Strategic judgment of “Hit” chemicals by FCCS.

<p>A. When a chemical has an inhibitory effect on FtsZ dimerization even in the presence of GTP, the cross-correlation amplitude remains low. These inhibitory chemicals are included in the subset in the red circle (B). C. After addition of GTP, cross-correlation amplitude is increased if the chemical does not affect FtsZ (D). A and C were evaluated by the “binding ratio” analyzed by FCCS. The magenta and green lines indicate the autocorrelation functions of FtsZ^K175D_N-terminal-EGFP and FtsZ_C-terminal-mCherry, respectively. The black line indicates the cross-correlation function. E. Fluorescent intensity (EGFP and/or mCherry) changed when FtsZ fragments were precipitated by addition of a chemical. Chemicals that induced intensity changes of more than 20% when DMSO was added are included in the subset in the brown circle (F). G. Diffusion of FtsZ fragments was slower by aggregation. Chemicals that increased the slow fraction by more than 20% in 2-component fitting are included in the subset shown in the purple circle (H). Finally, the chemicals included in the subset shown only in red, were determined to be “true Hit” chemicals.