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 (Mr = 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 in the 1H NMR spectrum of the Pf1 nucleoprotein complex indicate that the C-terminal region of the protein subunits enjoys remarkable conformational flexibility in the complex. In contrast, the globular N-terminal domain of the protein subunits is rigidly held and does not contribute to the spectrum. The Fd gene 5 protein lacks this C-terminal flexible domain, and no distinct resonances can be observed in the 1H NMR spectrum when this protein is complexed to single-stranded viral DNA. Differential scanning calorimetry shows that the thermal stability of both the Pf1 and Fd gene 5 protein is increased by 8 degrees C in the complex with DNA, and the transition is highly cooperative. Removal of the C-terminal domain of the Pf1 gene 5 protein subunits has no appreciable effect either on the Tm of the DNA-protein complex or on the cooperative nature of the thermal transition. It is suggested that the C-terminal domain of the Pf1 gene 5 protein acts as a dynamic clamp which kinetically stabilizes the nucleoprotein complex.
Davis
K G
KG
Biophysics Laboratories, School of Biological Science, University of Portsmouth, U.K.
Plyte
S E
SE
Robertson
S R
SR
Cooper
A
A
Kneale
G G
GG
D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D004277:DNA, Single-Stranded / Q000737:chemistry; D004279:DNA, Viral / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004926:Escherichia coli / Q000821:virology; D017912:Inovirus / Q000737:chemistry* / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009698:Nucleoproteins / Q000737:chemistry*; D011550:Pseudomonas aeruginosa / Q000821:virology; D013816:Thermodynamics; D014764:Viral Proteins / Q000737:chemistry*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009698:Nucleoproteins; D014764:Viral Proteins; C057816:gene 5 protein, Enterobacteria phage M13; C067863:gene 5 protein, Pseudomonas phage Pf1