Ultrafast Structural Dynamics of BlsA, a Photoreceptor from the Pathogenic Bacterium Acinetobacter baumannii

Abstract

Acinetobacter baumannii is an important human pathogen that can form biofilms and persist under harsh environmental conditions. Biofilm formation and virulence are modulated by blue light, which is thought to be regulated by a BLUF protein, BlsA. To understand the molecular mechanism of light sensing, we have used steady-state and ultrafast vibrational spectroscopy to compare the photoactivation mechanism of BlsA to the BLUF photosensor AppA from Rhodobacter sphaeroides. Although similar photocycles are observed, vibrational data together with homology modeling identify significant differences in the β5 strand in BlsA caused by photoactivation, which are proposed to be directly linked to downstream signaling.

Signaling-State Formation Mechanism of a BLUF Protein PapB from the Purple BacteriumRhodopseudomonas palustrisStudied by Femtosecond Time-Resolved Absorption Spectroscopy

“…25,27−29 One is the signaling-state formation directly from the S 1 state (S 1 → signaling), which was observed for AppA, BlrB, and BlsA. 25,28,29,31 This type of signaling-state formation proceeds in a H/D isotope independent manner. 25,33 The other is the signaling-state formation through FADH • (i.e., S 1 → FADH • → signaling).…”

“…33 Recent studies of other BLUF sensors, BlrB (from Rhodobacter sphaeroides) and BlsA (from Acinetobacter baumannii), have also shown that the signaling states are directly formed from the S 1 state. 25,31,36 On the other hand, the time-resolved absorption study of Slr1694 (alternative name: SyPixD) from cyanobacterium Synechocystis showed that the neutral flavin semiquinone FADH radical (FADH • ) is generated as an intermediate during the signalingstate formation. 27 This implies that not merely ET but protoncoupled ET takes place in Slr1694.…”

Integration of Temperature and Blue‐Light Sensing in Acinetobacter baumannii Through the BlsA Sensor

“…Indeed, blue‐light absorption in the dark state (dBlsA), referred to as the dark‐adapted conformation of the photoreceptor, triggers a photocycle to yield the signaling state (sBlsA), which typically shows a ~10–12 nm red‐shift of the absorption band indicating that BlsA is an active photoreceptor . The photocycle of dBlsA was later studied by ultra‐fast time‐resolved infrared (TRIR) spectroscopy, and it was shown that the signaling state of sBlsA is directly populated from the S 1 excited state of FAD in few ps . The formation of the signaling state of BLUFs occurs in the subnanosecond regime, and nowadays it is accepted that the ultra‐short lifetime of the S 1 state together with low fluorescence quantum yield (<0.01) are a consequence of a prompt electron‐transfer (ET) process from a nearby Tyr residue .…”

“…The surprising fact is that in the signaling state of BLUFs, the FAD undergoes subtle structural rearrangements but strong enough to trigger a significant change of the protein conformation for signal transduction . For BlsA, the β 5 strand containing the N‐terminal residue Trp92 was proposed to move upon light‐induced formation of sBlsA, initiating the biological signal transduction probably by formation of a complex with the downstream target protein . After photoactivation, the signaling state of BLUFs relaxes back in the time window of few seconds to several minutes depending on the protein , suggesting that moderate to large thermal activation is required.…”

Photoreceptors in Chemotrophic Prokaryotes: The Case of Acinetobacter spp. Revisited

“…those relevant for structural dynamics in proteins) differ in at least one key aspect: The altered H‐bonding around the glutamine that occurs upon photoactivation of the FMN chromophore strengthens rather than weakens the H‐bonding in the BlsA β ‐sheet. This was revealed by the change of sign of the β ‐strand marker mode in static IR‐difference spectroscopy studies of these two proteins. This suggests that also the BlsA photoreceptor functions such that its activation by blue light leads to formation of a complex with a downstream target (protein) rather than that it would lead to complex dissociation.…”