Site-directed mutagenesis to study the role of specific amino acids in the ligand binding domain of PPARs.

Nico Mitro, Federica Gilardi, Marco Giudici, Cristina Godio, Elena Scotti, Maurizio Crestani

Laboratorio Giovanni Galli di Biochimica e Biologia Molecolare del Metabolismo-Spettrometria di Massa, Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, Italy.

Methods in molecular biology (Clifton, N.J.) 2013

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The role of certain amino acids in the interactions of ligands with their cognate nuclear receptors is usually achieved by the resolution of the crystal structure of the receptor complexed with the ligand. As a complementary functional approach, site-directed mutagenesis, a technique broadly used in molecular biology, allows the assessment of the role of a specific amino acid in determining the interaction with a specific ligand. This method makes it possible to evaluate several mutations of a key amino acid for ligand bind ing and to determine the relationship between protein structure and ligand interaction. Here, we describe an application of this technique to evaluate different point mutations on the transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ) in the absence or presence of chemically different ligands.

Citation

Nico Mitro, Federica Gilardi, Marco Giudici, Cristina Godio, Elena Scotti, Maurizio Crestani. Site-directed mutagenesis to study the role of specific amino acids in the ligand binding domain of PPARs. Methods in molecular biology (Clifton, N.J.). 2013;952:137-44