Untersuchung der intramolekularen Signaltransduktion eines Blaulichtrezeptors

PixD (Slr1694) ist ein Photorezeptor, der den sensors of blue light using FAD (BLUF) Proteinen zugeordnet wurde. Die Übertragung des Stimulus auf das Apoprotein erfolgt in dieser Proteinfamilie über eine Neuordnung des Wasserstoffbrückennetzwerkes um den Kofaktor, in das die strikt konservierten Reste Tyrosin-8 (Y8), Glutamin-50 (Q50) und möglicherweise das semi-konservierte Tryptophan-91 (W91) involviert sind. Ziel dieser Arbeit war es, weitere Hinweise auf die Wasserstoffbrückenkonfiguration der Flavinbindetasche in Dunkel- und Lichtzustand zu erhalten, um eine bessere Vorstellung von der Stabilisierung des Lichtzustandes zu bekommen und mögliche Wege der Signaltransduktion an die Proteinoberfläche einzugrenzen. Die Ergebnisse weisen darauf hin, dass sich lichtaktivierte Interaktionsänderungen zwischen Apoprotein und Chromophor auf die Neubildung einer Wasserstoffbrücke zum Flavin C4-Carbonyl beschränken. In Übereinstimmung zu früheren Analysen liegt das Q50 im Dunkelzustand in seiner Amid-Form vor. Sein Seitenkettencarbonyl ist im Lichtzustand vermutlich zu Y8 ausgerichtet, wobei Hinweise auf eine Amid-Imidsäure-Tautomerisierung des Glutamins in PixD gefunden wurden. Eine selektive Isotopenmarkierung des Tryptophan-91 zeigte Anzeichen für eine Verlängerung des β5-Stranges, die wahrscheinlich ein zentrales Element der Signalweiterleitung an die Proteinoberfläche darstellt. Möglicherweise erstreckt sich das Wasserstoffbrückennetzwerk dabei bis in die über dem beta5-Strang liegende alpha-Helix. Wird es gestört, scheint das Proteininnere für Imidazol zugänglich gemacht zu werden, wo es die Aktivierungsenergie für die Rückkehr in den Dunkelzustand beeinflusst. Auch Substitutionen des H73 im gegenüberliegenden Eckstrang des beta-Faltblattes beeinflussten die Geschwindigkeit der Dunkelrelaxation von PixD. Sie veränderten die IR-Absorption gegenüber dem Wildtyp jedoch nicht und unterstützen die Theorie einer Protonenleitung über das benachbarte H72.The photoreceptor PixD (Slr1694) belongs to the sensors of blue light using FAD (BLUF) protein family. These photoreceptors propagate the signal by a rearrangement of hydrogen bonds surrounding the cofactor, involving the highly conserved residues tyrosine-8 (Y8), glutamine 50 (Q50) and perhaps the semi-conserved tryptophan-91 (W91). One aim of the presented work was to gain a deeper insight into the hydrogen bond configuration of the flavin binding pocket in the light and dark state conformations. Thereby, knowledge of the stabilization mechanisms for the light state and the signal propagation to the protein surface could be acquired. The results indicate a restriction of light induced changes in hydrogen bonding of the flavin to its C4 carbonyl. In agreement with former studies, the Q50 forms the amide isomer in the dark state. Its side chain carbonyl group most likely points towards Y8 in the active protein. Besides, the results support an amide-imidic acid-tautomerization of Q50 in PixD. A selective isotope labeling of the tryptophan-91 localized at the beginning of an edge strand of the beta sheet indicates an elongation of the secondary structure that may represent a central element of the signal propagation to the protein surface. The secondary structure is possibly connected with an alpha helix located above the beta5 strand by hydrogen bonds. A disturbance of this interaction probably allows the base catalyst imidazole to enter the protein core. Substitutions of H73 in the opposing edge beta strand changed the rate of the PixD dark relaxation as well. However, they had no visible effect on the infrared absorbance compared to the wild type and hence support a putative involvement of the neighbouring H72 in proton transfer reactions

Upgrading a microplate reader for photobiology and all-optical experiments

Automation can vastly reduce the cost of experimental labor and thus facilitate high experimental throughput, but little off-the-shelf hardware for the automation of illumination experiments is commercially available. Here, we use inexpensive open-source electronics to add programmable illumination capabilities to a multimode microplate reader. We deploy this setup to characterize light-triggered phenomena in three different sensory photoreceptors. First, we study the photoactivation of Arabidopsis thaliana phytochrome B by light of different wavelengths. Second, we investigate the dark-state recovery kinetics of the Synechocystis sp. blue-light sensor Slr1694 at multiple temperatures and imidazole concentrations; while the kinetics of the W91F mutant of Slr1694 are strongly accelerated by imidazole, the wild-type protein is hardly affected. Third, we determine the light response of the Beggiatoa sp. photoactivatable adenylate cyclase bPAC in Chinese hamster ovary cells. bPAC is activated by blue light in dose-dependent manner with a half-maximal intensity of 0.58 mW cm−2; intracellular cAMP spikes generated upon bPAC activation decay with a half time of about 5 minutes after light switch-off. Taken together, we present a setup which is easily assembled and which thus offers a facile approach to conducting illumination experiments at high throughput, reproducibility and fidelity.Peer Reviewe

A set of engineered Escherichia coli expression strains for selective isotope and reactivity labeling of amino acid side chains and flavin cofactors.

Biological reactions are facilitated by delicate molecular interactions between proteins, cofactors and substrates. To study and understand their dynamic interactions researchers have to take great care not to influence or distort the object of study. As a non-invasive alternative to a site-directed mutagenesis approach, selective isotope labeling in combination with vibrational spectroscopy may be employed to directly identify structural transitions in wild type proteins. Here we present a set of customized Escherichia coli expression strains, suitable for replacing both the flavin cofactor and/or selective amino acids with isotope enriched or chemically modified substrates. For flavin labeling we report optimized auxotrophic strains with significantly enhanced flavin uptake properties. Labeled protein biosynthesis using these strains was achieved in optimized cultivation procedures using high cell density fermentation. Finally, we demonstrate how this approach is used for a clear assignment of vibrational spectroscopic difference signals of apoprotein and cofactor of a flavin containing photoreceptor of the BLUF (Blue Light receptors Using FAD) family

Spectroscopic and Computational Observation of Glutamine Tautomerization in the Blue Light Sensing Using Flavin Domain Photoreaction

Blue light sensing using flavin (BLUF) domains constitute a family of flavin-binding photoreceptors of bacteria and eukaryotic algae. BLUF photoactivation proceeds via a light-driven hydrogen-bond switch among flavin adenine dinucleotide (FAD) and glutamine and tyrosine side chains, whereby FAD undergoes electron and proton transfer with tyrosine and is subsequently re-oxidized by a hydrogen back-shuttle in picoseconds, constituting an important model system to understand proton-coupled electron transfer in biology. The specific structure of the hydrogen-bond patterns and the prevalence of glutamine tautomeric states in dark-adapted (DA) and light-activated (LA) states have remained controversial. Here, we present a combined femtosecond stimulated Raman spectroscopy (FSRS), computational chemistry, and site-selective isotope labeling Fourier-transform infrared spectroscopy (FTIR) study of the Slr1694 BLUF domain. FSRS showed distinct vibrational bands from the FADS1 singlet excited state. We observed small but significant shifts in the excited-state vibrational frequency patterns of the DA and LA states, indicating that these frequencies constitute a sensitive probe for the hydrogen-bond arrangement around FAD. Excited-state model calculations utilizing four different realizations of hydrogen bond patterns and glutamine tautomeric states were consistent with a BLUF reaction model that involved glutamine tautomerization to imidic acid, accompanied by a rotation of its side chain. A combined FTIR and double-isotope labeling study, with 13C labeling of FAD and 15N labeling of glutamine, identified the glutamine imidic acid C═N stretch vibration in the LA state and the Gln C═O in the DA state. Hence, our study provides support for glutamine tautomerization and side-chain rotation in the BLUF photoreaction

Typical profile of a high cell density fermentation experiment.

<p>In the biomass phase (1.) cells are cultivated to high cell densities. The stirrer speed (red) is gradually increased in order to keep the pO₂ level (black) at above 30%. After the stirrer reaches its maximum speed the pO₂ level drops to ~0%. Consumption of primary nutrients is indicated by a rise of pO₂ (2.). Complete depletion of also secondary metabolites is recognized by a steep rise in pO₂ (3.). This event is used to set up expression conditions, in this case by lowering the temperature from 25°C (grey). After reaching the desired temperature fresh (labeled) substrates are pumped into the culture and the pO₂ level starts to decline again (4). At the same time protein production is induced by addition of an inducing agent (dotted grey line). After all substrates are consumed the pO₂ level rises again and thereby indicates the end of the expression phase. </p

Selective unlabeling of the flavin chromophore (A, light grey) upon uniform ¹³C labeling of the protein (dark grey and black) using the riboflavin auxotrophic strain CpXribF.

<p>Light-minus-dark FTIR difference spectra of unlabeled Slr1694 (grey) and apoprotein ¹³C-labeled Slr1694 (black) in H₂O are presented in B and C. The close-up of the amide frequency range shows a downshift of secondary structural changes as well as coupling of flavin and protein modes (C). </p

Amino acid and cofactor specific isotope labeling using custom-made auxotrophic expression strains (A) in a high cell density fermentation setup (B).

<p>CmpX13 is rendered auxotrophic for selected amino acid and/or cofactor synthesis pathways. The resulting expression strains are cultivated under controlled conditions to achieve the highest cell density under complete consumption of unlabeled substrates. Subsequently labeled substrates are fed and protein production is induced.</p

Cassette for constitutive expression of flavokinases in <i>E. coli</i> (A).

<p>The enhanced flavokinase activity of the strains CpXFMN and CpXribF containing the respective expression cassette reduces the intracellular amount of free riboflavin by accumulation of FAD and FMN, respectively. Thereby the transport equilibrium is shifted towards a higher uptake (B). CpXFMN and CpXribF accordingly show an enhanced growth at riboflavin concentrations below 10 µM as compared to the riboflavin auxotroph CmpX131 (C).</p