COMPARISON OF PF1 AND FD GENE-5 PROTEINS AND THEIR SINGLE-STRANDED-DNA COMPLEXES BY NMR-SPECTROSCOPY AND DIFFERENTIAL SCANNING CALORIMETRY

The Pf1 gene 5 protein forms a large helical nucleoprotein complex (M( r) 3.1 x 10(7)) with single-stranded viral DNA, from which a 32 amino acid sequence rich in alanine, proline, and glutamine residues can be removed from the C-terminus by limited proteolysis. Sharp resonances i n the H-1 NMR spectrum of the Pf1 nucleoprotein complex indicate that the C-teminal region of the protein subunits enjoys remarkable conform ational flexibility in the complex. In contrast, the globular N-termin al domain of the protein subunits is rigidly held and does not contrib ute to the spectrum. The Fd gene 5 protein lacks this C-terminal flexi ble domain, and no distinct resonances can be observed in the LH NMR s pectrum when this protein is complexed to single-stranded viral DNA. D ifferential scanning calorimetry shows that the thermal stability of b oth the Pf1 and Fd gene 5 protein is increased by 8 degrees C in the c omplex with DNA, and the transition is highly cooperative. Removal of the C-terminal domain of the Pf1 gene 5 protein subunits has no apprec iable effect either on the T-m of the DNA-protein complex or on the co operative nature of the thermal transition. It is suggested that the C -terminal domain of the Pf1 gene 5 protein acts as a dynamic clamp whi ch kinetically stabilizes the nucleoprotein complex.