Spectroscopic studies of the AppA BLUF domain from Rhodobacter sphaeroides: addressing movement of tryptophan 104 in the signaling state

Abstract

Previous crystallographic studies of the AppA BLUF domain indicated that Trp104 is capable of undertaking alternate conformations depending on the length of the BLUF domain. A BLUF domain containing a C-terminal deletion (AppA1-126) reveals that Trp104 is partially solvent exposed while a BLUF domain containing a slightly longer carboxyl terminal region (AppA17-133) shows that Trp104 is deeply buried. This observation has led to a model proposing that Trp104 moves from a deeply buried position in the dark state to a solvent-exposed position in the light excited state. In this study we investigated whether there is indeed movement of Trp104 upon light excitation using a combination of NMR and absorption spectroscopy, steady-state fluorescence, and acrylamide quenching of tryptophan fluorescence. Our results indicate that AppA17-133 and AppA1-126 contain Trp104 in distinct alternate conformations in solution and that light absorption by the flavin causes partial movement/uncovering of Trp104. However, we conclude that light exposure does not cause dramatic change of Trp104 from "Trp-in" to "Trp-out" conformations (or vice versa) upon light absorption. These results do not support a model of Trp104 movement as a key output signal.

Figure 1

Comparing the three structures of the AppA BLUF domain. Top:Crystallographic structures ofAppA17–133 (green) 1YRX(5) and AppA124 C20S (blue) 2IYG (7). Bottom: NMR structure of AppA5–125 2BUN (4). Side chains important for the photocycle and discussed in this work are labeled.

Figure 2

(A) Chemical shift perturbation map of AppA1–126 vs AppA17–133 represents the combined ¹H and ¹⁵N chemical shift, where Δδ (ppm)=(Δδ²_H + (Δδ_N/7)²)^1/2 (31). (B) Representation of the NMR structure of AppA5–125 (4) with larger chemical shift differences between AppA1–126 andAppA17–133 highlighted. The asterisk indicates the position of V17 that would define the N-terminus inAppA17–133. Yellow highlights local perturbations that occur in the immediate vicinity of V17. In green are residues that exhibit chemical shift differences in the α2 helix.

Figure 3

Normalized fluorescence emission spectra of AppA and its various clones: (a) AppA126, (b) AppA17–133, and (c) full-length AppA. D represents dark-adapted protein (solid lines), and L represents light-excited protein (dashed lines). (d) Nonnormalized fluorescence spectra of dark-adapted full-length AppA, AppA133wt, and AppA126wt using protein of the same concentration (A₂₈₀= 0.2).

Figure 4

Normalized fluorescence emission spectra of AppA mutants: (a) W302A, (b) W64F 1–126, and (c) W64F 17–133. D represents dark-adapted protein (solid lines), and L represents light-excited protein (dashed lines).

Figure 5

Quenching of tryptophan fluorescence with acrylamide: (a) AppA1–126, (b) AppA17–133, (c) AppA126W64F, (d) AppA17–133 W64F, (e) full-length AppA WT, and (f) AppA W302F. Solid lines, dark state; dashed lines, light excited state.

Figure 6

Fluorescence emission spectra and acrylamide quenching of W104A(a), M106A (b), andW104M-M106W(c) mutants in AppA126 clone.