This possibly alters the orientation of the STAS domains which subsequently leads to a change of binding properties to potential interaction partners. It has been suggested that an interaction partner for the STAS domain could be nucleoside triphosphates. This assumption was originally based on MK-0683 sequence homology to other STAS domains, in particular to SPOIIAA from Bacillus subtilis or the STAS domain of Rv1739c from Mycobacterium tuberculosis that have both been shown to bind NTPs. Another hint for a potential GTP-binding capability of YtvA is the existence of two classical GTP-binding motifs, DXXG and NKXD, within its STAS domain. It has been shown that beside other conserved regions both motifs are jointly responsible for the interaction of GTP with G-proteins. Further evidence for NTP binding of the STAS domain of YtvA was provided by Buttani et al. that used a fluorescence assay to investigate the binding of GTP to YtvA and found a binding constant of KD = 38 mM for illuminated YtvA and a increased affinity after dark reversion. This assay has since been used to study the effect of mutations on the activation mechanism in YtvA. More recently, however, Nakasone et al. could not confirm that GTP binds to YtvA but found that the fluorescence labeled GTP analog used in the assays binds unspecifically to YtvA. The aim of the investigations described here was originally to determine the precise binding site for GTP on YtvA using heteronuclear NMR. After a repeated failure to detect any binding we used fluorescence spectroscopy and NMR binding assays to investigate not only the binding of GTP to YtvA and to the isolated STAS domain of YtvA but also that of the fluorescent analogue, BODIPY-GTP. We can show that while BODIPY-GTP does in fact bind to YtvA via the fluorescent dye in an unspecific manner, GTP does not show any binding to either protein and that thus YtvA function does not involve GTP binding. Thus the binding of BODIPY-GTP appears to be confined to the Ja-helix and the STAS domain but in a rather unspecific manner. Their assumption being based on the comparison of BODIPY-GTP binding to different truncated versions of the LOV domain part of the protein. Such truncations may alter the hydrophobicity of the resulting surface leading to interactions with hydrophobic ligands which may explain why binding to the LOV domain was detected while the interaction seems to be confined to the STAS domain and the Jahelix in our experiments. As indicated in the ITC experiments we detect a similar effect when truncating the protein: the binding of BODIPY-GTP to YtvA-STAS differs from that to YtvA. We know from further NMR experiments performed with full length YtvA that the Ja-helix and the STAS domain are more flexible than the LOV domain. This could explain why the two former parts of the protein are more accessible to unspecific hydrophobic interactions while the LOV domain – being a relatively rigid and tight binding dimer – does not expose any hydrophobic patches as easily as the linker and the STAS domain during their movements. More importantly, however, the NMR experiments with protein detection described above indicate again that no binding of GTP to YtvA is taking place.