Red, Orange, Green: Light- and Temperature-Dependent Color Tuning in a Cyanobacteriochrome

Abstract

Cyanobacteriochromes (CBCRs) are photoreceptor proteins that photoconvert between two parent states and thereby regulate various biological processes. An intriguing property is their variable ultraviolet−visible (UV−vis) absorption that covers the entire spectral range from the far-red to the near-UV region and thus makes CBCRs promising candidates for optogenetic applications. Here, we have studied Slr1393, a CBCR that photoswitches between red-and green-absorbing states (Pr and Pg, respectively). Using UV−vis absorption, fluorescence, and resonance Raman (RR) spectroscopy, a further orange-absorbing state O 600 that is in thermal equilibrium with Pr was identified. The different absorption properties of the three states were attributed to the different lengths of the conjugated π-electron system of the phycocyanobilin chromophore. In agreement with available crystal structures and supported by quantum mechanics/molecular mechanics (QM/MM) calculations, the most extended conjugation holds for Pr whereas it is substantially reduced in Pg. Here, the two outer pyrrole rings D and A are twisted out of the plane defined by inner pyrrole rings B and C. For the O 600 state, the comparison of the experimental RR spectra with QM/MM-calculated spectra indicates a partially distorted ZZZssa geometry in which ring A is twisted while ring D and the adjacent methine bridge display essentially the same geometry as Pr. The quantitative analysis of temperature-dependent spectra yields an enthalpy barrier of ∼30 kJ/mol for the transition from Pr to O 600 . This reaction is associated with the movement of a conserved tryptophan residue from the chromophore binding pocket to a solvent-exposed position.

Needles in a haystack: H-bonding in an optogenetic protein observed with isotope labeling and 2D-IR spectroscopy

“…This result is in good agreement with a previous resonance Raman study of the related CBCR Slr-g3, where it was shown that the Trp sub-states are not sensed by other vibrational modes associated with ring D, but strongly by ring A. 16 The published FTIR spectra of other red/green CBCRs differ quite substantially for C O A , where the other proteins only show a weak bleach band in the FTIR difference spectra, 25,35–37 and only AmI-g2 displays the sharp positive feature. In line with the assignment of the two C O A bands in the Pr state, we conclude that in the Pg state of AmI-g2, the C O A must be located in a hydrophobic environment that does not allow H-bonding.…”

“…Like in all other proteins, this region is dominated by the amide I vibrations from the protein backbone (termed I′ in deuterated samples), but also contains C O stretching vibrations from amino acid side chains 28 and vibrational modes from the light-sensitive co-factor. 16 These contributions can be distinguished from each other by isotope labeling parts of the system and comparing the IR spectra. Here, we 13 C 15 N-labeled the entire AmI-g2 apo-protein and incooperated isotope-normal PCB.…”

“… 14,23 Resonance Raman spectra of Slr1393g3 in the Pr state could be decomposed into the two sub-states, confirming the two-state nature for a third member of the red/green lineage and providing insight into the differences of PCB configuration and fluorescence properties of the two states. 16 These two sub-states are observed in the 2D-IR spectra at 1720 (H-bond) and 1736 cm −1 (no H-bond). Interestingly, the H-bonded shoulder is heterogeneously broadened and maintains a high CLS of ca.…”

Nanosecond protein dynamics in a red/green cyanobacteriochrome revealed by transient IR spectroscopy

“…Slr-g3 was expressed and assembled with PCB in Escherichia coli BL21 cells in darkness. The holo-Slr-g3 expressing cell line [24] was a generous gift from the lab of Thomas Friedrich (TU Berlin). The protein was purified under native conditions via Ni-affinity chromatography and a His 6 -Tag N-terminal to the Slr-g3 domain and desalted using a Sephadex HiPrep 26/10 column (GE Healthcare Bio-Sciences, Uppsala, Sweden) into a final buffer containing 50 mM Tris (pH 7.8 or 7.4 for subsequent D 2 O exchange), 300 mM NaCl and 5 mM EDTA.…”

“…In the Pg state on the other hand, one methine bridge is isomerized (15E ), and the outer pyrrole rings A and D are twisted out of plane, leading to an effective reduction in conjugation length and thus the characteristic hypsochromic shift of the absorption maximum (Fig. 1D) [21][22][23][24]. The Pg and Pr states differ also with respect to the protein structure, e.g.…”