goal of the current project is to understand the folding process and explore the role of subunit interactions to test the hypothesis that the native dimer conformation of MDH governs the correct folding of the enzyme subunits in order to impose the observed spatial asymmetry of the subunits and facilitate active site communication across the interface thought to be involved in both overall catalysis and regulation. Clustal analysis identified a number of interface conserved residues and 4 mutants, H90Q, E256Q, S266A and L269A were constructed. The wild type protein and various mutants were expressed in E.Coli and purified using NiNTA Chromotography using the engineered His‐Tag. Purity was established by SDS‐Page or MALDI‐tof. Quaternary structure was established by size exclusion chromatography. Reversible denaturation of wild type and mutants was studied by fluorescence spectroscopy and CD to determine tertiary and secondary structures respectively, with activity loss and regain followed by enzymatic assay. The protein unfolding/folding dynamics were also probed using limited proteolysis in conjunction with mass spectrometry.