A Q63E Rhodobacter sphaeroides AppA BLUF domain mutant is locked in a pseudo-light-excited signaling state

Abstract

The AppA BLUF photoreceptor from Rhodobacter sphaeroides contains a conserved key residue, Gln63, that is thought to undergo a shift in hydrogen-bonding interactions when a bound flavin is light excited. In this study we have characterized two substitution mutants of Gln63 (Q63E, Q63L) in the context of two constructs of the BLUF domain that have differing lengths, AppA1-126 and AppA17-133. Q63L mutations in both constructs exhibit a blue-shifted flavin absorption spectrum as well as a loss of the photocycle. Altered fluorescence emission and fluorescence quenching of the Q63L mutant indicate significant perturbations of hydrogen bonding to the flavin and surrounding amino acids which is confirmed by (1)H-(15)N HSQC NMR spectroscopy. The Q63E substitution mutant is constitutively locked in a lit signaling state as evidenced by a permanent 3 nm red shift of the flavin absorption, quenching of flavin fluorescence emission, analysis of (1)H-(15)N HSQC spectra, and the inability of full-length AppA Q63E to bind to the PpsR repressor. The significance of these findings on the mechanism of light-induced output signaling is discussed.

Figure 1

Absorption spectra of AppA1–126 wt at dark and light (A) and Q63E (B, dash line) and Q63L mutants (B, solid line).

Figure 2

Normalized fluorescence emission spectra. A) AppA1–126wt (Insert: AppA full-length); B) AppA17–133 WT; C) AppA1–126 Q63E and Q63L; (D), AppA17–133 Q63E and Q63L. All samples were measured at dark (D) and light (L) conditions.

Figure 3

Quenching of tryptophan fluorescence with acrylamide. Samples are freshly purified and diluted to A₂₈₀= 0.02 (A) AppA1–126 WT and AppA full-length; (B) AppA17–133 WT; (C) AppA1–126 Q63E and Q63L; (D) AppA17–133 Q63E and Q63L; all samples are measured at dark and light conditions. Ksv values are indicated.

Figure 4

Superposition of the ¹H-¹⁵N HSQC spectra of wild-type (red crosspeaks) and Q63E (blue crosspeaks) AppA1–126 (A) and AppA17–133 (C). The chemical shift perturbation maps of AppA1–126 (B) and AppA1–133 Q63E (D) vs. WT represents the combined ¹H and ¹⁵N chemical shift, where Δδppm = √(Δδ²_H + (Δδ_N/7)²) (36).

Figure 5

Superposition of the ¹H-¹⁵N HSQC spectra of the wild-type (red crosspeaks) and Q63L (blue crosspeaks) in AppA1–126 (A) and AppA17–133 (B); Q63L (blue) vs. Q63E (red) in AppA1–126 (C) and AppA17–133 (D).

Figure 6

Size-exclusion chromatography profile (Sephacryl 200) of AppA WT-PpsR, AppA Q63L-PpsR, and AppA Q63E- PpsR, performed at dark and reducing conditions (5 mM DTT), with 10× excess of AppA. Bottom: SDS-PAGE of chromatography peaks (as indicated) including PpsR, AppA WT, Q63L and Q63E purified proteins as controls. AppA full-length protein often contains two bands as the unstable C-terminal (20–30 amino acids) gets easily degraded.

Figure 7

Ribbon views of the structural differences between wild-type and Q63E AppA17–133 from NMR.chemical shift perturbations. Colors are ramped on a grey ribbon diagram of the structure of AppA17–133 based on the Δδ ppm as follows; magenta, 0.05 < Δδ < 0.1 and red, 0.1 < Δδ < 0.60 (see Figure 4).