Histidine Protonation Controls Structural Heterogeneity in the Cyanobacteriochrome AnPixJg2

Abstract

Cyanobacteriochromes are compact and spectrally diverse photoreceptor proteins that bind a linear tetrapyrrole as a chromophore. They show photochromicity by having two stable states that can be interconverted by the photoisomerization of the chromophore. Hence, these photochemical properties make them an attractive target for biotechnological applications. However, their application is impeded by structural heterogeneity that reduces the yield of the photoconversion. The heterogeneity can originate either from the chromophore structure or the protein environment. Here, we study the origin of the heterogeneity in AnPixJg2, a representative member of the red/green cyanobacteriochrome family, that has a red absorbing parental state and a green absorbing photoproduct state. Using molecular dynamics simulations and umbrella sampling we have identified the protonation state of a conserved histidine residue as a trigger for structural heterogeneity. When the histidine is in a neutral form, the chromophore structure is homogenous, while in a positively charged form, the chromophore is heterogeneous with two different conformations. We have identified a correlation between the protonation of the histidine and the structural heterogeneity of the chromophore by detailed characterization of the interactions in the protein binding site. Our findings reconcile seemingly contradicting spectroscopic studies that attribute the heterogeneity to different sources. Furthermore, we predict that circular dichroism can be used as a diagnostic tool to distinguish different substates.

Comparison of the Forward and Reverse Photocycle Dynamics of Two Highly Similar Canonical Red/Green Cyanobacteriochromes Reveals Unexpected Differences

“…In addition to the Tyr rotamers, hydrogen-bonding heterogeneity has been previously suggested for the dark-adapted state of AnPixJg2. , On the basis of MD simulations, Scarbath-Evers et al proposed the existence of two 15 Z P r substates of AnPixJg2 that differ primarily in their D-ring configurations, i.e., α-facial substate I or β-facial substate II. These substates exhibit altered hydrogen bonding networks with the protein mostly involving carbonyl and nitrogen atoms in the D-ring and the B-ring propionate.…”

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

“…There is some progress, especially from NMR and theoretical studies that ascribe these observations for example mixed conformations of tryptophan residues 13–15 or the protonation of histidine residues. 23 However NMR has intrinisically slow time resolution, and there are up to now no experimental studies that access the ultrafast dynamics of the electronic ground states. Here, we employ two dimensional infrared (2D-IR) spectroscopy in combination with isotope editing to isolate two vibrational modes that provide site-specific information about their direct environment within the protein in the Pr and Pg states.…”

Protein–chromophore interactions controlling photoisomerization in red/green cyanobacteriochromes

“…NpR6012g4 is a well-characterized protein with a stable photoproduct state [36,45,54,55,90,96,98,[107][108][109][110], but the equivalent domain (AnPixJg4) instead gives rise to a very unstable photoproduct and hence can function as a sensor of light intensity [46]. AnPixJg2 is also well-characterized [33,46,65,85,[111][112][113][114][115][116], whereas NpR6012g2 is unstable and is only amenable to basic characterization [45]. AnPixJg3 is inactive, whereas NpR6012g3 undergoes facile photoconversion [45,85].…”