Light-Induced Structural Changes in the Active Site of the BLUF Domain in AppA by Raman Spectroscopy

Unno, Masashi; Sano, Rikiya; Masuda, Shinji; Ono, Tetsuya; Yamauchi, Seigo

doi:10.1021/jp0522664

Cited by 94 publications

(174 citation statements)

References 25 publications

Supporting

Mentioning

150

Contrasting

Order By: Relevance

“…FTIR showed that light exposure weakens the carbonyl bonds at C2 and C4 of the flavin, but suggested much larger changes in the protein itself (21,22). Stronger hydrogen bonding between the flavin O4 atom and the neighboring glutamine residue can explain the spectral shifts (13,(21)(22)(23) and is reflected in changes at the exterior of the BLUF domain, near the last β-strand and the surface tryptophan. This change leads to different downstream effects in different proteins; for example in BlrP1, it affects the coordination of essential metal ions at the active site of the neighboring domain (7).…”

Section: Significancementioning

confidence: 99%

“…Different BLUF proteins show very different rates of return to the dark state, which occurs within a few seconds in some cases and a few minutes in others (4,21). FTIR showed that light exposure weakens the carbonyl bonds at C2 and C4 of the flavin, but suggested much larger changes in the protein itself (21,22). Stronger hydrogen bonding between the flavin O4 atom and the neighboring glutamine residue can explain the spectral shifts (13,(21)(22)(23) and is reflected in changes at the exterior of the BLUF domain, near the last β-strand and the surface tryptophan.…”

Section: Significancementioning

confidence: 99%

See 1 more Smart Citation

Molecular mechanism of photoactivation of a light-regulated adenylate cyclase

Ohki

Sato‐Tomita

Matsunaga

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) detects light through a flavin chromophore within the N-terminal BLUF domain. BLUF domains have been found in a number of different light-activated proteins, but with different relative orientations. The two BLUF domains of OaPAC are found in close contact with each other, forming a coiled coil at their interface. Crystallization does not impede the activity switching of the enzyme, but flash cooling the crystals to cryogenic temperatures prevents the signature spectral changes that occur on photoactivation/deactivation. High-resolution crystallographic analysis of OaPAC in the fully activated state has been achieved by cryocooling the crystals immediately after light exposure. Comparison of the isomorphous light-and dark-state structures shows that the active site undergoes minimal changes, yet enzyme activity may increase up to 50-fold, depending on conditions. The OaPAC models will assist the development of simple, direct means to raise the cyclic AMP levels of living cells by light, and other tools for optogenetics.cAMP | BLUF domain | optogenetics | photoactivation | allostery

show abstract

Section: Significancementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Molecular mechanism of photoactivation of a light-regulated adenylate cyclase

Ohki

Sato‐Tomita

Matsunaga

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…The molecular identities of the BLUF dark and photoactivated states remain controversial as X-ray and NMR structures have given different orientations for the conserved glutamine in the dark (i.e., with the amino group hydrogen bonded to the conserved tyrosine or to FAD C 4 dO; see Figure 1A,B). A similar issue holds for W91 and M93, which were found either close to FAD or exposed to the solvent (4-8, 12, 13).The current opinion on the photoactivation of the BLUF domains as observed by steady-state FTIR (14-16), Raman (17,18),, and quantumchemical calculations (26-29) includes light-induced radical pair formation by electron and proton transfer from an essential tyrosine side chain (Y8) to the flavin cofactor on the picosecond time scale. Subsequently, hydrogen bond rearrangement takes place, possibly involving ∼180°rotation of the amide group of an essential glutamine side chain (Q50) which is followed…”

mentioning

confidence: 92%

The Role of Key Amino Acids in the Photoactivation Pathway of the Synechocystis Slr1694 BLUF Domain

et al. 2009

View full text Add to dashboard Cite

BLUF (blue light sensing using FAD) domains belong to a novel group of blue light sensing receptor proteins found in microorganisms. We have assessed the role of specific aromatic and polar residues in the Synechocystis Slr1694 BLUF protein by investigating site-directed mutants with substitutions Y8W, W91F, and S28A. The W91F and S28A mutants formed the red-shifted signaling state upon blue light illumination, whereas in the Y8W mutant, signaling state formation was abolished. The W91F mutant shows photoactivation dynamics that involve the successive formation of FAD anionic and neutral semiquinone radicals on a picosecond time scale, followed by radical pair recombination to result in the long-lived signaling state in less than 100 ps. The photoactivation dynamics and quantum yield of signaling state formation were essentially identical to those of wild type, which indicates that only one significant light-driven electron transfer pathway is available in Slr1694, involving electron transfer from Y8 to FAD without notable contribution of W91. In the S28A mutant, the photoactivation dynamics and quantum yield of signaling state formation as well as dark recovery were essentially the same as in wild type. Thus, S28 does not play an essential role in the initial hydrogen bond switching reaction in Slr1694 beyond an influence on the absorption spectrum. In the Y8W mutant, two deactivation branches upon excitation were identified: the first involves a neutral semiquinone FADH • that was formed in ∼1 ps and recombines in 10 ps and is tentatively assigned to a FADH • -W8 • radical pair. The second deactivation branch forms FADH • in 8 ps and evolves to FAD •-in 200 ps, which recombines to the ground state in about 4 ns. In the latter branch, W8 is tentatively assigned as the FAD redox partner as well. Overall, the results are consistent with a photoactivation mechanism for BLUF domains where signaling state formation proceeds via light-driven electron and proton transfer from Y8 to FAD, followed by a hydrogen bond rearrangement and radical pair recombination.Blue light photoreceptors using flavin cofactors have been the focus of recent research because of their novel mechanisms of photoactivation in contrast to "classical" photoreceptors like phytochromes and rhodopsins. Especially members of the BLUF 1 (blue light photoreceptors using FAD) family (1-3) show an especially intriguing light-induced proton network rearrangement resulting in a 10-15 nm red-shifted spectrum of the signaling state. The BLUF domain shows a ferredoxin-like fold consisting of a five-stranded β-sheet with two R-helices packed on one side of the sheet, with the isoalloxazine ring of flavin adenine dinucleotide (FAD) positioned between the two R-helices (4-12). FAD is noncovalently bound to the protein through a number of hydrogen bonds and hydrophobic interactions. Figure 1 shows the three-dimensional structure of the Synechocystis Slr1694 BLUF domain (also known as PixD) in its proposed dark and light states, with the FAD binding pock...

show abstract

“…In contrast, flavincontaining BLUF (blue-light using flavin adenine dinucleotide (FAD)) (2) proteins show a different photoactivation mechanism that is not accompanied by prominent structural changes in the chromophore (3). In BLUF proteins, the signaling state is characterized by a light-induced~10 nm red shift of the ultraviolet-visible (UV-vis) absorption spectrum (4-10) and an~16 cm À1 downshift of the C4¼O stretching mode of the flavin ring (11)(12)(13). These relatively minor spectroscopic changes are characteristic of BLUF proteins.…”

Section: Introductionmentioning

confidence: 99%

Structural Refinement of a Key Tryptophan Residue in the BLUF Photoreceptor AppA by Ultraviolet Resonance Raman Spectroscopy

Unno¹,

Kikuchi²,

Masuda³

et al. 2010

AIP Conference Proceedings

Self Cite

View full text Add to dashboard Cite

The flavin-adenine-dinucleotide-binding BLUF domain constitutes a new class of blue-light receptors, and the N-terminal domain of AppA is a representative of this family. The BLUF domain is of special interest because it uses a rigid flavin rather than an isomerizable chromophore, such as a rhodopsin or phytochrome, for its light-activation process. Crystal and solution structures of several BLUF domains were recently obtained, and their overall structures are consistent. However, there is a key ambiguity regarding the position of a conserved tryptophan (Trp-104 in AppA), in that this residue was found either close to flavin (Trp in conformation) or exposed to the solvent (Trp out conformation). The location of Trp-104 is a crucial factor in understanding the photocycle mechanism of BLUF domains, because this residue has been shown to play an essential role in the activation of AppA. In this study, we demonstrated a Trp in conformation for the BLUF domain of AppA through direct observation of the vibrational spectrum of Trp-104 by ultraviolet resonance Raman spectroscopy, and also observed light-induced conformational and environmental changes in Trp-104. This study provides a structural basis for future investigations of the photocycle mechanism of BLUF proteins.

show abstract

Light-Induced Structural Changes in the Active Site of the BLUF Domain in AppA by Raman Spectroscopy

Cited by 94 publications

References 25 publications

Molecular mechanism of photoactivation of a light-regulated adenylate cyclase

Molecular mechanism of photoactivation of a light-regulated adenylate cyclase

The Role of Key Amino Acids in the Photoactivation Pathway of the Synechocystis Slr1694 BLUF Domain

Structural Refinement of a Key Tryptophan Residue in the BLUF Photoreceptor AppA by Ultraviolet Resonance Raman Spectroscopy

Contact Info

Product

Resources

About