Symmetry and secondary structure of the replication terminator protein of Bacillus subtilis: sedimentation equilibrium and circular dichroic, infrared, and NMR spectroscopic studies.
We have used analytical ultracentrifugation in combination with a number of spectroscopic techniques to analyze the symmetry and secondary structure of the DNA-binding replication terminator protein (RTP) of Bacillus subtilis. Sedimentation equilibrium studies confirm that RTP is a dimer in solution under the conditions used for spectroscopic analysis, whereas the number of cross peaks displayed in 1H-15N HSQC NMR spectra of uniformly 15N-labeled RTP are consistent with the primary structure of the monomer. These two results in combination lead to the conclusion that RTP is a symmetric dimer in solution. Circular dichroic and Fourier-transform infrared spectra reveal, in contrast to the results obtained from a number of commonly used secondary structure prediction algorithms, that RTP contains 20-30% alpha-helical and 40-50% beta-sheet/beta-turn secondary structure and that the conformation of the protein remains unchanged over the pH range 5-8. It is proposed on the basis of protein folding-class prediction algorithms, in combination with various physical properties of RTP, that it belongs to the alpha + beta protein-folding class.
Department of Biochemistry, University of Sydney, NSW, Australia.
A V
AV
Kralicek
N A
NA
Vesper
G B
GB
Ralston
R G
RG
Wake
G F
GF
King
D000465:Algorithms; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification; D004926:Escherichia coli; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D017550:Spectroscopy, Fourier Transform Infrared
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; C063112:rtP protein, Bacillus subtilis