Ligand-induced conformational changes in cyclic nucleotide phosphodiesterases and cyclic nucleotide-dependent protein kinases.
Three methods have been used to assess the conformational effects associated 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 should be applicable to other proteins and to other types of modification such as phosphorylation. The procedures use either ion-exchange chromatography, size-exclusion chromatography, or native gel electrophoresis 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 relative compactness (Stokes radius) of the protein molecule. The combined 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 proteins and to proteins that are available in limited quantities. Conformational changes due to stable modifications of proteins can be potentially examined in crude extracts of intact cells. Each of the methods can be tailored to optimize resolution of a particular protein under a variety of conditions. Activity measurements, Coomassie brilliant blue or silver staining of gels, radioautography, or Western blot analysis can be used for detection of the protein.
Francis
S H
SH
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615, USA.
Chu
D M
DM
Thomas
M K
MK
Beasley
A
A
Grimes
K
K
Busch
J L
JL
Turko
I V
IV
Haik
T L
TL
Corbin
J D
JD
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry*; D000818:Animals; D001011:Aorta / Q000201:enzymology; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006152:Cyclic GMP / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry* / Q000302:isolation & purification; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D004591:Electrophoresis, Polyacrylamide Gel; D008024:Ligands; D008168:Lung / Q000201:enzymology; D008970:Molecular Weight; D011487:Protein Conformation / Q000187:drug effects*; D011994:Recombinant Proteins / Q000737:chemistry
D008024:Ligands; D011994:Recombinant Proteins; D017869:Cyclic GMP-Dependent Protein Kinases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D006152:Cyclic GMP; D015056:1-Methyl-3-isobutylxanthine