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Abstract The lack of a theory capable of connecting the amino acid sequence of a light-absorbing protein with its fluorescence brightness is hampering the development of tools for understanding neuronal communications. Here we demonstrate that a theory can be established by constructing quantum chemical models of a set of Archaerhodopsin reporters in their electronically excited state. We found that the experimentally observed increase in fluorescence quantum yield is proportional to the computed decrease in energy difference between the fluorescent state and a nearby photoisomerization channel leading to an exotic diradical of the protein chromophore. This finding will ultimately support the development of technologies for searching novel fluorescent rhodopsin variants and unveil electrostatic changes that make light emission brighter and brighter.
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Electrostatic control of photoisomerization pathways in proteins
Rotation around a specific bond after photoexcitation is central to vision and emerging opportunities in optogenetics, super-resolution microscopy, and photoactive molecular devices. Competing roles for steric and electrostatic effects that govern bond-specific photoisomerization have been widely discussed, the latter originating from chromophore charge transfer upon excitation. We systematically altered the electrostatic properties of the green fluorescent protein chromophore in a photoswitchable variant, Dronpa2, using amber suppression to introduce electron-donating and electron-withdrawing groups to the phenolate ring. Through analysis of the absorption (color), fluorescence quantum yield, and energy barriers to ground- and excited-state isomerization, we evaluate the contributions of sterics and electrostatics quantitatively and demonstrate how electrostatic effects bias the pathway of chromophore photoisomerization, leading to a generalized framework to guide protein design.
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- Award ID(s):
- 1740645
- PAR ID:
- 10211374
- Date Published:
- Journal Name:
- Science
- Volume:
- 367
- Issue:
- 6473
- ISSN:
- 0036-8075
- Page Range / eLocation ID:
- 76 to 79
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Rhodopsins are light-responsive proteins forming two vast and evolutionary distinct superfamilies whose functions are invariably triggered by the photoisomerization of a single retinal chromophore. In 2018 a third widespread superfamily of rhodopsins called heliorhodopsins was discovered using functional metagenomics. Heliorhodopsins, with their markedly different structural features with respect to the animal and microbial superfamilies, offer an opportunity to study how evolution has manipulated the chromophore photoisomerization to achieve adaptation. One question is related to the mechanism of such a reaction and how it differs from that of animal and microbial rhodopsins. To address this question, we use hundreds of quantum-classical trajectories to simulate the spectroscopically documented picosecond light-induced dynamics of a heliorhodopsin from the archaea thermoplasmatales archaeon (TaHeR). We show that, consistently with the observations, the trajectories reveal two excited state decay channels. However, inconsistently with previous hypotheses, only one channel is associated with the –C13QC14– rotation of microbial rhodopsins while the second channel is characterized by the –C11QC12– rotation typical of animal rhodopsins. The fact that such –C11QC12– rotation is aborted upon decay and ground state relaxation, explains why illumination of TaHeR only produces the 13-cis isomer with a low quantum efficiency. We argue that the documented lack of regioselectivity in double-bond excited state twisting motion is the result of an ‘‘adaptation’’ that could be completely lost via specific residue substitutions modulating the steric hindrance experienced along the isomerization motion.more » « less
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null (Ed.)Light-absorbing chromophores in photoreceptors contain a π-electron system and are intrinsically planar molecules. However, within a protein environment these cofactors often become nonplanar and chiral in a manner that is widely believed to be functionally important. When the same chromophore is out-of-plane distorted in opposite directions in different members of a protein family, such conformers become a set of enantiomers. In techniques using chiral optical spectroscopy such as Raman optical activity (ROA), such proteins are expected to show opposite signs in their spectra. Here we use two microbial rhodopsins, Gloeobacter rhodopsin and sodium ion pump rhodopsin (NaR), to provide the first experimental and theoretical evidence that the twist direction of the retinal chromophore indeed determines the sign of the ROA spectrum. We disrupt the hydrogen bond responsible for the distortion of the retinal in NaR and show that the sign of the ROA signals of this nonfunctional mutant is flipped. The reported ROA spectra are monosignate, a property that has been seen for a variety of photoreceptors, which we attribute to an energetically favorable gradual curvature of the chromophore.more » « less
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The versatile functions of fluorescent proteins (FPs) as fluorescence biomarkers depend on their intrinsic chromophores interacting with the protein environment. Besides X-ray crystallography, vibrational spectroscopy represents a highly valuable tool for characterizing the chromophore structure and revealing the roles of chromophore–environment interactions. In this work, we aim to benchmark the ground-state vibrational signatures of a series of FPs with emission colors spanning from green, yellow, orange, to red, as well as the solvated model chromophores for some of these FPs, using wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) in conjunction with quantum calculations. We systematically analyzed and discussed four factors underlying the vibrational properties of FP chromophores: sidechain structure, conjugation structure, chromophore conformation, and the protein environment. A prominent bond-stretching mode characteristic of the quinoidal resonance structure is found to be conserved in most FPs and model chromophores investigated, which can be used as a vibrational marker to interpret chromophore–environment interactions and structural effects on the electronic properties of the chromophore. The fundamental insights gained for these light-sensing units (e.g., protein active sites) substantiate the unique and powerful capability of wavelength-tunable FSRS in delineating FP chromophore properties with high sensitivity and resolution in solution and protein matrices. The comprehensive characterization for various FPs across a colorful palette could also serve as a solid foundation for future spectroscopic studies and the rational engineering of FPs with diverse and improved functions.more » « less
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UV-Vis transient absorption (TA) spectroscopy is used to carry out a systematic investigation of the ultrafast CC double photoisomerization dynamics and quantum yield of each isomer of a set of six chromophores based on the same retinal-inspired, indanylidene pyrrolinium (IP) molecular framework. All compounds undergo a sub-picosecond photoisomerization, and can be categorized within two photoisomerization scenarios. Scenario I corresponds to compounds which display the signatures of a vibrationally coherent reactive motion through the conical intersection, with different degrees of vibrational coherence. Qualitatively distinct TA signatures are observed for other compounds which are therefore proposed to obey scenario II, referring to an intermediate regime between scenario I and a thermally-equilibrated, fully stochastic photoreaction. Remarkably, the photoisomerization scenario is observed to correlate with the computed distortion from planarity of the ground state equilibrium geometry, reflecting the torsional strain that would be released after photoexcitation. The most planar compounds – i.e. those having a CC double bond pre-twist of less than 10° – obey scenario II, while compounds obeying scenario I have larger pre-twists. The most pre-twisted compounds (>15°) show pronounced oscillatory signatures of a reaction-induced, low-frequency vibrational wavepacket observed in the S 0 photoproduct and assigned to the torsion mode of the reaction coordinate, thus mimicking the vibrationally coherent photoisomerization dynamics of the rhodopsin protein. Importantly, the systematic comparison of all photoisomerization quantum yields does however not reveal any correlation with observables such as excited state life time, vibrational coherence, absorption wavelengths or degree of pre-twisting.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1126/science.aax1898
