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 H-1-N-15 HSQC NMR spectra of uniformly N-15-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.