## 3 Discussion  
Two tryptophan residues at the positions 14 and 86 of CRP contribute to the intrinsic fluorescence. [PERSON] et al. suggested the predominance of fluorescence by W14 [25], which might attenuate the complexity due to the presence of multiple fluorophores. [PERSON] et al. reported that cGMP bound to CRP competitively with cAMP had a similar affinity, but the effect of their binding on CRP deferred. The cGMP binding properties remained unchanged in the presence of double-stranded deoxyribonucleic acid (dsDNA), while cAMP bound more efficiently to dsDNA-bound CRP [8]. This is the first report showing ppGpp binding to CRP, suggesting that the effect of ppGpp binding to CRP may be similar to cGMP-binding and that the gene expression by CRP may be further modulated by ppGpp binding to CRP under starvation conditions. Surprisingly, ATP bound to CRP with a similar affinity as cAMP. In glucose-fed, exponentially growing _Escherichia coli_, the cellular concentrations of ATP and cAMP were 9600 uM and 35 uM, respectively [26]. Since over-estimation of cAMP is well-documented [7], it can be assumed that the difference in ATP and cAMP concentrations may be substantially higher. To overcome the huge difference in concentration, the binding affinity for cAMP must increase significantly in the presence of DNA and RNA polymerase. Otherwise, cAMP will never have a chance to compete with ATP. [PERSON] et al. reported that H-NS stimulated stringently controlled bacterial genes [27]. _E. coli_ lacking both H-NS and its paralog, StpA, showed a lower expression of CRP and reduced growth. The growth rate was restored in the mutant with impaired ppGpp synthesis; overexpression of CRP partially compensated the reduced growth. Although their study links CRP with the (p)ppGpp regulons in _E. coli_, this study, demonstrating direct interactions of ppGpp to H-NS and CRP, seems to be unrelated to their studies. The cellular concentrations of ppGpp reaches millimolar levels under starvation conditions [28; 29]. Therefore, the cellular ppGpp concentration under starvation conditions may be 10-20 times higher than the dissociation constant (80 \(\pm\) 11 uM), and the binding site on H-NS may be fully occupied by ppGpp under physiological conditions. Based on the cellular concentration of ppGpp and the dissociation constants, the binding affinities of ppGpp to H-NS and CRP may be considered as'strong' ones. CRP is a dimeric protein and each CRP monomer binds with 2 molecules of cAMP. The first binding of cAMP, with an approximate \(K_{D}\) of 20 uM, activates CRP. The second binding with syn-conformation of cAMP, however, down-regulates CRP. The \(K_{D}\) for the  
Figure 4: Interaction of ppGpp with N-NS. (**a**) The ppGpp binding site proposed by a molecular modeling study. The interactions between ppGpp and H-NS are shown as dotted lines. (**b**) Interacting residues at the binding site. S2, E3, K6, and N10 interact with phosphate groups of ppGpp. second site is around 2 mM [30]. The binding site of cAMP on H-NS, with eight-fold higher affinity than the second cAMP site on CRP, appears to be occupied fully only when the second binding site on CRP is occupied by cAMP. Recent developments in proteomics report larger numbers of proteins for analysis. For example, one study utilizing a photo-crosslinking ppGpp analog has captured 299 proteins as ppGpp binding proteins [22]. To analyze the binding affinities of those proteins for ppGpp, a rapid and reliable method may be required. Fluorescence spectroscopic analysis, used to determine ligand binding, has been introduced over 30 years ago. However, intrinsic fluorescence of proteins may be considered too weak and its utilization for ligand binding study has not been explored much. This study demonstrates that the fluorescence intensities of CRP and H-NS are significant in that the binding of various ligands can be measured without the labelling of proteins with fluorophores.