LIGAND-INDUCED CONFORMATIONAL-CHANGES IN CYCLIC-NUCLEOTIDE PHOSPHODIESTERASES AND CYCLIC NUCLEOTIDE-DEPENDENT PROTEIN-KINASES

Three methods have been used to assess the conformational effects asso ciated with ligand binding to two unrelated cyclic nucleotide receptor proteins: the cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE or PDE5A) and the cGMP-dependent protein kinase (PKG). The methods sho uld be applicable to other proteins and to other types of modification such as phosphorylation. The procedures use either ion-exchange chrom atography, size-exclusion chromatography, or native gel electrophoresi s of these proteins in the absence and presence of regulatory ligands. Measurements from these respective approaches allow documentation of changes in the quaternary structure, surface electronegativity, and re lative compactness (Stokes radius) of the protein molecule. The combin ed data allow the changes in protein conformation to be quantitated in terms of alterations in the axial ratio or length/width dimension of the molecule. The methods can be applied to partially purified protein s and to proteins that are available in limited quantities. Conformati onal changes due to stable modifications of proteins can be potentiall y examined in crude extracts of intact cells. Each of the methods can be tailored to optimize resolution of a particular protein under a var iety of conditions. Activity measurements, Coomassie brilliant blue or silver staining of gels, radioautography, or Western blot analysis ca n be used for detection of the protein. (C) 1998 Academic Press.