Seeing the light with BLUF proteins

Abstract

First described about 15 years ago, BLUF (Blue Light Using Flavin) domains are light-triggered switches that control enzyme activity or gene expression in response to blue light, remaining activated for seconds or even minutes after stimulation. The conserved, ferredoxin-like fold holds a flavin chromophore that captures the light and somehow triggers downstream events. BLUF proteins are found in both prokaryotes and eukaryotes and have a variety of architectures and oligomeric forms, but the BLUF domain itself seems to have a well-preserved structure and mechanism that have been the focus of intense study for a number of years. Crystallographic and NMR structures of BLUF domains have been solved, but the conflicting models have led to considerable debate about the atomic details of photo-activation. Advanced spectroscopic and computational methods have been used to analyse the early events after photon absorption, but these too have led to widely differing conclusions. New structural models are improving our understanding of the details of the mechanism and may lead to novel tailor-made tools for optogenetics.

Keywords: Allostery; Flavin; Optogenetics; Photo-activation.

Fig. 1

A sequence alignment of the six BLUF (Blue Light Using Flavin) domains for which independent experimental models have been deposited in the Protein Data Bank. The conserved residues, shown on a red background, are clustered around the flavin chromophore. Secondary structure elements from the crystal structure of OaPAC, a photo-activated adenyl cyclase (PAC) from Oscillatoria acuminata, are indicated with coils and arrows to indicate α-helices and β-strands, respectively, and turns are indicated by T. Tyr 8, Gln 48, Met 92 and Trp 90 of OaPAC form the quartet of residues that attract the most interest in functional studies. Asn 30 forms hydrogen bonds directly with the flavin. This figure was created with the program Espript (Robert and Gouet 2014)

Fig. 2

Hydrogen bonds formed by conserved residues with the flavin chromophore in the BLUF domain of OaPAC. The figure shows a ribbon diagram of the N-terminal 100 residues of the protein, with helix 1 omitted for clarity. The ribbon is coloured from blue to red (N to C terminal end). The flavin mononucleotide (FMN) is shown as a stick model, with carbon atoms coloured yellow, oxygen red and nitrogen blue. The O4 of the flavin receives hydrogen bonds from both Asn 30 and Gln 48, two absolutely conserved side-chains in the BLUF family

Fig. 3

Ribbon diagram of the BLUF domain of OaPAC, coloured as in Fig. 2. The side-chain of Trp 90 is shown as a stick model, and the prominent kink in the backbone between this residue and the β5-strand is clearly visible. Residues at this point play an important role in signalling at the domain surface the changes that occur around the buried flavin upon photo-excitation

Fig. 4

A schematic diagram showing the movement of an electron from the conserved tyrosine upon photo-excitation, giving a bi-radical form. Rearrangement of the hydrogen bonding between the protein and prosthetic group allows the tyrosine to lose a hydrogen atom and the flavin to gain one, while at the same time Gln 48 isomerises to the imidic form

Fig. 5

Ribbon diagram of the complete OaPAC dimer. Each subunit contains 366 residues, consisting of a BLUF domain, helical region (coiled-coil) and C-terminal adenylate cyclase domain. One dimer is coloured light blue, and the other is shown in dark blue (N-terminus) to red (C-terminus). The flavin molecules are shown as red stick models. Activation of the adenylate cyclase domains by light involves signal transduction from the flavin through the central coiled-coil to the active sites