Design, preparation, and characterization of mixed dimers of inducible nitric oxide synthase oxygenase domains.
A limitation of site-directed mutagenesis of homodimeric proteins is that both subunits will carry the same mutation. We have devised a way to prepare mixed dimers, in which only one chain bears a desired mutation, or each chain can bear a different mutation. Using the inducible nitric oxide oxygenase domain as a model, our strategy focused on the co-expression of two differentially tagged versions of the oxygenase domain, with isolation of the desired mixed dimer in two chromatography steps. We evaluated expression vectors encoding polyhistidine (His(6)), cellulose binding domain, glutathione-S-transferase, and polyglutamate (Glu(7))-tagged versions of the oxygenase domain for satisfactory levels of soluble protein expression and for their ability to form mixed dimers. The combination of His(6)- and Glu(7)-tagged subunits was successful in both respects, and the mixed dimers could be separated from either form of homodimer by sequential immobilized metal affinity chromatography and anion exchange chromatography. The UV-Vis spectrum, substrate binding properties, and enzymatic activity were not altered in the mixed dimer wild-type (His(6)/Glu(7)) compared to the two homodimers (His(6)/His(6) and Glu(7)/Glu(7)). We then characterized a mixed dimer variant in which one chain contained an E371A substitution (which prevents binding of the substrate L-arginine) while the other subunit was left unaltered. This species binds L-arginine and has about one-half the activity of the wild-type homodimer. Mutants known to destabilize the iNOS dimer (E411A, D454A, and W188F) were also investigated; in these cases co-expression with the wild-type subunit did not lead to the formation of stable mixed dimers.
McDonald
Jennifer K
JK
Chemistry Department, Trent University, 1600 West Bank Drive, Peterborough, Ont., Canada K9J 7B8.
Taylor
Carol M
CM
Rafferty
Steven
S
D020134:Catalytic Domain; D019281:Dimerization; D008958:Models, Molecular; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013057:Spectrum Analysis
D010446:Peptide Fragments; D011994:Recombinant Proteins; D010105:Oxygenases; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II