On the Binding of BODIPY‐GTP by the Photosensory Protein YtvA from the Common Soil Bacterium Bacillus subtilis^†

Abstract

The YtvA protein, which is one of the proteins that comprises the network carrying out the signal transfer inducing the general stress response in Bacillus subtilis, is composed of an N-terminal LOV domain (that binds a flavin [FMN]) and a C-terminal STAS domain. This latter domain shows sequence features typical for a nucleotide (NTP) binding protein. It has been proposed (FEBS Lett., 580 [2006], 3818) that BODIPY-GTP can be used as a reporter for nucleotide binding to this site and that activation of the LOV domain by blue light is reflected in an alteration of the BODIPY-GTP fluorescence. Here we confirm that BODIPY-GTP indeed binds to YtvA, but rather nonspecifically, and not limited to the STAS domain. Blue-light modulation of fluorescence emission of YtvA-bound BODIPY-GTP is observed both in the full-length YtvA protein and in a truncated protein composed of the LOV-domain plus the LOV-STAS linker region (YtvA(1-147)) as a light-induced decrease in fluorescence emission. The isolated LOV domain (i.e. without the linker region) does not show such BODIPY-GTP fluorescence changes. Dialysis experiments have confirmed the blue-light-induced release of BODIPY-GTP from YtvA.

Blue News Update: BODIPY-GTP Binds to the Blue-Light Receptor YtvA While GTP Does Not

“…After addition of YtvA or YtvA-STAS to the fluorophore solution the relative fluorescence was significantly increased. This is in agreement with the results presented in the literature so far [25], [29]. Since BODIPY is very hydrophobic, however, interactions of BODIPY with hydrophobic regions on the protein surface could be the reason for binding.…”

“…This assay has since been used to study the effect of mutations on the activation mechanism in YtvA [25]–[28]. More recently, however, Nakasone et al could not confirm that GTP binds to YtvA but found that the fluorescence labeled GTP analog (BODIPY-GTP) used in the assays binds unspecifically to YtvA [29]. The aim of the investigations described here was originally to determine the precise binding site for GTP on YtvA using heteronuclear NMR.…”

STAS Domain Structure and Function

“…3A) by assessing the impact of directed mutagenesis on in vivo activation of B [20] and by assessment of alterations in 4-bora-3a,4a-diaza-s-indacene (BODIPY)-GTP TR fluorescence enhancement [21]. However, a recent report indicates that some recombinant YtvA LOV constructs lacking the STAS domain similarly enhance BODIPY-GTP TR fluorescence in a manner insensitive to competition by added nucleotide triphosphates, but suggestive of predominant actions of the hydrophobic BODIPY moiety [25]. In addition, GTPase activity of the STAS domain has not yet been detected.…”

“…3A). Studies demonstrating YtvA STAS domain-mediated enhancement and red-shift of BODIPY TR -GTP fluorescence in the illuminated state, with further slight enhancement and redshift during thermally driven recovery in the dark [24,22] have suggested a guanine nucleotide binding function of the STAS domain with conformational sensitivity to FMN-suggest that the light-induced conformational change may be more modest than depicted in this schematic drawing [25].…”

LOV and BLUF flavoproteins’ regulatory photoreceptors of microorganisms and photosensory actuators in optogenetic systems

“…Кроме того, фотоактивиро-ванный YtvA in vivo действует как положительный регулятор транскрипционного фактора общего стресса σB [23]. Поскольку мутации, нарушающие связывание ГТФ в STAS-домене или междоменное распространение светоиндуцированного сигнала в фоторецепторе, подавляют активированную синим светом σB-зависимую транскрипцию in vivo, счита-ется, что связывание ГТФ необходимо для прояв-ления функциональной активности YtvA [23][24][25].…”