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Engineering Tryptophan Residues into Glyoxysomal Malate Dehydrogenase as Probes of Structure and Function
Author(s) -
Johnson Adam,
Bell Ellis
Publication year - 2006
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.20.4.a53-b
Subject(s) - förster resonance energy transfer , tryptophan , allosteric regulation , chemistry , biochemistry , mutant , biophysics , fluorescence , enzyme , biology , amino acid , physics , quantum mechanics , gene
Resonance energy transfer measurements between two points on a protein are exquisitely sensitive to changes in the overall conformation of the protein. Glyoxysomal Malate Dehydrogenase, which natively lacks any tryptophans but has been engineered to contain a single tryptophan, has been used to demonstrate that resonance energy transfer measurements between the single tryptophan and the reduced cofactor NADH. This represents a significant tool in the ability to ascertain conformational integrity in mutants and conformational changes induced by allosteric ligands binding to distinct sites in the oligomer. In malate dehydrogenase a single tryptophan has been engineered into the protein on a flexible loop near the active site. This mutant has been characterized by initial rate kinetics and fluorescence spectroscopy, and has the potential for resonance energy transfer, but with different efficiencies to the active site bound reduced cofactor. Further trytophan mutants have been designed, adding single trytophan residues at L110, V189, L229, and I319 using QuikChange® Site‐Directed Mutagenesis. The position of these mutations is important due to the distance and orientation dependent nature of the FRET being observed. Further experiments characterizing the kinetics and flexibility of these mutants will also need to be done to better characterize the FRET occurring. Preliminary results suggest that resonance energy transfer from the catalytic product NADH to the tryptophan residue, combined with a FRET system utilizing a fluorescent protein, may eventually be useful in determining catalytic activity of the protein and could be applied to in vivo activity measurements. This work is supported by NSF Grant MCB 0448905 to EB.