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Engineering Cysteine Residues into Glyoxysomal Malate Dehydrogenase to Study Subunit Interactions Using Fluorescence and AFM
Author(s) -
Drake Mary Jane,
Bell Ellis
Publication year - 2011
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.25.1_supplement.725.1
Subject(s) - cysteine , dimer , protein subunit , chemistry , förster resonance energy transfer , guanidine , protein engineering , biophysics , malate dehydrogenase , crystallography , fluorescence , biochemistry , enzyme , organic chemistry , biology , physics , quantum mechanics , gene
Despite the fact that Malate Dehydrogenase was one of the first multisubunit enzymes to have its structure determined by x ray crystallography many questions remain about the mechanism and role of subunit interactions in this protein. To provide a quantitative framework to analyze engineered interface mutants, a single cysteine residue has been engineered into the subunit on the outer edge, furthest from the interface and shown to be reactive [the five native cysteine residues all appear buried and inactive in the native form of the enzyme]. Random labeling of the 2 cysteines per dimer with Alexa Fluor 488/AF647 [Ro approximately 52A] or AF546/AF647 [Ro approximately 63A] is being used to allow resonance energy transfer measurements between the two halves of the dimer to probe ligand induced changes over long distances and effects on dimer stability assessed using mild guanidine hydrochloride dissociation. In an alternative approach preliminary data has been obtained focused on using the engineered sulfhydryl groups to immobilize individual monomers (confirmed by tapping mode analysis) on the gold tip and stage of an atomic force microscope to allow pulling type experiments to probe the effects of ligands on the stability of the dimer interface relative to the protein structure using analysis of the resultant force curves. This work is supported by NSF Grant MCB 0448905 to EB