TAILIEUCHUNG - Báo cáo khoa học: The hinge region operates as a stability switch in cGMP-dependent protein kinase Ia

The molecular mechanism of cGMP-dependent protein kinase activation by its allosteric regulator cyclic-3¢,5¢-guanosine monophosphate (cGMP) has been intensely studied. However, the structural as well as thermo-dynamic changes upon binding of cGMP to type I cGMP-dependent protein kinase are not fully understood. | ễFEBS Journal The hinge region operates as a stability switch in cGMP-dependent protein kinase Ia Arjen Scholten1 2 Hendrik FuB1 Albert J. R. Heck2 and Wolfgang R. Dostmann1 1 Department of Pharmacology College of Medicine University of Vermont Burlington VT USA 2 Department of Biomolecular Mass Spectrometry Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences Utrecht University the Netherlands Keywords cGMP cGMP-dependent protein kinase Ia limited proteolysis mass spectrometry tryptophan fluorescence Correspondence W. R. Dostmann Department of Pharmacology College of Medicine University of Vermont 149 Beaumont Avenue Burlington VT 05405 USA Fax 1 802 6564523 Tel 1 802 6560381 E-mail Present address University of Ulster Schoolof Biomedical Sciences Cromore Road Coleraine BT52 1SA UK Received 19 September 2006 revised 28 January 2007 accepted 1 March 2007 The molecular mechanism of cGMP-dependent protein kinase activation by its allosteric regulator cyclic-3 5 -guanosine monophosphate cGMP has been intensely studied. However the structural as well as thermodynamic changes upon binding of cGMP to type I cGMP-dependent protein kinase are not fully understood. Here we report a cGMP-induced shift of Gibbs free enthalpy AAGD of kJ-mol-1 as determined from changes in tryptophan fluorescence using urea-induced unfolding for bovine PKG Ia. However this apparent increase in overall stability specifically excluded the N-terminal region of the kinase. Analyses of tryptic cleavage patterns using liquid chromatography-coupled ESI-TOF mass spectrometry and SDS PAGE revealed that cGMP binding destabilizes the N-terminus at the hinge region centered around residue 77 while the C-terminus was protected from degradation. Furthermore two recombi-nantly expressed mutants the deletion fragment D1-77 and the trypsin resistant mutant Arg77Leu R77L revealed that the labile nature of the N-terminus is primarily associated .

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