科研之友
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摘要
The RI alpha subunit of cAMP-dependent protein kinase is maintained as an asymmetric dimer by a dimerization motif at the N terminus, Based on resistance to proteolysis and expression as a discrete domain in Escherichia coli, this motif is defined as residues 12-61. This motif is chemically, kinetically, and thermally stable, The two endogenous interchain disulfide bonds between Cys(16) and Cys(37) in RI alpha are extremely resistant to reduction even in 8 M urea, indicating that they are well shielded from the reducing environment of the cell. The disulfide bonds were present in recombinant RI alpha as well as when the dimerization domain alone was expressed in E. coli, emphasizing the unusual stability of this motif and the disulfide bonds. Although 100 mM, dithiothreitol was sufficient to reduce the disulfide bonds, it did not abolish dimerization. In addition, a stable dimer also still formed when Cys(37) was replaced with His, confirming unambiguously the original antiparallel alignment of the disulfide bonds, Thus, both in vitro and in vivo, disulfide bonds are not required for dimerization. Circular dichroism of the dimerization domain indicated a high content of a thermostable alpha-helix. Based on the CD data, trypsin resistance of the fragment, location of the disulfide bonds, and amphipathic helix predictions, potential models are discussed, A new alignment of the dimerization domains of RI, RII, and cGMP-dependent protein kinase elucidates fundamental similarities as well as significant differences among these three domains.
